TIHIIE WNilWIE ~§JI'Ir"V ((])IF 'II' IE .TI.AQ IF>101R II.lI tCA.'ll'lICON NO. 5721 November 1, 1957 Studies in the GENETICS OF DROSOPHILA IX. ARTICLES ON GENETICS, TAXONOMY, CYTOLOGY, AND RADIATION Edited by MARSHALL R. WHEELER ASSOCIATE PROFESSOR OF ZOOLOGY THE UNIVERSITY OF TEXAS THE UNIVERSITY OF TEXAS AUSTIN Copies of this publication may be procured for $2.00 each from the University Press, The University of Texas, Austin, Texas Studies in the GENETICS OF DROSOPHILA IX. ARTICLES ON GENETICS, TAXONOMY, CYTOLOGY, AND RADIATION Edited by MARSHALL R. WHEELER ASSOCIATE PROFESSOR OF ZOOLOGY THE UNIVERSITY OF TEXAS THE UNIVERSITY OF TEXAS AUSTIN The benefits of education and of useful knowledge, generally diffused through a community, are essential to the preserva­tion of a free government. SAM HOUSTON Cultivated mind is the guardian genius of Democracy, and while guided and controlled by virtue, the noblest attribute of man. It is the only dictator that freemen acknowledge, and the only security which freemen desire. MIRABEAU B. LAMAR PUBLISHED BY THE UNIVERSITY TWICE A MONTH. ENTERED AS SECOND-CLASS MATTER ON MARCH I2, I9I3, AT THE POST OFFICE AT AUSTIN, TEXAS, UNDER THE ACT OF AUGUST 24, I912 Contents I. A Study of lnterspecific Hybridization Between Members of the Tripunctata Group of Drosophila . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 J. T. PATTERSON II. Intraspecific Relationships of Drosophila croczna Patterson and Mainland from . Three Localities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 WILLIAM B. HEED III. Thirteen New Species in the Genus Drosophila from the Neotropical Region 17 WILLIAM B. H EED AND MARSHALL R. WHEELER IV. Drosophila insularis, A New Sibling Species of the Willistoni Group . . . . . . . . . 39 THEODOSIUS DoBZHANSKY, LEE EHRMAN, AND OLGA PAVLOVSKY V. Morphological Differences Between Sibling Species of Drosophila . . . . . . 48 B. SPASSKY VI. Ecological and Distributional Notes on the Drosophilidae (Diptera) of El Salvador. 62 WILLIAM B. HEED VII. Taxonomic and Distributional Studies of Nearctic and Neotropical Drosophilidae. . 79 MARSHALL R. WHEELER VIII. A Parthenogenetic Strain of Drosophila mangabeirai Malogolowkin .. ........... 11 5 H. L. CARSON, M . R. WHEELER, AND w. B. HEED IX. A Preliminary Note on the Cardini Group of Drosophila in the Lesser Antilles . .. 123 WILLIAM B. HEED X. Chromosomal Studies of Several Species of Drosophila . . . . . . . . . . . . . . . . . . . . . . . . . 125 FRANCESE. CLAYTON AND MARVIN WASSERMAN XI. Further Studies on the Repleta Group ..... . . .. . .. . .......... . .. .. ........ . . . 132 MARVIN WASSERMAN AND FLORENCE D. WILSON XII. Studies on Experimental Populations of Drosophila arizonensis and Drosophila mojavensis ............................ ..... ...... .............. .. 157 LAWRENCE E. METTLER XIII. An Attempt to Detect Hybrid Matings Between D. mulleri and D. aldrichi under Natural Conditions .............. . . .. . ....... . . . .. . , .............. 182 WILLIAM B. HEED XIV. Genetic Studies on Drosophila mulleri. I. The Genetic Analysis of a Population .. . 186· wARREN P. SPENCER XV. Genetic Studies on Drosophila mulleri. II. Linkage maps of the X and Chromo­some II with Special Reference to Gene and Chromosome Homologies . . . 206: wARREN P. SPENCER XVI. Relationships between Species Groups of the Subgenus Pholadoris, Drosophila (Diptera: Drosophilidae) . ............ . ..... .. . . . ........ . ... . .... 218: WHARTON B. MATHER XVII. Genetic Relationships of Four Drosophila Species from Australia (Diptera: Dro­sophilidae) . ..... ... .... .. .. ... ..... . .. . .. . .. . ... .. .. . ........... 22~ WHARTON B. MATHER XVIII. A New Drosophilid from Australia (Diptera) ..... ...... ..................... 226 MARSHALL R. WHEELER XIX. Genetic Variation of Isoxanthopterin Content in Drosophila melanogaster . . . . . . . . 229 FORBES W. ROBERTSON AND HUGH s. FORREST XX. The Production of Attached-X Chromosomes in Drosophila hydei ............... 238 THOMAS G . GREGG XXL Effects of Irradiation on the Screw-worm, Callitroga hominivorax (Coq.) . 246 GAIL KAUFMAN AND MARVIN WASSERMAN XXII. Genetic Studies of Irradiated Natural Populations of Drosophila . .... .. .. ....... 260 WILSON S. STONE, MARSHALL R. WHEELER, WARREN P. SPENCER, FLORENCE D. WILSON, JUNE T. NEUENSCHWANDER, THOMAS G . GREGG, ROBERT L. SEECOF, CALVIN L. WARD -: ·. Preface This bulletin constitutes No. IX of the series of publications originated by Dr. J. T. Patterson in 1940. The 22 articles appearing here were contributed by the genetics staff, students, and visiting investigators. Intensive investigations into the fields of taxonomy, species relationships, and evolution in Drosophila have been carried out since 1938, when Dr. Patterson began his studies of the populations on Capt. Aldrich's farm outside Austin. During the intervening years, collecting was extended from Texas to the remain­der of the United States, and to Mexico. The work of our collaborators in Brazil indicated that Drosophila species in Central America and the islands of the Caribbean should be especially valuable in studies of evolution and relationship; accordingly, William B. Heed spent 1953-54 in San Salvador as a guest of the Instituto Tropical de Investigaciones Cientificas, Universidad Autonoma de El Salvador, where a tremendous wealth of Drosophila material was collected. We wish to thank Professor Adolph Meyer-Abich, Founder of the Institute, for helping arrange Mr. Heed's stay there. We also express our thanks to Dr. Aris­tides Palacios, Director General, and Dr. Helmut Meyer-Abich for their great help. Mr. 0 . C. Harper, Midland, Texas, kindly provided funds with which Mr. Heed extended his collections into Honduras and Nicaragua. Our work has now expanded into the entire Caribbean area, and the expenses of the field collecting have been provided for by a grant from the National Sci­ence Foundation. The first 11 articles in this bulletin are based wholly or in part on material collected on expeditions financed by this grant. Our collectors have been Dr. W. B. Heed (principal collector), Dr. Marvin Wasserman, Dr. H. L. Carson. Mr. Hugo Hoenigsberg, and Mrs. Marta Breuer. Collections have been made in South America for us by Dr. Th. Dobzhansky, Dr. C. Pavan, Dr. Danko Brncic, and Mrs. Breuer. The basic genetics laboratory is supported by the Rockefeller Foundation and The University of Texas; research in biochemical genetics is supported in part by the Welch Foundation. The Atomic Energy Commission, through grant AT­( 40-1 )-1323, has provided funds and support for the investigation of the Dro­sophila populations of the Marshall Islands. In addition, a great many people have contributed their time and knowledge in helping our collectors. A simple listing of these people seems hardly adequate, but we want them all to know that the wonderful cooperation and friendliness which they have shown has been most sincerely appreciated. We are especially indebted to members of the Rockefeller Foundation in Colombia and Trinidad, and to the staff of the United Fruit Company in Colombia and Honduras, for their very great assistance. In Honduras, we wish to thank Dr. Wilson Popenoe, Director, and Dr. Louis Williams, Escuela Agricola de Panamericana, Tegucigalpa; Dr. N. C. Thornton, Director, and Dr. Robert Roberts, of the Tropical Research Dept., United Fruit Co., La Lima; and Dr. Robert P. Armour, Director, Research Dept., Lancetilla. In Nicaragua, we acknowledge the help of Mr. Raymond White, Director, Es­tacion Experimental Agricola del Recreo, El Recreo, and Mr. A. R. McBirney, Compania Minera la India, Managua. In Costa Rica, we wish to thank Dr. Jorge Leon, Chief Botanist, lnstituto ln­teramericano de Ciencias Agricolas, Turrialba, and Dr. Bernal Fernandez, San Jose, a former student of The University of Texas, for his generous personal hospitality. In Panama, Mr. James Zetek, Former Director, and Dr. Carl Koford, Resi­dent Naturalist, Canal Zone Biological Area (Barro Colorado Island) were unusually helpful. In Colombia, Mr. Hugo Hoenigsberg, Senior, of Barranquilla, helped our col­lectors in many ways, and the following members of the Rockefeller Founda­tion's Agricultural Program must also be acknowledged: Dr. Lewis M. Roberts, Director, Bogota; Dr. U. J. Grant, Local Director, Medellin; and Mt. Rafael Bravo, Facultad de Agronomia, Palmira. On Trinidad, Dr. Wilbur Downs of the Trinidad Regional Virus Laboratory, Port of Spain, very graciously supplied laboratory facilities and other comforts. In Venezuela, Mr. Walter Arp, Resident Director of the Estacione Biologia de Henry Pittier, Rancho Grande, Maracay, was of considerable help, as were Mr. William H. Phelps, and Mr. William H. Phelps, Jr., of the Coleccion Orni­tologica Phelps, Caracas, and Dr. Pablo Cova Garcia, Jefe Sec. Estudios Especi­ales, Division de Malariologia, Maracay, has cooperated by sending a number of Venezuelan Drosophila to our laboratory. On Puerto Rico, Dr. Irving Fox, School of Tropical Medicine, San Juan; Dr. George Wolcott, Agricultural Experiment Station, Rio Piedras; Dr. John B. Grant, Professor of Public Health and Medical Care, University of Puerto Rico, San Juan; and Dr. H. E. Warmke, U. S. Department of Agriculture, Federal Experiment Station, Mayaguez, have all been most helpful. On Jamaica, we wish to thank Dr. Thomas H. Farr, Science Museum, King­ ston for his help. Finally, we wish to express our gratitude to our collaborators in South Amer­ ica, not only for the collections which they have made for us, but also for their great help in solving some of the problems in identification and relationship. Dr. Crodowaldo Pavan, Dr. A. B. da Cunha, Mrs. Marta Breuer and Mr. L. E. Magalhaes of the University of Sao Paulo, Brazil, have been especially helpful in this respect, as has Dr. Danko Brncic of the University of Chile, Santiago. MARSHALL R. WHEELER, for the Genetics Group Austin, Texas August 8, 1957. I. A Study of lnterspecific Hybridization Between Members of the Tripunctata Group of Drosophila J.T.PATTERSON INTRODUCTION At the time these tests were begun in June 1955, 31 species belonging to the tripunctata group had been described. All of these are included in the important monograph by Frota-Pessoa (1954), who established four subgroups, I, II, III, and IV. We had among our laboratory stocks eight of the 31 described forms, plus one newly described species (D. paramediostriata) . In addition two unde­scribed species were also in stock. These two have been given names as follows: D. trapeza, D. trifiloides, and they are described elsewhere in this volume. These made a total of 11 available species for this study. In Table 1 the 11 species are listed, together with the localities at which the original flies had been collected, and the names of the collectors. TABLE 1 Subgroups and Species Collecting Localities Collectors II. medionotata Frota-Pessoa 1954 La Palma. El Salvador H eed II. mediopunctata Dobzhansky and Pavan 1943 Itanhaen, Sao Paulo, Brazil Nona ta II. unipunctata Patterson and Mainland 1943 La Palma, El Salvador H eed III. crocina Patterson and M ainland 1944 San Salvador, El Salvador Heed III. mediostriata Duda 19Z5 San Salvador, El Salvador Heed III. paramediostriata Townsend and Wheeler 1955 Rio Piedras, Puerto Rico Townsend III. trapeza H eed and Wheeler (this bulletin) La Lima, Honduras Heed III. trifiloides Wheeler (this bulletin) La Lima, H onduras Heed IV. albicans Frota-Pessoa 1954 San Salvador, El Salvador Heed IV. metzii Sturtevant 19Z1 San Salvador, El Salvador Heed IV. tripunctata Loew 186Z Gulfport, Mississippi Wheeler Past experience with hybridization tests among Drosophila species has shown that small mass matings are more effective in obtaining hybrids than are pair matings. Consequently, we used mass matings of about 20 pairs per each large food vial. Three such cultures were established for each cross, making a total of about 60 pairs per test, and since 11 species were employed, the total number of reciprocal crosses will be 110. The males and females to be used were separated within 24 hours after the culture vials had been cleared of all flies. They were then aged for five days be­fore making the crosses. After five days the flies were transferred to fresh food vials, and at the end of this period, 20 females from each cross were dissected and their reproductive tracts examined for the presence of sperm. All of the remain­ing flies were then changed to a fresh food vial and kept long enough to make it possible to determine whether the cross was fertile or incompatible. At first both the corn-meal and banana-agar media were used, but since the latter gave the better results, it was employed for all the tests. HYBRIDIZATION TESTS The results obtained for the 110 crosses are tabulated in Table 2, and reveal that they fall into three categories, as follows: first, crosses which produced no hybrids; second, crosses which also yielded no hybrids but the dissections re­vealed that some few females had been inseminated; and third, crosses which gave some form of hybridization. The first group included 97 of the 110 crosses, or slightly over 88% of the total. These 97 contained 11,644 flies, which is the equivalent of 5,822 pairs. TABLE 2 H y bridization tests Crosses Pairs Females Males tested Hybrids Remarks I. albicans X crocina 57 none incompatible cross 2. crocina > + none 5 & incompatible cross incompatible cross incompatible cross incompatible cross incompatible cross incompatible cross Fi X Fi fertile incompatible cross 70. 71. metzii X m ediostriata mediostriata X paramediostriata 65 70 none larvae incompatible cross F1 X F., fertile Z6'i'+ZZ& 7Z. paramediostriata X mediostriata 70 larvae F, X Fi fertile 5 'i' + H 73. 74. 75. mediostriata X trapeza trapeza X mediostriata mediostriata X trifiloides 60 60 60 none none none incompatible cross incompatible cross incompatible cross 76. trifiloides X mediostriata 65 none incompatible cross 77. 78. 79. 80. 81 . 8Z. 83. 84. 85. medios:riata X tripunctata tripunctata X mediostriata mediostriata X unipunclala unipunctata X mediostriata metzii X paramediostriata paramediostriata X metzii metzii X trapeza trapeza X metzii metzii X trifiloides 60 60 63 60 60 60 60 60 57 none none none none none none none none none incompatible cross incompatible cross incompatible cross incompatible cross incompatible cross incompatible cross incompatible cross incompatible cross incompatible cross 86. trifiloides X metzii 60 none incompatible cross 87. metzii X tripunctata 60 larvae no adults produced pupae 88. 89. 90. tripunctata X metzii metzii X unipunctata unipunctata X metzii 60 60 60 none none none incompatible cross incompatible cross incompatible cross 91. paramediostriata X trapeza 60 none incompatible cross 9Z. trapeza X paramediostriata 60 none incompatible cross 93. 94. paramediostriata X trifiloides trifiloides X paramediostriata 60 60 none none incompatible cross incompatible cross 95. 96. paramediostriata X tripunctata tripunctata X paramediostriata 58 60 none none incompatible cross incompatible cross 97. 98. paramediostriata X unipunctala unipunctata X paramediostriata 60 54 none none incompatible cross incompatible cross 99. trapeza X trifiloides 60 none incompatible cross 100. trifiloides X trapeza 60 none incompatible cross 101. trapeza X tripunctata 60 none incompatible cross The University of Texas Publication T ABLE 2.----Continued Hybridization tests Crosses Pairs Females M:iles tested Hybrids Remarks 10Z. tripunctata X trapeza 60 none incompatible cross 103. trapeza X unipunctata 60 none incompatible cross 104. unipunclata X trapeza 60 none incompatible cross 105. trifiloides X tripunctata 60 none incompatible cross 106. tripunctata X trifiloides 60 none incompatible cross 107. trifiloides X unipunctata 66 none incompatible cross 108. unipunctata X trifiloides 60 none incompatible cross 109. tripunctata X unipunctata 60 none incompatible cross 110. unipunctata X tripunctata 60 none incompatible cross The second group included but six crosses, which are indicated in the last col­umn of Table 2. In Cross 5 (albicans c;> and 13 J 5, plus several non-viable larvae. The F1 males mated to both types of parental females produced 16 c;> c;> and 19 J 5 to mediostriata females, and none to paramediostriata females, not even non-viable larvae. The reciprocal Cross 72 (paramediostriata c;> X mediostriata J ) gave many non-viable larvae and only nine adults, five c;> c;> and four 5 J. The F1 flies were all used for inbred tests and produced 35 pupae, from which 13 females and 11 males emerged. In Cross 87 (metzii c;> X tripunctata J) many non-viable larvae and some pupae appeared in the culture, but no adults emerged from these pupae. In Table 3 are summarized the results of the 110 reciprocal crosses. The table was constructed in order to facilitate the discussion and conclusions. The species included in Frota-Pessoa's subgroup I were not available for study. Only three species were available from his subgroup II, five from subgroup III, and three from subgroup IV. The symbols "Hy" and "In" represent the seven cases of hy­bridization and six of insemination, respectively. The numbers following these symbols refer to the numbers of the crosses as given in Table 2. Among the reciprocal crosses within subgroup II, only one yielded hybrids (Hy-68), and none gave an insemination. The matings of the females of these three species to the males of the five of subgroup III gave a single case of in­semination (ln-61), which was found in the cross between mediopunctata fe­males to trapeza males. The females of subgroup II mated to the males of sub­group IV gave two cases of hybrids (Hy-6, Hy-57) and one of ~nsemination (In-20). It is interesting to note that medionotata females did not cross with any of the males of the other ten species, while the females of mediopunctata crossed to the males of three other species, and those of unipunctata mated to the males of two other species. The females of subgroup III crossed to the males of subgroup II yielded a single case of hybridization. This was from trapeza females to the males of medionotata (Hy-48). The matings within subgroup III produced two cases of hybrids and one of insemination. The hybrids were from the reciprocal crosses between the two related species mediostriata and paramediostriata (Hy-71, Hy­72). The single case of insemination was from the cross of crocina females to trifiloides males (ln-33). In subgroup IV the females of albicans mated to the males of mediopunctata produced a single case of insemination (In-5). There were no successful crosses between members of subgroups IV and III. But in crosses within subgroup IV one case of hybridization (Hy-87) and two of insemination occurred (ln-10, ln-17). These results indicate that the species of these two subgroups are com­pletely isolated from each other, for in a total of thirty crosses not a single case of either hybridization or insemination was obtained. CONCLUSIONS One may' conclude from the results obtained in the cross tests that these 11 species of the tripunctata group are rather highly isolated from one another. There are, however, two cases in which the members of two species are rather - to TABLE 3 MALES ~ FEMALES II. medionotata II. mediopunctata Subgroup II medio­medio­no ta ta punctata 0 0 uni­punctata 0 0 crocina 0 0 Subgroup III medio­paramedio­striata stria ta 0 0 0 0 trapeza 0 In-61 tri­filoides 0 0 Subgroup IV albicans metzii 0 0 Hy-6 Hy-57 tri­punctata 0 0 t:! ~ ..... §..... ..... ~ II. unipunctata III. crocina 0 0 Hy-68 0 0 0 ... 0 0 0 0 0 0 · o In-33 In­20 0 0 0 0 0 .Q. ~ III. mediostriata 0 0 0 0 Hy-7t 0 0 0 0 0 ~ III. paramediostriata 0 0 0 0 Hy-72 0 0 0 0 0 ~ III. trapeza Hy­4·8 0 0 0 0 0 0 0 0 0 ~ III. trifiloides IV. albicans 0 0 0 In-5 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 In-17 ~ ........... ~ IV. metzii 0 0 0 0 0 0 0 0 In-10 Hy-87 s· IV. tripunctata 0 0 0 0 0 0 0 0 0 0 ~ similar morphologically, with the possibility that gene exchanges could ocwt. The first of these include mediopunctata and unipunctata, which show consider­able resemblance to each other, but the cross was fertile only when unipunctata represented the female parent. The F1 hybrids were fertile in the inbred test, and partially fertile in the backcrosses. There is one striking difference in the two species in the behavior of the larvae during pupation. The larvae of medio­punctata pupate at the surface of the food, while those of unipunctata pupate within masses of food which they had carried up on the inner surface of the vial. The second pair of species between which gene exchanges could occur includes mediostriata and paramediostriata, which are very similar morphologically. The reciprocal crosses were fertile, but went better when mediostriata was employed as the female parent. The F, flies of both crosses were fertile, as indicated above. There is also a possibility of gene exchange in a third case. This is Cross 6 (D. mediopunctata ~ X D. albicans ~) in which the F1 male was fertile in backcrosses. The final point of interest is the degree of isolation that exists between the dif­ferent species. The results obtained in the tests show that several isolating mecha­nisms operate to prevent the exchange of genes. The most important of these is sexual isolation, or the failure of the sexes to mate. Of the 110 reciprocal crosses, 97, or slightly more than 88%, were incompatible. The writer never observed a single copulating pair in all of these cultures. Occasionally a male would per­form feeble courtship movements, but in every instance the female gave no response. A second type of isolating mechanism is present in Crosses 5, 10, 17, 20, 33, and 61. In each cross the female had been inseminated, but in f'ach case the sperm had become inactivated or even killed in the reproductive tract of the alien female. This type of isolation has been termed sperm or gametic mortality. :\ third type of isolating mechanism is represented by crosses in which the larvae are non-v~able and fail to pupate, or pupate without producing adults (Cross 87). Even when hybrids are produced the cultures may contain dead larvae (Crosses 71, 72) . This form of isolation has been termed zygotic mortality. Finally, a fourth isolating mechanism is revealed in matings producing only a few hybrids, such as seen in Crosses 6, 48, and 57, as well as in matings yielding larger numbers of progeny, in which some of the F, flies exhibited hybrid sterility. Geographical isolation must be an important means of preventing gene ex­changes between the different members of the group. At present we do not have enough accurate data on distribution to justify an extended discussion of the subject. However, we do know that D. tripunctata Loew is the only species of the group thus far collected in the United States, although Dr. W. B. Heed has collected a closely related form in Costa Rica, Panama, and Colombia. REFERENCES Dobzhansky, Th. and C. Pavan. 1943. Studies on Brazilian species of Drosophila. Biol. Facul. Fil. Cien. e. Letr. Univ. Sao Paulo. No. 36, Biol. Geral., 4: 7-72. Duda, 0. 1925. Die costaricanischen Drosophiliden (Diptera) des Ungarischen National Museums zu Budapest. Ann. Mus. Nat. Hung., 22: 149-229. The University of Texas Publication Frota-Pessoa, 0 . 1954. Revision of the tripunctata group of Drosophila with descriptions of fifteen new species (Drosophilidae, Diptera). Arquivos do Museu Paranaense, 10: 253-330. Loew, H. 1862. Diptera Americae septentrionalis indigena. Berl. ent. Zeit., 6: 185-232. Patterson, J; T., and G. B. Mainland in Patterson. 1943. The Drosophilidae of the Southwest. Univ. of Texas Puhl., 4313:7-203. Patterson, J. T., and G. B. Mainland. 1944. The Drosophilidae of Mexico. Univ. of Tex. Puhl., 4445: 9-101. Sturtevant, A. H. 1921. The North American species of Drosophila. Carneg. Inst. Wash. Puhl 301. 150 pp. Townsend, J. I., and Marshall R. Wheeler. 1955. Notes on Puerto Rican Drosophilidae, including descriptions ,'}f two new species of Drosophila. Jour. Agric. Univ. Puerto Rico, 39: 57--64. II. lntraspecific Relationships of Drosophila crocina Patterson and Mainland from Three Localities WILLIAM B. HEED1 The distribution of Drosophila crocina Patterson and Mainland extends from central Mexico to northern South America. Drosophila campestris Burla is quite possibly a synonoym; if this is so, then the distribution is extended into Brazil. D. crocina has also been taken on all of the islands of the Greater Antilles. The species may be recognized by the presence of a row of short bristly spines along the inner surface of the fore femora and by the clouding of the anterior and posterior crossveins. It is a member of the tripunctata group of the subgenus Drosophila. D. crocina is variable in abdominal pattern from one region to another. The most outstanding variation is the darkened body of the island forms. Strains from three widely separated localities, showing different degrees of variation, were tested for their ability to cross with one another. The strains used are as follows: No. H15.8, from El Salvador; No. H109.17, from Trinidad; and No. H131.2, from Puerto Rico. Other less obvious morphological differences became evident in the strains chosen for testing; these differences are difficult to measure, but may be summarized as follows: El Salvador Trinidad Puerto Rico abdomen brownish yellow brownish yellow brownish gray mesonotum yellowish brown brown grayish brown crossveins clouded strongly clouded clouded wings paler grayish paler eye size regular larger regular On the basis of these characters, it may be seen that the Trinidad strain is unique in three respects, the Puerto Rico strain in two, and the El Salvador strain in one. In another sense, the Trinidad and Puerto Rico strains differ from each other in five features, Trinidad and El Salvador in four, and El Salvador and Puerto Rico in two. The morphological differences coincide with the results of the hybridization tests given below. Table 1 shows the results from pair mating tests of the three strains, mated in all combinations. We have used the letter "S" for El Salvador, "T" for Trinidad TABLE 1 Pair matings of D. crocina from three localities <;? ~ No. pairs tested Percent fertile Total F, <;? <;? ~ ~ Average No. F, per fertile<;? F, s x s 22 50 691 366 325 62.8 yes T X T 22 64 952 486 466 68.0 yes P x P 23 26 403 207 1Y6 67.2 yes S x T 24 42 820 393 417 82.0 few S x P 30 33 965 51 1 454 96.5 yes T x S 21 0 . . . . .. T x P 21 14 239 136 103 79.7 no P xS 27 0 P x T 20 5 79 36 43 79.0 no Zoological Laboratory, Univ. Pennsylvania, Philadelphia. and "P" for Puerto Rico. The F1 from these matings were mass mated to test for fur,ther fertility. Table 2 shows the results from mass matings of the three strains; the F1 from these matings were further mass mated to derive an F2• When no F2 generation was produced, the first date of emergence of the F1 is re­corded to show the number of days they were under test. TABLE 2 Mass matings of D. crocina from three localities Average Earliest Number Date Total no. date F, Date ) ; wing length a bout 2.0mm. ( J ),2.3mm. ( <;> ). Internal characters of imagines and genitalia. Anterior Malpighian tubes branched basally near gut; posterior tubes apposed without continuous lumen. Testes with about 8 very pale yellow outer coils and 4--5 white inner coils. Sperm pump diverticula short, about half greatest diameter of pump. Forceps with 6-8 primary teeth, 5-6 strong secondary teeth on upper surface and 6-8 marginal bristles. Toe usually with 3 long bristles; 2--6 bristles on genital arch in back of toe, sometimes continuous with toe bristles. Head of penis arrow-head shaped with 2 horn-like processes pointing back toward the apodeme. Shells of hypandrium very narrow. Spermathecae pear-shaped, about 0.13 mm. long, yellow brown in color; inner duct funnel-shaped. Ventral receptacle with about 60 coils. Ovipositor plate with about 19-21 teeth, apex rounded. Other characteristics, relationship, and distribution. Eggs. With 2 pairs of thin filaments. Larvae. Posterior spiracles not black; do not skip. Puparia. Red-brown; anterior spiracle with about 12 gray branches, the horn, including spiracles, about 1,4 length of puparium. Chrorrwsomes. Five pairs of rods and one pair of large dots. The longest rod pair represents the X chromosomes; the Y is a rod about % the length of the X. Salivary preparations show 5 long arms and the dot chromosome. Determinations are by Dr. Clayton, using stock No. H29.16 from San Salvador, El Salvador, and by Dr. Mettler, using stock No. H167.1 from La Lola, Costa Rica. Relationship. Belongs in subgroup III of the tripunctata group of the sub­genus Drosophila. Distribution. A widely distributed but relatively uncommon species of the Neotropical region. Collected by the senior author and other Texas collectors (1952-56) in Vera Cruz, Mexico, El Salvador, Honduras, Costa Rica, Nicaragua, Colombia, Venezuela and Trinidad at elevations from sea-level to 5000 feet; also collected at the Agricultural Experiment Station, Rio Piedras, Puerto Rico, and at Kingston, Jamaica, B.W.I., in Feb., 1956. Types. Holotype male, 2 ~ and 3 9 paratypes, from stock No. H167.1 from La Lola, Costa Rica. Notes. D. trapeza is attracted to banana bait but it is never found in great quantities in any collection. The largest number taken on any day was 22 indi­viduals ( 4.8% of the total collection) collected over zapote fruits (Calocarpum) at San Salvador in May. It has been reared in El Salvador from the following plants: Helosis (Balanophoraceae), Inga preussii Harms (Leguminosae) , Cas­tilla gummifera Pittier (Moraceae), and Brosimum terrabanum Pittier (Moraceae) . Distinguishing characters are: the typical trapezoidal mark on 6th tergite of males, the small delicate paramedian triangles on 6th tegite of females, and the slight markings of the pleura. In general view, the head seems somewhat too small for the size of the body. Drosophila mediodiffusa Heed and Wheeler, new species. External characters of imagines. ~ . Arista with 5-6 dorsal and 3 ventral branches in addition to the fork. An­tennae and front brown; few frontal hairs. Proclinate orbital about % length posterior reclinate; anterior reclinate 1,4 proclinate. Only one strong oral, the 2nd about % as long. Carina prominent, a bit longer than 3rd antenna! segment. Face whitish but not strikingly so. Greatest width of cheek about Ys greatest diameter of eye, extremely narrow directly below the eye. Eyes scarlet with silvery pile. Palpi with 8-10 strong bristles on lateral margin and 30-40 shorter bristles ventrally. Acrostichal hairs in 6 rows; no prescutellars. Anterior scutellars straight or slightly divergent. Mesonotum shining brown, scutellum darker. Pleura very light tan to nearly whitish. Anterior sternopleural about 0.3 length posterior, the middle bristle usually shorter than anterior. Legs tan; numerous recurved hairs on fore tarsi; flexor and lateral extensor surfaces of fore femur with several long bristles; several strong bristles near base of hind metatarsus. Apical bristles on 1st and 2nd tibiae, preapicals on all three. Wings grayish, posterior crossvein clouded, anterior crossvein dark or slightly The University of Texas Publication clouded. Costal index about 3.3; 4th vein index about 1.7; 5x index about 1.2; 4c index about 0.8. Two prominent bristles at apex of 1st costal section; 3rd sec­tion with heavy bristles on the basal % . Vein 2L bends to costa near its tip. Abdomen shining tan with grayish shading dorsally which is darker on tergites 2 and 3. Body length (etherized) about2.1 mm.; wing, 2.0 mm. Female. Palpi with 4-5 strong bristles on lateral margin and about 12 shorter bristles on ventral surface. A few recurved hairs on fore tarsi. Abdomen shining tan with a diffuse gray band as broad as the tergite on 2nd and 3rd segments which terminate at the angle; the remainder of the abdomen varies from a gray­ish dorsum with a slight clear midstripe, to shining tan. Body length (etherized) about 2.5 mm.; wing, 2.3 mm. Internal characters of imagines and genitalia. Anterior Malpighian tubes branched at about the basal 1/ 10; posterior tubules with a continuous lumen. Testes with 3 pale yellow outer coils and 2 white inner coils. Sperm pump diverticula slightly longer than greatest diameter of the pump. Forceps with 6-8 primary teeth in a straight row, 4-6 long bristles on upper sur­face and 4-9 short marginal bristles. No bristles on toe; head of penis with 2 prominent horn-like processes extending back toward the apodeme. Spermatheca chitinized, brown, the width about equalling the length; inner duct usually with uniform diameter but sometimes the distal portion is widened into a cup. Ventral receptacle with only about 6 irregular coils. Ovipositor plate with 23-28 teeth. Other characteristics, relationship, and distribution. Eggs. With 2 pairs of thin filaments. Puparia. Red-brown; anterior spiracle with 15-16 white branches, the horns, including spiracles, about 14 length of puparium. Larvae. Posterior spiracles yellow; do not skip. Chromosomes. Five pairs of rods and one pair of dots; the Xis about twice as long as the autosomes and as the Y; one autosomal pair has satellites on the centromere end. The Y is a rod and the dot pair is very small. The analysis was made by Dr. Mettler using stock No. H130.6 from Puerto Rico. Relationship. Belongs in subgroup IV of the tripunctata group of the sub­genus Drosophila; it is most similar to albescens and tristriata. Distribution. Collected by the senior author in January and February, 1956, in Haiti, Jamaica, and Puerto Rico. Mrs. Marta Breuer also collected the species in Cuba in January, 1956. Types. Holotype male and 6 paratypes of both sexes, descendants of stock No. H129.16 from El Yunque Resort Area, Caribbean National Forest, Puerto Rico at about 2000 feet elevation. Notes. D. mediodiffusa comes readily to fruit baits and is easily cultured on banana medium. The species is named for the diffuse, indistinct abdominal pattern. Drosophila medioparva Heed and Wheeler, new species. External characters of imagines. 5 , !i? • Arista with 7 dorsal and 3 ventral branches in addition tq the fork. Face white; carina reaching nearly to level of vibrissae, round~d at tip, not sulcate. Antennae, cheeks and front brown. Proclinate orbital about % length posterior reclinate; anterior reclinate about 14, length proclinate. One strong oral; greatest cheek width about Ys greatest diameter of eye, extremely narrow directly below eye. Eyes red with pale pile. Palpi with about 4 long bristles along antero-lateral margin, gradually decreasing in size posteriorly. Acrostichal hairs in 6 rows; no prescutellars. Anterior scutellars divergent. Mesonoturn and scutellum semi-shining brown; pleura and legs paler. Anterior and mid-sternopleurals about Jf2 length posterior one. Fore femur usually with 5 prominent bristles on lateral extensor surface and 3 on flexor surface. Several recurved hairs on fore tarsus. No heavy black bristles at base of hind metatarsus. Apical bristles on 1st and 2nd tibiae, preapicals on all three. Wings with grayish cast; posterior crossvein clouded. Costal index about 4.0; 4th vein index about 1.5; 5x index about 1.4; 4c index about 0.6. One prominent bristle at apex of 1st costal section; 3rd section with heavy bristles on the basal Jf2. Abdomen brownish yellow with black apical bands on tergites 2-4 which are incompletely interrupted on 2-3 and usually completely interrupted on 4th. The widest band is on the 3rd tergite where it is about half the tergite width. All bands fade at the angle of the tergites. Tergite 5 usually without markings, but rarely with a pair of faint, small, paramedian spots. Tergite 6 with a large round median spot that extends almost the width of the tergite. Sexes similar. Body length (pinned specimens) about 2.2 mm.; wing length about 2.3 mm. (all measurements made on males). Internal characters of imagines and genitalia. Posterior Malpighian tubes apposed but apparently without a continuous lumen. Testes with 8-9 outer yellowish coils and 3-4 inner coils. Sperm pump diverticula short, about % length of pump. Forceps with 8 primary teeth in a curved row, and 12 marginal bristles that come around onto upper surface. Toe large with about 8 bristles. Gonapophysis without conspicuous bristle. Basal part of anal plate with a straight row of about 6 small, well-spaced bristles. Spermathecae elongate, about 0.18 mm. long and 0.1 mm. wide; inner duct tubular with a median constriction. Ventral receptacle with about 40 coils. Ovipositor plate with 21 teeth, the apex rounded. Other characteristics, relationship, and distribution. Eggs. With 2 pairs of thin filaments. Larvae. Posterior spiracles black. Puparia and chromosomes. Unknown. Relationship. Belongs in subgroup IV of the tripunctata group of the sub­genus Drosophila. It is quite similar to D. albicans in the male genitalia, but the two species are easily distinguished on other features. Distribution. Collections were made by the senior author and Drs. Carson and Wasserman in July and August, 1956, as follows: Costa Rica: Turrialba, 2000'; La Lola, 128'; Panama: Cerro La Campana, 60 klm. southwest of Panama City, 2700'; Colombia: El Recuerdo, 24 klm. southeast of Santa Marta, 4000'. Mr. Hoenigsberg assisted in the last locality. Types. Holotype male and 3 paratype males, No. H154.10, from Turrialba, Costa Rica. The University of Texas Publication Notes. Collected over hule fruits (Castilla) in Costa Rica and on rotting celery cabbage in Panama. It was not common in any collection. Two males were reared by Dr. Wasserman from fruit of Anthurium (Araceae). We have been unable to raise this species on banana medium as yet. The species may be recognized by its small size, white face, large round spot on 6th tergite, rather large eye, and semi-shining mesonotum. The only striking difference in male genitalia from albicans is the arrangement of small bristles along the base of the anal plate, since in albicans these are formed into a closely­set row of 10 or more bristles. Drosophila spinatermina Heed and Wheeler, new species. External characters of imagines. J , S' . Arista with 5-6 dorsal and 2-3 ventral branches in addition to the terminal fork. Face and 3rd antennal joint tan; front dull brown with paler areas at bases of orbitals and in ocellar triangle, the latter extending as a thin line to anterior margin. Proclinate orbital about % length posterior reclinate; anterior reclinate about Vi. proclinate. Two strong orals of equal length. Carina promi­ nent with rounded edges, not sulcate. Greatest cheek width about Vi. greatest di­ ameter of eye, about 1;6 directly below eye. Eyes bright red with light pile. Palpi with 2-3 long bristles on antero-lateral margin and about 20 shorter ones on ventral surface and margin. Acrostichal hairs in 6 rows; no prescutellars. Anterior scutellars divergent. Mesonotum and scutellum subshining brown; pleura brown. Anterior sterno­ pleural about Yz length posterior; middle bristle about half length anterior one. Legs light brown; a few recurved hairs on fore tarsus. Lateral extensor surface of fore femur with 4 long bristles; flexor surface with 3 long bristles. Apical bristles on 1st and 2nd tibiae, preapicals on all three. Wings clear; posterior crossvein clouded. Tip of 3L usually with slight shading. Costa} index about 2.7; 4th vein index about 1.7; 5x index about 1.5; 4c index about 0.9. Two prominent bristles at apex of 1st costal section; 3rd section with heavy bristles on the basal Yz. Vein 2L bends toward costa at its tip, and 3L usually bends toward 4th at its tip, the apex of the wing appearing rounded. Abdomen shining yellow with very narrow dark brown posterior bands on 2nd and 3rd tergites that are interrupted in the midline and which extend to the angle of the tergite. Remainder of abdomen without marks. Body length (etherized) a bout 2. 0 mm. ( 6 ) , 2. 4 mm. ( s> ) ; wing length a bout 1.9 mm. ( J ), 2.1 mm. ( s> ). Internal characters of imagines and genitalia. Anterior Malpighian tubes branched at about the basal 1/ 10; posterior tubes with continuous lumen. Testes with about 7 thin outer coils and 4 thicker inner coils; white. Sperm pump diverticula about half length of pump itself. Anal plate with a single prominent spine on lower tip; toe with 4 bristles; forceps with a row of 8-9 primary teeth, 5-8 secondary teeth and 8-9 marginal bristles which continue around to upper surface. Head of penis rather large and complex, in a filiform envelope. Spermathecae dark brown, oval, the greatest width %. the length; inner duct broadly funnel-shaped, narrowing gradually to a constriction near base with a slight bulblike expansion just before the point of exit. Ventral receptacle with over 50 coils. Ovipositor plate with 15-17 teeth, the tip somewhat blunt. Other characteristics, relationship, and distribution. Eggs. With 4 thin filaments, the apical pair thinner and shorter than the basal pair. Larvae. Posterior spiracles yellow; do not skip. Puparia. Red-brown; anterior spiracle with 24-26 thin yellow branches; horns, including spiracles, about % length of puparium. Chromosomes. Three pairs of rods, one pair of V's and one pair of dots. One rod pair is longer than the other two, and longer than the arms of the V. Determination made by Dr. Mettler on stock No. H106.1 from Port of Spain, Trinidad. Relationship. A rather atypical member of subgroup IV the tripunctata group of the subgenus Drosophila. It resembles tripunctata in some respects, including the low costal index, darkening at apex of 3L, small size, and shape of ovipositor plate. It resembles members of the cardini group in having a spine on the lower tip of the male anal plate and large secondary teeth on the forceps. Distribution. Known from two individuals (and their descendants) col­lected by the senior author while sweeping terrestrial aroids near the Queen's Savannah, Port of Spain, Trinidad in December, 1955. Types. Holotype male, 3 i and 4 'i? paratypes, descendants from stock No. H106.1 from Port of Spain, Trinidad. Notes. Some individuals from the stock show a doubling of ocellar bristles on one or both sides, and a few females have the proclinate and posterior reclinate bristles doubled on one side or the other. Such abnormalities were found in about 30% of the 52 individuals examined. Drosophila tristriata Heed and Wheeler, new species. External characters of imagines. ~ , 'i? • Arista with 5-6 dorsal and 3 ventral branches in addition to the fork. Antennae and front brown; few frontal hairs. Proclinate orbital % posterior reclinate; anterior reclinate 1,4 proclinate. One strong oral, 2nd about half length 1st. Carina widened below, not sulcate. Face whitish in females, tan to whitish in males; on pinned specimens the face turns rather brownish. Greatest cheek width about 1/ 7 greatest diameter of eye; cheek exceedingly narrow directly below eye. Eyes red with silvery pile. Male palpi with about 8-10 strong bristles on lateral margin and 15 shorter bristles on ventral sudace. Female palpi with 4-6 strong bristles on lateral margin and about 9 shorter bristles on ventral sur­face. Acrostichal hairs in 6 rows; no prescutellars. Anterior scutellars slightly di­vergent. Mesonotum and scutellum shining brown; pleura whitish. Anterior sternopleural %-Yz posterior; middle one 1,4-Yz anterior. Legs whitish tan; re­curved hairs on fore tarsi. Flexor surface and lateral extensor surface of fore fe­mur with 3-5 long bristles each. Several long yellow bristles present on base of hind metatarsus. Apical bristles on 1st and 2nd tibiae, preapicals on all three. The University of Texas Publication Wings grayish; posterior crossvein clouded, anterior crossvein dark to slightly clouded. Costal index about 3.2; 4th vein index about 1.6; 5x index about 1.3; 4c index about 0.7. One prominent bristle at apex of 1st costal section; 3rd section with heavy bristles on the basal ¥2 or slightly less. Vein 2L bends to costa at its tip and is slightly darkened apically. Abdomen shining yellow with posterior black bands on 2nd and 3rd tergites that fade at the angle and are usually diffusely interrupted medianly, but some­times with diffuse mid-dorsal extensions. Tergites 4-6 in male with median longi­tudinal stripes that usually extend the length of the tergite; last 3 segments sometimes with narrow posterior bands in male, quite variable in females, some­times with median stripes as in males, or with only median dots, or without mark­ings. A narrow posterior band usually present on 4th when median markings are present. Body length (etherized) about 2.0 mm. ( J ), 2.5 mm. ( c;>); wing length about2.0mm. ( J ),2.2mm. ( c;> ). Internal characters of imagines and genitalia. Anterior Malpighian tubes branched basally near gut; posterior tubes with continuous lumen. Testes with 3 outer yellow coils and 2 inner white coils. Sperm pump diverticula about %. greatest diameter of the pump. Genital arch with well developed anterior process; forceps with 8-9 primary teeth in a straight row, usually 6 long pale bristles on upper surface and 6 long marginal bristles. Toe with 1-2 bristles subapically; lower tip of anal plate with a loose tuft of about 10 hairs. Head of penis rounded, with a lateral pair of short rounded chitinized processes (ventral view). The median hornlike projection connecting the arms of the hypandrium, between the forceps, unusually narrow. Spermatheca round, brown; inner duct with uniform diameter. Ventral re­ ceptacle with about 15 irregular coils. Ovipositor plate with 22-27 teeth, the apex fairly broad. Other characteristics, relationship, and distribution. Eggs. With 2 pairs of thin filaments. Larvae. Posterior spiracles yellow; do not skip. Puparia. Yellowish brown; anterior spiracle with about 16 whitish branches; horns, including spiracles, about~ length of puparium. Chromosomes. Five pairs of rods and one pair of dots; one rod pair is nearly twice the length of the others and bears satellites; two pairs of shorter rods also bear satellites. The X and Y were not recognized. The analysis was made by Dr. Wasserman using stock No. H109.22 from Port of Spain, Trinidad. Relationship. Belongs in subgroup IV of the tripunctata group of the sub­ genus Drosophila and is most similar to albescens and mediodiffusa. It may be distinguished from mediodifjusa by the presence of a rather strong 2nd oral, abdominal stripes (when present) and characteristic genitalia. D. albescens differs from the other two by the nearly transparent mesonotum and absence of sperm pump diverticula, and from mediodiffusa by the absence of prominent hornlike processes on the head of the penis; it differs from tristriata also in the absence of bristles on the toe, and the lack of an unusually narrowed connection between the forceps. Studies in the Genetics of Drosophila Distribution.-Collected by the senior author in El Salvador: San Salvador, throughout the year, 1953-54; Honduras: La Lima; April 1954; Trinidad: Port of Spain, Dec. 1955; Puerto Rico: Rio Piedras Experiment Station, Jan. 1956; and Jamaica: Bath and Kingston, Feb. 1956. Types.-Holotype male, 3 5 and 7 <;> paratypes, from collection No. H109.22 from the vicinity of Port of Spain, Trinidad. Notes.-Near Port of Spain, Trinidad the species was collected in a cacao finca on the fallen red flowers of Erythrina micropteryx Poepp. (Leguminosae) where, on Dec. 24, 1955, the species made up 7.5% of the collection of 374 flies, and on Dec. 26, 1955, this species accounted for 6.5 % of the 369 flies collected. Drosophila neomorpha Heed and Wheeler, new species. This new member of the cardini group is most similar to polymorpha and parthenogenetica; it occurs in both a dark and a light form. External characters of imagines. CS , 'i' . Arista with 5 dorsal and 2 ventral branches in addition to the fork. Face, cheeks and antennae light tan; two orals of equal length; carina fairly broad, reaching below 3rd antenna! joint, not sulcate. Proclinate orbital equal to length of posterior reclinate; anterior reclinate about V5 the other two. Front tan; base of orbitals and ocellar triangle shining, the latter eJ..tending forward to level of orbital bases. Few frontal hairs present. Greatest width of cheek about V5 greatest diameter of eye; eye red with light colored pile. Palpi of male with 2-3 long bristles on antero-lateral margin and 25-30 only slightly shorter ones ( % length long o.nes) on anterior and ventral surfaces. Palpi of female with one strong bristle subapically and about 16 shorter ones (%length of long bristle) on an­terior and ventral surface. Acrostichal hairs in 6 rows; no prescutellars. Anterior scutellars convergent. Mesonotum, scutellum and pleura shining yellowish-brown; legs and halteres lighter. Anterior sternopleural about % posterior; mid-sternopleural about % anterior. . Fore femur with one prominent bristle on basal lateral extensor surface; flexor surface with 1-2 prominent bristles about equal to greatest width of femur in length and 1-2 shorter bristles about Yz-% length of longer ones in addition to several still shorter ones. Posterior surface of fore femur fairly densely haired, especially in male. Numerous recurved hairs on fore tibia and tarsus; fore meta­tarsus with one, sometimes two, prominent bristles at base. Apical bristles on 1st and 2nd tibiae, preapicals on all three. Wings clear, crossveins not clouded. Costa! index about 3.2; 4th vein index about 1.7; 5x index about 1.5; -4c index about 0.8. One strong and one weak bristle at apex of 1st costal section, this area slightly darkened; 3rd costal section with heavy bristles on the basal Yz. Abdomen shining brovynish yellow with polymorphic pattern. The light and dark forms of both sexes will be described, but several grades of intermediates exist. Light form, male: narrow posterior black bands on tergites 2-4, interrupted medianly, fading at the angle; 5-6 sometimes without markings or with 5th The University of Texas Publication segment only with a suggestion of a posterior band; 6 with a pair of small para­median spots at posterior border. Genital arch darkened. Dark form, male: tergites 2--4 with shining black posterior bands, interrupted medianly and extended basally to preceding tergite at the angle but failing to reach lateral margin; 5 shining black except for a narrow dorsal interruption, a central narrow area along edge of preceding segment and a narrow area along lateral margin; 6 shining black except along lateral margin. Light form, female: tergites 2--4 with medianly interrupted black bands about % width of tergite; the bands stop rather abruptly at the angle and continue toward but fail to reach lateral margin as a much narrower line or band. Inter­rupted band of tergite 5 very narrow, almost reaching lateral margin; 6 with only a suggestion of a band. Segments 3-6 with an extra narrow mark on anterior border slightly beyond the angle. Genital arch darkened. Dark form, female: bands become progressively wider posteriorly until seg­ments 5-6 where they are slightly more than half the width of the tergite; at the angle the bands of tergites 3-6 extend forward to basal border; 6 with non­interrupted band with paramedian extensions that reach base; area around anal plates darkened. Body length (etherized) about 2.5 mm. ( J ), 2.8 mm. ( g); wing length about 2.1 mm. ( t ),2.4mm. ( g ). Internal characters of imagines and genitalia. Anterior Malpighian tubes branched at their distal Y6; posterior tubes apposed, without a continuous lumen. Testes with about 6 outer yellow coils and 3 thicker inner white coils. Sperm pump diverticula about twice the greatest diameter of the pump. Forceps with 7-8 primary teeth, 8-10 strong secondary teeth and about 8 marginal bristles that come around onto upper surface. Lower tip of anal plate with 3 strong spine-like bristles that are thicker and shorter than the usual anal plate bristles. Hypandrium with one long bristle; base of genital arch with 1-2 long bristles. Spermatheca vase-shaped, the inner duct broadly funnel-shaped in its upper %. Ventral receptacle with about 70 coils. Ovipositor plate with 18-22 teeth. Other characteristics, relationship, and distribution. Eggs.-With 2 pairs of thin filaments. Larvae.-The larvae skip. Puparia.-Reddish brown; anterior spiracle with about 18 grayish branches; horns, including spiracles, about %length of puparium. Chromosomes.-Two pairs of V's, one pair of rods, and a pair of dots. One V­pair has slightly unequal arms; the Xis a rod with a satellite on the centromere end and is slightly longer than an arm of the metacentric V. The Y is slightly shorter than the X and lacks a satellite. Salivary chromosomes show 5 long arms and the dot; the X has much heterochromatin at the centromere end. No in­versions were seen in 5 preparations. Analysis made by Dr. Mettler using stock No. H80.8 from Barro Colorado Island, Canal Zone. Relationship.-Belongs to the cardini group of the subgenus Drosophila and is most similar to D. parthenogenetica and D. polymorpha. Distribution.-Collected at Chapulhuacan, Hidalgo, Mexico (Wasserman and Heed, June 1952); Lancetilla, Honduras (Heed, April 1954); Barro Colorado Island, Canal Zone (Heed, Oct. 1955) (Heed, Wasserman, Carson, Aug. 1956); ' Cerro La Campana, Panama (Heed, Wasserman, Carson, Aug. 1956); Arima. Valley, Trinidad (Heed, Dec. 1955). Types.-Holotype male, 2 t, 2 ~ paratypes, descendants from stock No. H80.8 from Barro Colorado Island, Canal Zone; 1 t , 1 ~ paratype from stock No. H107.21 from Arima Valley, Trinidad. Notes.-D. neomorpha may be distinguished from polymorpha and partheno-­genetica by the following combination of characters: long bristles on flexor sur­face of fore femur, strong spine-like bristles on lower tip of male anal plate, and long secondary bristles on palpi. Dr. Harrison D. Stalker, Washington University, St. Louis, Missouri, tested this species for parthenogenetic tendencies; a total of 14,278 eggs was examined from 40 virgin females. Seven eggs began development but died before hatching; a single larva hatched from the egg but died within 24 hours. No adults were produced by parthenogenesis. Drosophila nigrodunni Heed and Wheeler, new species. External characters of imagines. t. Arista with 5 dorsal and 2-3 ventral branches in addition to the fork. Front dull brown; bases of orbitals and ocellar triangle shining brown; few frontal hairs. Proclinate orbital equal to length of posterior reclinate; anterior reclinate % other two. Two moderately strong orals of equal length. Carina prominent, % as broad as 3rd antenna! joint, fused to mouth margin. Face brown; greatest cheek width about Vs greatest diameter of eye, about 1/ 7 directly below eye. Eyes garnet with short, pale pile. Palpi with 6-8 strong bristles on anterior distal margin and about 20-25 shorter bristles on ventral surface. Acrostichal hairs in 6 rows; no prescutellars. Anterior scutellars convergent. Mesonotum subshining grayish brown, darker on scutellum and pleura. Anterior1 sternopleural about % posterior; middle bristle Yz length anterior one. Legs tan; numerous recurved hairs on fore tarsus. Flexor surface of fore femur with 2-4 long bristles; lateral extensor surface with 1-3 long bristles. Apical bristles on 1st and 2nd tibiae, preapicals on all three. Wings clear; posterior crossvein dark but not clouded. Costal index about 3.2; . 4th vein index about 1.8; 5x index about 1.6; 4c index about 0.8. Two prominent bristles at apex of 1st costal section; 3rd section with heavy bristles on the basal half. Vein 2L bends to costa at apex. Abdomen generally shining brownish black with a tan pattern dorsally. Ter­ gite 2 with a faint posterior gray band vaguely interrupted, and a tan spot an­ teriorly at the angle; 3-4 with darker wide posterior bands, usually interrupted, and with narrow paramedian extensions reaching basal border. Entire lateral area from angle of tergite brownish black. The pattern thus obtained is a pair of dorsal rectangular tan areas on 3-4; 5-6 shining black. Body length (etherized) about 2.2 mm.; wing, 2.0 mm. ~. Palpi with 4-6 strong bristles on anterior distal margin and about 10-16 shorter bristles on ventral surface; few recurved hairs on fore tarsus. Abdomen with shining black bands with definite brownish cast on 2-3. Paired rectangula~ dorsal tan areas sometimes on all tergites. Posterior half of 5th and all of 6th usually entirely black. Body length about 2.5 mm.; wing, 2.1 mm. Internal characters of imagines and genitalia. Anterior Malpighian tube branched at its distal V6; posterior tubes apposed without a continuous lumen. Testes white, with 5 outer and 3 inner coils. Sperm pump diverticula slightly longer than greatest diameter of pump. Forceps with 6-7 primary teeth in a straight row, 5-8 secondary teeth and 8-10 marginal bristles. Spermathecae chitinized dark brown with a slight indentation at distal tip; inner duct funnel-shaped. Ovipositor plate with 18-21 teeth. Other characteristics, relationship, and distribution. Eggs.-With 2 pairs of thin filaments. Larvae.-The larvae skip Puparium.-Brown, redder with age; anterior spiracle with 9-10 branches; horns including spiracles, about 115 length of puparium. Chromosomes.-Two pairs of V's, one pair of rods and one pair of dots. The rods are slightly longer than an arm of the V's. Analysis made by Dr. Mettler using stock No. H119.4 from Barbados. Relationship.-Belongs to the cardini group of the subgenus Drosophila and is most similar to D. dunni. Distribution.-Barbados, British West Indies. Collected by the senior author over fruit bait in Turner's Hall Woods, and in several orchards. Types.-Holotype male, 4 & , 3 ~ paratypes, descendants from stock No. H119.4 collected Jan. 1956, at Grant's Castle, Barbados. Drosophila aracea Heed and Wheeler, new species. This interesting new species cannot, as yet, be assigned to any of the estab­lished subgenera of Drosophila. External characters of imagines. & , ~ . Arista with 3 dorsal and 2 ventral branches of moderate length in addition to the terminal fork. Face, cheeks and antennae brown; carina of moder­ate length, rounded. One strong oral, 2nd much weaker, lateral to 1st rather than ventral to it. Front brown, about as broad as long; frontal hairs present. Base of orbitals and ocellar triangle shining brown. Proclinate and posterior reclinate orbitals usually nearly of equal length but somewhat variable; anterior reclinate about 14 proclinate and laterad of the other two. Postverticals about % (some­times equal) length of two long orbitals. Eye castillian red with light colored pile; eyes round. Cheeks wide, the greatest width about% diameter of eye. Palpi with 3 long bristles on antero-lateral margin; numerous shorter bristles on ventral surface. Acrostichal hairs in about 6 irregular rows; no prescutellars. Anterior scu­tellars convergent; posterior scutellars usually directed upwards at a 45 ° angle. Usual anterior.dorsocentrals about % length posterior ones and placed rather far forward 04-115 distance from transverse suture to scutellum), the distance be­tween anterior dorsocentrals equalling that from anterior to posterior pair so that the two pairs form a square. Anteriorly in each dorsocentral row, immediately behind transverse suture, there is a shorter bristle, a little larger than acrostichal Studies in the Genetics of Drosophila hairs and about half length of anterior dorsocentrals. Other hairs in the dorso­central row may sometimes be enlarged and bristle-like. Mesonoturn, scutellum and pleura shining light brown; halteres and legs paler brown. No middle sterno­pleural; anterior one about Y3 posterior. Fore femur with 5 prominent bristles on flexor surface and 4 prominent bristles on lateral extensor surface. Recurved hairs on all tibiae and tarsi. First 4 segments of all tarsi with 2-3 stouter and longer apical bristles; distal tarsal joint of all legs somewhat enlarged, with large empodium and pulvilli. Hairs and bristles on all legs somewhat elongated. Apical bristle developed on 2nd tibia only, preapicals on all three. Wings clear, veins yellow. Costal index about 3.0; 4th vein index about 1.5; 4c index about 0.8; 5x index about 1.1. One prominent bristle at apex of 1st costal section; 3rd section with heavy bristles on the basal ~ or less. The double row of recurved hairs along costa enlarged more than usual in the genus, giving the wing a pectinate appearance. Abdomen considerably wider than thorax, brownish yellow with black apical bands that are widest at the angle of the tergite and fade away just before reach­ing lateral margins. Bands of tergites 2-5 usually with slight mid-dorsal ex­tensions, also sometimes incompletely interrupted here. Sixth tergite with a faint, incomplete band. Posterior borders of tergites 5-6 lined with long thin bristles. Sexes similar. Body length (etherized) about 3.1-3.5 mm.; wings, 2.8 mm. Internal characters of imagines and genitalia. Anterior and posterior Malpighian tubes branched basally near gut, the latter with continuous lumen. Testes with 10-15 bright yellow outer coils and 1-2 thicker white inner coils. Sperm pump diverticula long, about 8~10 times the length of the pump. Anal plates fused ventrally by a strong chitinized bow; for­ceps with 8-10 primary teeth, 5 marginal bristles and 3-6 bristles on upper surface. Toe short and wide, the surface thickly covered with hairs. Base of geni­tal arch with an extended thin pitted shell of about the same size and shape as the hypandrium shell, and which overlies the toe and forceps. Arch of hypan­drium short, not extending beyond shells, these with one median bristle. Head of penis acuminate, without special processes. Spermathecae lightly chitinized, almost round. Ventral receptacle with about 120 tight coils. Ovipositor plate with one row of 23-24 teeth, broad until the very tip where a small extended apex bears the first 5 teeth, a long bristle between teeth 6-7. Other characteristics, relationship, and distribution. Eggs.-With 2 pairs of flattened filaments which taper to a point and which are equal in length and equal to egg length. Larvae.-Posterior spiracles yellow; do not skip. Puparium.-Red-brown; horns, including spiracles, about Y3 length of pupa­rium; each anterior spiracle with 22-24 yellow branches. Chromosomes.-There are 3 pairs of chromosomes: one pair of rods with a subterminal constriction, one pair of large J's, and one pair of V's. Salivary chromosomes show 5 long arms and a dot chromosome; several inversions were seen. The chromocenter is small and very little heterochromatin is present. The University of Texas Publication Determined by Dr. Clayton using stocks No. H46.28 (El Salvador) and H57.69 (Nicaragua). Relationship.-Of uncertain relationship in the genus Drosophila. Distribution.-Collected by the senior author as follows: El Salvador: near San Salvador, 2000', Oct. 1953, Feb., May, June and Aug. 1954; San Marcos Lempa, 150', May 1954; Honduras: La Lima, 100', March 1954; Nicaragua: Santa Maria de Ostuma, 11 klm. north of Matagalpa, 4500', July 1954; Costa Rica: San Jose, 3000', Oct. 1955. Types.-Holotype male, 5 ~, 4 '? paratypes, descendants from stock No. H57.69 from Santa Maria de Ostuma, Nicaragua. Notes.-D. aracea has been collected only from inside the white spathes of aroid flowers (Araceae), where they were feeding and laying eggs on the sticky yellow gynecium at the base of the spadix. A total of 49 individuals has been reared from flowers of the genus Xanthosoma and 10 came from flowers of Syn­gonium in El Salvador. The species seems to prefer fresh water swamps where "elephant ears" (Xanthosoma) are abundant, but it has also been taken in a wet cloud forest in Nicaragua from aroid flowers. In both flowers and laboratory culture vials, individuals have a habit of "flock­ing" together in one place; the species is reared fairly readily on banana medium. Distinguishing characters are: forward position of anterior dorsocentrals; small pair of additional dorsocentral bristles; elongated recurved hairs on costa; en­larged terminal tarsal segment, empodium and pulvilli; flower feeder. REFERENCES Frota-Pessoa, 0 . 1954. Revision of the tripunctata group of Drosophila with description of fifteen new species (Drosophilidae, Diptera) . Arquivos do museu paranaense Curitiba 10 (Art. 6): Z53-330. Heed, W . B. 1957. Ecological and distril:jutional notes on the Drosophilidae (Diptera) of El Salvador. This bulletin. Salles, H. 1947. Sohre a genitalia dos Drosophilidios (Diptera): I. Drosophila melanogaster e D. simulans. Sum. Bras. Biol. 1(15):311-383. IV. Drosophila insularis, a New Sibling Species of the Willistoni Group 1 1 1 THEODOSIUS DOBZHANSKY , LEE EHRMAN , AND OLGA PAVLOVSKY INTRODUCTION For about two centuries, insect species names were given to single specimens, or to series of specimens, of dried remains of insect bodies, preserved on pins in museum drawers. The chief criterion of validity of such "species" was that the specimens be distinguishable without undue trouble, preferably with the aid of no more than a simple hand lens, from other pinned specimens. The ne­cessity of such "alpha taxonomy" for a preliminary orientation in the otherwise baffling and bewildering diversity of insect forms can hardly be questioned. "Alpha taxonomy" continues to be necessary in little known groups and in little known faunae. However, after the taxonomic foundation has been laid, the methods of the so­called "new systematics" are made use of. New systematics studies species not as series of specimens but as biological phenomena. The "alpha taxonomists" have already had the intuition of the existence in nature, among sexually re­producing and cross-fertilizing organisms, of very significant biological units. These are Mendelian populations, or groups of such populations, within which the gene exchange normally occurs, but between which it is impeded or pre­vented by reproductive isolating mechanisms. The remarkable fact is that these reproductively isolated groups of populations coincide, in most cases, with the species of taxonomists. They coincide in most and yet not in all cases; the repro­ductive isolation, which is the touchstone of biological species in sexual organ­isms, is strongly but not absolutely correlated with easy morphological recog­nizability, which is the touchstone of "good" species of "alpha taxonomy." The chief interest of systematics of Drosophila lies in that most of the insects belonging to this genus are singularly amenable to methods of investigation which shed light on species as biological phenomena. Most Drosophilae can be easily and cheaply maintained in laboratory cultures. A variety of tests disclos­i:qg the presence or absence of certain forms of reproductive isolation can readily be imposed. As material for ecological studies in nature Drosophila proved rather intractable; nevertheless, some ecological data can also be secured. In Drosophila one is, then, able in most instances to evaluate the degree of correspondence be­tween the species of "alpha taxonomy" and biological species. It is perhaps gratifying that by and large this correspondence amounts to identity, as shown most convincingly particularly by Patterson and Stone (1952) in their admirable work on the evolution of Drosophila. And yet some note­worthy exceptions have been found (see Patterson and Stone, page 180). The exceptions concern mainly sibling species. Sibling species have been defined by Mayr (1942) as species "which are morphologically very similar and indistin­guishable, but which possess specific biological characteristics and are repro­ductively isolated." It would be premature at present to decide whether sibling i Dept. Zoology, Columbia Univ., New York City. spe~ies are more frequent among Drosophila than among other insects, although takmg the data at their face value this would appear to be so: The work of Sonne­born and his school has shown that sibling species (there dubbed "varieties") are much more numerous among certain infusorians than they are among Dro­sophila. However that may be, and despite some disingenuous suggestions that have been made that Drosophila systematics should be kept within the bounds of "alpha taxonomy," the study of sibling species is certainly important because of the light which it sheds on the nature of species in general. The group of sibling species related to D. willistoni is perhaps one of the most interesting among those known in the genus. . THE WILLISTON/ SPECIES GROUP The willistoni species group is a subdivision of the sub-genus Sophophora of the genus Drosophila. Patterson and Stone (1952, pages 51 and 52) list nine species, and three further species have since been added to the group. They are all native in the Neotropical faunal region, although D. willistoni reaches south­ern Florida, and D. nebulosa occurs in southern Texas and has been found as far north as Nebraska, doubtless an accidental introduction by man. According to Burla et al. (1949), the sibling species related to D. willistoni are four in number, namely D. willistoni Sturtevant, D. paulistorum Dobzhansky and Pavan, D. equinoxialis Dobzhansky, and D. tropicalis Burla and da Cunha. A fifth sibling. Drosophila insularis Dobzhansky may now be added. The siblings are morphologically very close. Although Burla et al. (1949) have listed several minor differences, both external and internal, identification of single specimens, even living ones, used to be practically out of the question. The situation has changed thanks to Spassky's discovery of slight but constant differences in the external male genitalia (Spassky, an article in this volume) . Male specimens may now be identified as to the species. As correctly pointed out by Spassky himself, this does not alter the status of the five species as sib­lings. The morphological differences between these species are still so small that no one would recognize them as distinct species if it were not for other relevant evidence. This evidence shows that the species are completely isolated repro­ductively. Burla et al. (1949) found that the four sibling species known to them fail to cross. Insemination is rarely observed even when females of one species are confined with males of another in the absence of conspecific males (except that D. paulistorum females of the strain used accepted males of the other species relatively easily, though much less so than their own males). However, regard­less of the frequency of insemination by foreign males, no hybrid larvae or adults have ever appeared. It remains unknown whether fertilization of eggs by foreign spermatozoa occurs at all, and if so at what stage the embryos die out. At least equally decisive as the above evidence is the evidence that no gene exchange between the sibling species takes place in nature. Some of the chromo­somes of the sibling species (especially the third) are distinguishable in the preparations of salivary gland cells, and the populations of each species contain species-characteristic inversions. If effective introgression were taking place, these cytological markers would occasionally be exchanged between the species. Rather extensive studies of natural populations failed to disclose a single indi­cation of anything of this sort (da Cunha and Dobzhansky 1954, and much un­published evidence). The geographic distribution of the sibling species is interesting. D. willistoni is distributed most widely from the Argentine Pampa to the West Indies, south­ern Florida, and north-central Mexico (San Luis Potosi), and from the eastern slope of the Andes in Peru and northern Argentina to the Atlantic. The distri­bution areas of the other siblings seemed to be included into each other, in the order willistoni-paulistorum-tropicalis-equinoxialis (Burla et al. 1949), but the study of the abundant material collected by the expeditions of the genetics group of the University of Texas in Central America and the West Indies discloses a somewhat different situation (Dobzhansky 1957). Namely, D. paulistorum is more at home in the south, while its nearest relative, D. equinoxialis, has a more northern distribution. The former species occurs from southern Brazil (Rio Grande do Sul) to Colombia, Panama, Costa Rica and Salvador; it is often the dominant species in superhumid rainforest regions of Brazil, but it seems to be entirely absent in the West Indies, except in Trinidad. D. equinoxialis is, on the contrary, rare in Brazil, but it is frequent or even dominant in Central America and on the islands of the Greater Antilles. The Lesser Antilles have only D. willistoni and D. insularis (see below). D. tropicalis extends from cen­~ral Brazil (Goyaz) where it is rare, to Central America and the Greater An­tilles, where it is common and locally even dominant. It is tempting to speculate that D. paulistorum and D. equinoxialis, and D. willistoni and D. tropicalis were at some time in the past respectively the southern and northern subspecies of their ancestral species. After having evolved complete reproductive isolation, the now full-fledged sibling species all became sympatric in a large territory, embracing the Amazon Valley, the northern part of the continent of South America, a part of Central America, and the Island of Trinidad. The newly discovered, the fifth, sibling species, D. insularis, may now be described. DROSOPHILA INSULARIS DOBZHANSKY, SPECIES NOVA Male and female: Arista with 10-11 branches, both numbers being about equally frequent. Front dusky yellow. Anterior orbital bristles shorter than the posterior, middle orbital one-third posterior. Two prominent oral bristles. Face yellow. Carina short and broad, not sulcate. Cheeks yellow, their greatest width about one-tenth greatest diameter of eye. Eyes bright red with a short brownish pile. Acrostichals in 6 to 8 rows, often quite irregular. Thorax dusky yellow, darker on the average and in flies of equal age than in Drosophila willistoni Sturtevant; pleurae lighter than the thorax. Anterior and middle sternopleurals at most half as long as the posterior and much thinner. Legs greyish yellow. Abdomen yellow with diffuse dark brown bands expanded in the middle and fading out laterally. Wings clear, proportions like in Drosophila willistoni but slightly darker in color, especially in old individuals. Length of body in laboratory-bred specimens: ~ 2.9-3.2 (mean 3.03), i 2.5­ 2.8 (mean ~.65) mm.; wings ~ 2.1-2.3 (mean 2.16), i 2.0-2.1 (mean 2.04) mm. Reproductive organs of the adults, eggs, larvae, and pupae like those in Oro­sophila willistoni, except for minute but constant differences in the external male genitalia which will be described in a separate publication by Mr. B. Spassky. Remarks. Closely related to Drosophila willistoni, from which it differs by a slightly larger body size and a darker pigmentation. These differences, though fairly clear in flies grown in similar environments, are not reliable for identi­fication of single specimens grown in diverse environments. However, the re­productive isolation between Drosophila insularis and Drosophila willistoni is, as shown below, complete. Geographic Distribution. In the sample of about 80 females of willistoni­like species collected on the Island of St. Kitts by Dr. W. B. Heed in January of 1956 and sent to the laboratory in Columbia University, 77 females which yield­ed progenies belonged to D. willistoni and 3 to D. insularis. The sample from the Island of St. Lucia collected by Dr. Heed in the same month contained 105 fertile females of D. willistoni and 1 of D. insularis. The samples from Trinidad, Bar­bados, or the Greater Antilles, contained no D. insularis. This species is probably confined to the Lesser Antilles, but how widespread it is on the islands of this chain cannot be decided from the data now available. Chromosomes. The metaphase group consists of two pairs of metacentric and one pair of acrocentric chromosomes. The "dots" are absent. This meta­phasic configuration is invariant in all known species of the willistoni group, and, with one unconfirmed exception, in all examined species of the second char­acteristically Neotropical group of the subgenus Sophophora, namely the saltans species group. The giant chromosomes in the salivary gland cells show slight but characteristic differences in the different sibling species of the willistoni group (Burla et al. 1949) . Most characteristic are the disc patterns in the distal por­tions of the third chromosomes (cf. Plate 1, page 304, in Burla et al., l.c.). The disc pattern of this chromosome in D. insularis is most nearly akin to that in D. tropicalis, except for the absence of the distinct light "bulb" located proxi­mally from the group of heavy doublets characteristic of the latter species. A further resemblance of D. insularis to D. tropicalis lies in the disc patterns of the right limbs of the X-chromosomes (XR), which have the characteristic "repeat" area in a more nearly subterminal position, while in D. willistoni it is in a sub­median position. SEXUAL ISOLATION As mentioned above, Burla et al. (1949) found that the four sibling species of the willistoni group known to them showed strong sexual isolation from each other. We have, consequently, studied the situation in this respect of the fifth sibling, D. insularis. The usual "multiple choice" method was used in the ex­periments, a mixture of females of two species being confined with males of one of them. ­ Newly hatched females and males were divided in groups of three. Three females of one species, three females of another, and three males conspecific with one of the kinds of females were placed togethed in small containers ("cream­ ers") and left undisturbed for 8-10 days at room temperature (about 22° C. with fluctuations of several degrees). To make the identification of the females of the sibling species easy, the tips of the wings were clipped either on the right or on the left side. The dissection of the females and the examination of the contents of their sperm receptacles under a microscope yielded the data summarized in Table 1. The isolation index was completed according to Stalker (1942). TABLE 1 Numbers of females tested and per cent found inseminated in various crosses of D. insularis with other sibling species Homogamic Heterogamic Isolation Males Females n % n % Index insularis insularis +paulistorum 95 96.8 94 1.06 0.976 insularis insularis +equinoxialis 166 95.1 169 6.47 0.873 insularis insularis +tropicalis 86 96.5 82. 4.57 0.903 insularis insularis +willistoni (St. Kitts) 180 92..3 180 3.8 0.879 insularis insularis +willistoni (Recife) 182. 94.5 182. 10.3 0.791 paulistorum paulistorum +insularis 159 96.85 155 5.03 0.901 equinoxialis equinoxialis +insularis 72. 100 7Z 8.33 0.846 tropicalis tropicalis +insularis 171 98.Z 171 8.76 0.836 willistoni willistoni (St. Kitts) +insularis 180 100 180 1.59 0.957 willistoni willistoni (Recife) +insularis 180 97.Z 180 13.3 0.759 It can be seen that the sexual isolation is quite strongly pronounced in all the crosses studied. And yet, cross-inseminations have been observed in every one of the ten combinations of heterospecific parents which have been tried. In the experiments of Burla et al. (1949) several of the crosses between the sibling species of the willistoni group showed no insemination at all, and only the crosses to paulistorum males have a considerable frequency of inseminations. There seems to be, therefore, a real difference between the behavior of D. in­sularis with respect to the other sibling species and that of these sibling species with respect to one another. There is one further fact that should be noted. The crosses insularis ~ X willistoni S and willistoni <;? X insularis S have been made using two different strains of the willistoni parent, namely a strain from the Island of St. Kitts and from Recife, Brazil. Now, D. insularis shares the islands of St. Kitts and St. Lucia only with D. willistoni and with no other species. The crosses D. insularis x D. willistoni from St. Kitts are, therefore, crosses be­tween strains which are sympatric in origin. The Recife strain of D. willistoni comes, on the contrary, from a locality quite remote from the islands in which D. insularis is known to live. The isolation coefficients in the crosses which in­volve the Recife strain are ostensibly, though not quite significantly, lower than those in the corresponding crosses with D. willistoni from St. Kitts. The chi­square test gives 0.05 < p<0.10 in both crosses. INTERSPECIFIC HYBRIDIZATION Systematic experiments have been made crossing D. insularis to the other four sibling species. All the hybrids so far obtained came from the crosses in which D. insularis was used as the female parent. It would, nevertheless, be premature to conclude that the reciprocal crosses are wholly incapable of pro­ducing viable hybrids, since perhaps an insufficient number of attempts have been made, and since insemination of females of other species by D. insularis males does occur (see a hove). The technique of the experiments was to place several (up to about a dozen) virgin females with approximately equal num­bers of males of a foreign species in a culture bottle. After a lapse of about a week, inspection disclosed the presence or absence of hybrid larvae. If hybrids were absent, the parents were transfered to fresh cultures, to give them another chance to produce hybrids. Flies of D. insularis from every one of the four St. Kitts strains were used in these experiments indiscriminately. The cross insularis c;? X tropicalis t (the strain from Palma, Goyaz, Brazil) yields hybrids most easily. Several hundred such hybrids have been examined from a number of different cultures. Hybrids of both sexes appear, the sex ratio being about 1: 1. The external morphology of the hybrids is apparently normal; study of the copulatory organs of hybrid males by Mr. Spassky (in press) showed these to be approximately intermediate between those of the parental species. The internal reproductive organs are also normal, except for the gonads themselves which are grossly abnormal. The testes of the males are reduced to tiny rudi­ments, smaller in size than the testes of fully grown larvae. No histological ex­amination has been made, except for observation under an intermediate magni­fication (about X 600) of a microscope of freshly dissected gonads flattened under a cover-slip. This examination shows nothing resembling normal sperma­togenesis. The remains of the testes consist of fairly large undifferentiated cells. The ovaries of the hybrid females are composed of normal-looking egg strings and ovarioles, in which the oocytes and the nurse cells are clearly differentiated. In the ovaries of hybrid females which are about a week old one finds abnormal eggs, with a formed chorion and with two filaments which are shorter than those in the eggs of either parental species. Such females were kept with males of both parental species, but, as expected, no progenies resulted. The hybrid females, like the hybrid males, are wholly sterile. The crosses insularis <;? X willistoni t (using strains of D. willistoni derived from flies collected on the Island of St. Kitts) succeed not quite as easily as those to tropicalis males. Nevertheless, hybrids of both sexes have been obtained and studied. Their characteristics are quite similar to those of the insularis <;? X tropicalis t hybrids described above. They are completely sterile. The crosses insularis c;? X equinoxialis t (using the strain from Teffe, Amazonas, Brazil) produce mostly no hybrids at all. However, one cross yielded several pupae which failed to hatch, and in another cross five hybrid females, and no males, were ob­tained. These hybrid females were kept for some time in a culture with males of both parental species, but proved to be sterile. The cross insularis <;? X paulis­tOrum t (a brown-eyed mutant strain) is most refractory. Some larvae have, however, been noted in one of the cultures, but they failed to produce adult hybrids. A most interesting peculiarity of the insularis <;? X tropicalis t and the in­ sularis <;? X willistoni t hybrids, which as far as we are aware has no analogue in any other interspecific hybrids, concerns the behavior of the chromosomes in the salivary gland cells of the hybrid larvae. This matter will be described in more detail elsewhere (Dobzhansky, in press in "Chromosoma"). The essence of the story is as follows. The heterochromatic parts adjacent to the centromeres in all the chromosomes come together in the hybrid cells to form a compact chromocenter which resembles the chromocenter of normal cells. However, the euchromatic parts of the chromosomes of the two species evince no attraction toward each other, and remain completely unpaired. The hybrid males, thus, have eight chromosomal strands radiating from the chromocenter. Two of these eight strands are the two arms of the X-chromosome. Female larvae must have 10 chromosomal strands, four of them being X-chromosomes, but no cell has been found in which all strands were identifiable. The lack of pairing of the euchro­matic chromosome strands certainly cannot be due to lack of linear homology, since the chromosomes remain easily recognizable as homologues by their disc patterns. Another interesting fact is that the X-chromosomes in the male larvae were clearly greater in average diameter than are the autosomes in the same cells, or than the X-chromosomes or the autosomes in female larvae. Anybody with any experience in studying the chromosomes in the salivary gland cells of Dro~ sophila larvae knows that the X-chromosome in male larvae (which is single)' differs in a paler color, but not in average diameter, from the X-chromosome of female larvae (which is double). The observations on the insularis 'i? X tro,picalis J hybrid larvae prove that this behavior of the X-chromosome de­pends upon the sex of the cell and not upon the chromosome being paired or unpaired. VARIATIONS IN THE GENE ARRANGEMENT IN° D. INSULARIS The sibling species of the willistoni group tend to be highly polymorphic with respect to the gene arrangements in their chromosomes. In fact, natural'poptthr­tions of D. willistoni show a diversity of chromosomal structures which is, as far as we know, greater than that recorded in any other species. Itis interesting that chromosomal polymorphism exists even in D. insularis, the populations of whi.Ch are isolated on islands. The four strains of D. insularis from St. Kitts were out-cros'sed to each other and to the strain from St. Lucia. As expected, the crosses went easily and the hybrids were fertile. Three different inversions exist in the St. Kitts population'. One of them includes the sub-terminal portion of the right limb of the second chromosome (II R) . The homologue of the heavily-banded "repeat" located in this chromosome, corresponding to the section 70 in the II R of D. willistoni (see the map in Dobzhansky, 1950) , is included in this inversion. The position of this landmark in the chromosome is more nearly subterminal in D. insularis than in D. willistoni. Another inversion has been found in the subbasal portion of the left limb of the second chromosome (II L). This inversion, like that in II R, is short, but it includes no conspicuous landmark, and its position in terms of the standard map in D. willistoni has not been determined. The third inversion has been found in the subterminal portion of the third chromsome (III). All three inversions exist within the St. Kitts population of D. insularis, and the only avail­able strain from St. Lucia happens to be free from inversions. The precise limits of the inversions in D. insularis in terms of the maps of the salivary gland chromosomes have as yet not been determined. A comparison of the inversions in the species with those recorded in the other four siblings has nevertheless been made, using the published descriptions and the unpublished notes. The inversions in D. insularis are not identical with any found in other species. Thus far, no inversion has been encountered in more than one of the five siblings. CONCLUSIONS AND SUMMARY Drosophila insularis resembles morphologically the other four sibling species of the willistoni group. It shows some slight differences in the external appear­ance, a difference in the structure of the male copulatory organs (discovered by Mr. B. Spassky), and differences in the disc patterns of the chromosomes in the salivary gland cells. Nevertheless, the reproductive isolation between any two of , ~e,five siblings is complete. Of the isolating mechanisms so far discovered, sexual isolation is probably most important in natural populations. Nevertheless, this isolation is not absolute, and cross-insemination has been observed under experi­ . mental conditions, especially when D. insularis females were exposed to males of the other species. Viable hybrids are produced particularly in the cross D. in­sularis 'i' X D. tropicalis t. These hybrids are, however, completely sterile in both sexes. D. insularis is the only one of the five sibling species which is capable of pro­ducing some hybrids with every one of the remaining four siblings. These latter have never been observed to produce any progenies in the intercrosses with each other. The hybrids are, however, completely sterile, thus making D. insularis a reproductively absolutely isolated species. Sexual isolation has been tested be­tween D. insularis and the other species with the aid of the multiple choice meth­od. A pronounced isolation has been observed in every cross, though perhaps less strong on the average than the sexual isolation between the four sibling species other than D. insularis, as studied by Burla et al. (1949). It is tempting to cor­relate the fact that the isolating mechanisms between these latter species are more nearly perfect than those between them and D. insularis with the geo­graphic distribution of these species. The four siblings other than D. insularis have their geographic distributions widely overlapping, so that they are sym­ .patric in extensive territories in South and Central America. D. insularis is, on the other hand, endemic on some of the islands of the Lesser Antilles, and the only other species which occurs there is D. willistoni. Another interesting fact in this connection is that this sexual isolation between D. willistoni and D. insularis is at least ostensibly stronger if the strains tested are sympatric in origin than when they are allopatric in origin. All these facts are consistent with the hypoth­esis that reproductive isolating mechanisms between populations of sympatric species may be perfected owing to pressure of natural selection. REFERENCES Burla, H., da Cunha, A. B., Cordeiro, A. R., Dobzhansky, Th., Malogolowkin, C., and C. Pavan, 1949. The willistoni group of sibling species of Drosophila. Evolution, 3:300-314. Da Cunha, A. B., and Th. Dobzhansky, 1954. A further study of chromosomal polymorphism in Drosophila willistoni in its relation to the environment. Evolution, 8: 119-134. pobzhansky, Th., 1950. The chromosomes of Drosophila willistoni. J. of Heredity, 41: 156-158. ----, 1957. Genetics of natural populations. XXVI. Chromosomal variability in island and continental populations of Drosophila willistoni from Central America and the West Indies. Evolution (in press). Studies in the Genetics of Drosophila ~----, 1957. The X-chromosome of the larval salivary glands of hybrids Drosophila in­sularis x Drosophila tropicalis. Chromosoma (in press). Levine, H., 1949. A new measure of sexual isolation. Evolution, 3:315-321. Patterson, J. T., and W . S. Stone, 1952. Evolution in the genus Drosophila. Macmillan, New York. Spassky, B., 1957. Morphological differences between sibling species of Drosophila. Univ. Tex. Puhl. (in this volume). Stalker, H. D., 1942. Sexual isolation in the species complex Drosophila virilis. Genetics, 27:238­ 257. . V. Morphological Differences Between Sibling Species of Drosophila 1 B. SPASSKY INTRODUCTION Mayr (1942) has· defined sibling species as "pairs or even larger groups of re­lated species which are so similar that they are considered as belonging to one species until a more satisfactory analysis clears up this mistake." The genus Dro­sophila is known to contain several groups of sibling species, which are sup­posedly indistinguishable in external morphology, and yet can be shown by ge­netic and other techniques to represent independent and separate biological species. Clearly, morphological similarity is a relative matter, dependent upon the methods of investigation used. What Mayr has called a "satisfactory analysis" needed to "clear up this mistake" of supposedly complete morphological identity of sibling species takes more effort in some than in other cases. The purpose of the present work is to show that some of the longest known and best studied groups of sibling species of Drosophila can be differentiated by inspection of the external genitalia of males, and that, using a sufficient magnification of a micro­scope, these species can be recognized in living individuals as well as in cleared preparations. Morphology of External Male Genitalia The external genitalia of Drosophila males have been described and figured in detail by Salles (1947), Malogolowkin (1948, 1951), and Hsu (1949). The nomenclature of these authors will be followed in the present investigation. The genital arch (GA in Figure 1, see also Figures 2 and 30) is a more or less crescent-shaped sclerite which, according to Dobzhansky (1928), corresponds to the fused eighth and ninth abdominal tergites. To it are attached the genital claspers (CL in Figures 1 and 11, see this part also in Figures 2--4, 11-18, and 30­32), which are a pair of plates armed with various kinds of teeth, spines, and bristles. Neither the genital arch nor the claspers enter the vagina of the female at copulation. It appears, however, the claspers function to hold the genitalia of the fem ale during copulation. In etherized copulating pairs of species of salt ans group observed with the aid of high magnification of dissecting microscope it can be seen that the relaxation of the claspers causes the genitalia of the pair to sepa­rate at the termination of the copulation. The inside margin of the genital arch delimits a depression in which are located the anal tubercle (AT, Figures 1 and 2) and the penis with its appendages. This depression may be called the genital chamber. The shape of the genital chamber is often characteristically different in closely related species, particularly in the sibling species of willistoni group (Figures 11-18). In copulating pairs it can be observed that the female genitalia fit accurately in the opening of the genital chamber of the male. The penis (Pin Figures 3, 5, 6, and 18, see also Figures 4, 8, 10, 12-18, 25­38) is an unpaired organ, often of species-characteristic shape and adorned with 1 Dept. Zoology, Columbia Univ., New York City. species-characteristic bristles or spines (see particularly Figures 33-38) . On either side of the penis lie two pairs of gonapophyses ( G), which can be dis­tinguished as anterior (AG) and posterior (PG) gonapophyses (Figures 3, 5, 6, see also Figures 4, 10, 25-29, 33-35). During the copulation, the gonapophyses enter the vagina together with the penis, and their rapid twitchings inside the vagina can be seen clearly in etherized pairs. T 4 0.0 0.1 0.2 . 0.3 FIGs. 1 and 3-Drosophila pseudoobscura. FIGs. Zand 4-D. persimilis. FIGs. 1 and 2-genital arches (GA), claspers (CL), anal tubercles (AT), and "toes" of the genital arch (T). FIGS. 3 and 4-side views of the penis-gonapophyses complex; CL-Claspers, PG-penis and gonapophyses, T-"toes". The scale in this and the following figures represents 0.3 mm. FIGs. 5-7-Drosophila pseudoobscura. FIGs. 8-10-D. persimilis. A-apodeme: AG-anterior gonapophyses; H-hypandrium; P-penis; PG-posterior gonapophyses. The penis together with the gonapophyses is articulated to the apodeme (A in Figures 5 and 6, also Figures 7-10, 25-29, 33-38). The apodeme is a heavily sclerotized endoskeletal rod, to which are attached the muscles which move the penis complex and the gonapophyses. The muscles which move the penis complex together with the apodeme are attached to the hypandrium, to which is articu­lated also the genital arch. The hypandrium (Hin Figure 5, see also Figures 1{}­24, 39-41) is a roughly triangular plate which may correspond to the eighth abdominal sternite (Dobzhansky, 1928). The form of the hypandrium is very important for distinguishing closely related species. In some species the hypan­drium has characteristic bristles or teeth (HT in Figure 11, see also Figures 12-­24, 39-41). For the purposes of the present study, it was found desirable to observe the male genitalia in living flies, as well as after maceration in hot 10% solution of KOH in water. Examination in macerated preparations alone can lead to serious errors, since in flies killed by etherization the penis-gonapophyses complex is usually pushed out, and a prolonged maceration sometimes dissolves some deli­cate parts and makes them invisible. The drawings in Figures 1-41 have all been made with the aid of a camera lucida in macerated preparations and at the same magnification of a microscope. The magnification is indicated for every group of figures by a scale, which represents 0.3 of a mm. Drosophila pseudoobscura and Drosophila persimilis Drosophila pseudoobscura Frolova and Drosophila persimilis Dobzhansky and Epling are a pair of sibling species which have been studied in perhaps the most detail. They were first distinguished by Lancefield ( 1929) because they produced sterile male hybrids when crossed; Lancefield regarded them as "two races or physiological species," although he found them to be morphologically indistin­guishable. Dobzhansky and his co-workers (a review in Dobzhansky and Epling, 1944) found that the two species have overlapping geographic distributions and show partial ecological and sexual reproductive isolation, sterility of F1 hybrid males, and hybrid breakdown in the progeny of the backcrosses of F1 hybrid fe­males to males of either parental species. These facts, as well as the presence of several chromosomal and physiological differences, led these authors to conclude that the process of speciation has reached a complete separation of the gene pools of the two species, and accordingly they proposed the suitable nomenclatorial rec­ognition of this fact. Subsequent studies confirmed the specific status of these forms, by showing that no gene exchange occurs where the species are sympatric. Mather and Dobzhansky (1939) and Reed, Williams, and Chadwick (1942) found that D. pseudoobscura and D. persimilis differ in the mean numbers of teeth in the sex combs, in certain dimensions of the wings, and in some other traits. However, all these differences are so slight that single individuals cannot be classified as to species. Rizki ( 1951) was the first to find a morphological dif­ ference which permitted such a classification. This difference concerns the shape of the penis. This organ is relatively longer and more parallel-sided in D. psuedo­ obscura than it is in D. persimilis. Cooper and Lewontin (personal communica­ tions) noted in 1954 a difference in the shape of the hypandrium and a possible difference in the number of teeth on the claspers in the two species. Studies in the Genetics of Drosophila The work of the present writer has confirmed the existence of a difference in the shape of the penis, which is relatively longer and gradually tapering towards the distal end in D. pseudoobscura (Figure 5) and is shorter but inflated at base in D. persimilis (Figure 10). The hypandrium of D. pseudoobscura is somewhat larger and its sides converge frontwards less rapidly than in D. persimilis (Fig­ures 5 and 10) . The apodeme of D. pseudoobscura (Figure 7) is longer and more nearly cylindrical, while in D. persimilis it is shorter, inflated at both ends and thinner in the middle. However, for practical identification of living specimens the above character­istics are not suitable, since they are clearly visible only in macerated and mounted preparations. A more convenient and more easily visible characteristic is the shape of the penis-gonapophyses complex, especially as seen in a lateral aspect (Figures 3, 4, 6, 8). A male fly to be classified as to species should be etherized and placed on his side on a plate under a binocular dissecting micro­scope; a needle is then placed lightly across his abdomen, and with the aid of a gentle pressure the penis complex is extruded. Care should be taken not to make the gonapophyses flare apart from the penis. In D. pseudoobscura (Figures 3, 6) the gonapophyses are bent sabre-like, while in D. persimilis they are straight ex­cept at the free ends which are bent ski-like (Figures 4, 8). With some practice, this method permits mistake-free identification; some hundreds of cultures of un­known species were classified by the present writer, and the classification was subsequently verified by Prof. Th. Dobzhansky by means of examination of the salivary gland chromosomes. The willistoni group of five sibling species of Drosophila Drosophila willistoni Sturtevant is a new name for the species described in 1896 by Williston from the island of St. Vincent in the West Indies under the preoccupied name of Drosophila pallida Williston. In 1943, Dobzhansky and Pavan noticed the existence of two externally very similar but non-interbreeding species resembling D. willistoni in southern Brazil. One of these is on the average larger in body size than the other. A wrong guess led to the larger species being described as a new species, Drosophila paulista Dobzhansky and Pavan, but when it was found that this is a synonym of D. willistoni, the smaller species was given the name of D. paulistorum Dobzhansky and Pavan. A third species, morphologi­cally very similar to the preceding two but reproductively completely isolated from them, was described from the Upper Amazon as Drosophila equinoxialis Dobzhansky (1946). Burla et al. (1949) added a fourth species, .Drosophila tropicalis Burla and da Cunha from Central Brazil, and reviewed the mor­phology, cytology, and genetics of the four sibling species. Several minute mor­phological differences have been recorded between the sibling species, involving the structure of the maxillary palpi, position of the orbital bristles, coloration of the ocelli, the shape of the vaginal plates, of the hypandrium, and of the chitinous spermatheca of the females. Nevertheless, the authors concluded that: "The variability is great enough to make identification of species in single individuals hazardous." This contrasts, however, with complete reproductive isolation of these species and the relative facility of their recognition by the structure of their chromosomes in the salivary glands cells. Malogolowkin (1952) made a more detailed study of the genitalia of males of the willistoni group, and discovered several additional characteristics which differentiate the species in cleared and macerated preparations. Further work brought to light the existence of morphologically cryptic but genetically explicit differentiation within the sibling species. Townsend ( 1954) described Drosophila tropicalis subsp. cubana Townsend from Cuba, which can be crossed to D. tropicalis tropicalis from Brazil, but which produces sterile male and fertile female hybrids. He also noticed some minute morphological differ­ences between the two subspecies, which however do not permit recognition of single individuals. Cordeiro (1952) found a difference in the shape of the hypan­drium between certain strains of D. paulistorum from northern and southern Brazil. These strains cross freely, and analysis of the F2 generation hybrids shows that the differences in the hypandrium shape observed between these strains of D. paulistorum have a rather simple genetic basis. The present writer (unpub­lished) found in 1955 that strains of D. paulistorum collected by Dr. W. B. Heed in Honduras differ from the Brazilian strains of this species. The crosses Hon­duras X Brazil (Piras­sununga) 3 produce hybrid sons which are completely sterile. The F1 hybrid females are fertile in backcrosses with both parental races. The reciprocal cross, Brazil <;> X Mexico 3 gives fertile hybrids of both sexes. Among the prosaltans-like flies collected at Pirassununga, state of Sao Paulo, Brazil, and sent to this laboratory through the courtesy of Professor C. Pavan, this writer discovered the existence of two distinct species. Although they appear very similarin most traits used in the conventional systematics of Drosophila, they proved to be completely isolated reproductively. Females of either of these were kept in culture bottles with males of the other species for a week or more, where­upon the females were dissected. Examination under the microscope disclosed no sperm at all in the ventral or in the chitinous receptacles. One of the forms from Pirassununga was found to cross easily and to produce fertile female progeny with strains of D. prosaltans either from Brazil or from Mexico. This form evi­dently is D. prosaltans. The other form has refused to cross not only to D. pro­saltans from Pirassununga but to the Mexican strains of the same species as well. It is evidently a separate species which is described below as D. austrosaltans. Male genitalia of species of saltans group seem to be more complex in structure than those in obscura and willistoni groups. The penis is very large relative to the two pairs of gonapophyses, and the latter appear as mere outgrowths on the penis (Figures 33-35). The penis itself carries denticulate processes of various kinds (Figures 34-38). The claspers (Figures 30-32) carry relatively large numbers of bristles of several kinds-peg-like, tooth-like, and hair-like, the numbers and ar­rangements of which are characteristic for different species. The large horn-like processes on the genital arch (the anterior claspers) have the same function in copulation in prosaltans as they have in the willistoni group. The hypandria (Figures 39-41 ) are very large and of characteristic shapes. In six of the strains of D. prosaltans kept in this laboratory the penis and the adjacent parts of the genital arch have, observed from the ventral side, the ap­pearance shown in Figure 30. All these strains are derived from flies. :Collected in various localities in Brazil (Bertioga in the state of Sao Paulo; Monjolinho, Goyaz; Barreiras, Bahia; Fordlandia and Marajo Island, Para; and I~ana, Ama­zonas). In the two northern strains (Guatemala and Chilpancingo, Mexico) these parts appear as shown in Figure 31, the head of the penis being triangular rather than persimmon-shaped. Finally, in D. austrosaltans (Figure 32) the head The University of Texas Publication 31 32 30 0.0 FIGs. 30--32---genital arch and the tips of the penis of-FrG. 30-Drosophila prosaltans from Brazil; FIG. 31-D. prosaltans from Mexico; F1G. 32---D. austrosaltans. Fies. 33-38-distal parts of the penis of-FrGs. 33 and 36-D. prosaltans from Brazil; FrGs. 34 and 37-D. prosaltans from Mexico, and FIGS. 35 and 38-D. austrosaltans. of the penis is pear-shaped. There exists also a characteristic difference in the color of the gonapophyses in living young males-they are amber yellow in Brazilian D. prosaltans, dark brown in D. prosaltans from Mexico and Guate­mala, and black in D. austrosaltans. In macerated and cleared preparations one can observe also extensive differ­ences in the shape and ornamentation of the penis and of the gonapophyses FIGs. 39-41-Hypandria of, Fie. 39-Drosophila austrosaltans. Fie. 40-D. prosaltans from Brazil, and FIG. 41-D. prosallans from Mexico. (Figures 33-38). In Brazilian D. prosaltans the tip of the penis carries minute teeth-like bristles, in those from Mexico and Guatemala it has numerous minute hair-like bristles, while in D. austrosaltans it is sparsely covered with hairs. In D. austrosaltans the body of the penis is covered with denticulate ridges resem­bling the edge of a saw (Figure 38). The hypandria of D. prosaltans are triangu­lar, with two pairs of processes, one of which carries a bristle (Figures 40 and 41). In D. austrosaltans the hypandrium is rounded off, and the processes are fused to form ear-like lobes which carry a bristle on their inner margins (Figure 39). A description of D. austrosaltans follows. Drosophila austrosaltans, Species Nova Female and Male.-Arista with 9-10 branches, 9 being the mode. Antennae, head, carina, cheeks, eyes, and palpi like in Drosophila prosaltans Duda (see a re-description of the latter species in Dobzhansky and Pavan, 1943 (pp. 17-19), except that the middle orbital bristle is a little longer relative to the anterior and posterior orbital, being about half as long as the latter. Thorax with its bristles, legs, and wings like in D. prosaltans, except that the pleurae on the average a little darker than in the latter species. Abdomen and the opaque areas on the posterior part of the sixth abdominal tergite in female like in D. prosaltans, the opaque areas a little broader. Measurements of 10 'i1 'i1 and 10 5 5 each of D. austrosaltans and of the Belem and Barreiras strains of D. prosaltans gave the following results: D. austrosaltans D. prosaltans Pirassununga Belem Barreiras Body Length, 'i' 3.12mm. 2.98 mm. 2.96 mm. " " J 2.43mm. 2.34 mm. 2.58 mm. Wing Length, 74.3 181.16 192.1 1 203.21 203.23 2378.2 i Costa Rica Panama Colom bia Venezuela Venezuela Cuba 74.3 x F F F s s 181.16 F x F F s s 192.1 1 F F x F s s 203.21 F F F x s s 203.23 s s s s x s 2378.2 s s s s s x F = fertile, producing an F, S = sterile; no F, Drosophila kallima Wheeler, new species. External characters of imagines. ~ , ~ . Most similar to schildi, differing most obviously in the wing pattern (Fig. 7). Front whitish yellow, brown around ocelli and bases of verticals; pro­clinate orbital % length posterior reclinate; anterior reclinate thin, hairlike, about ¥6 posterior. Face, antennae, clypeus except base, and palpi except base, all pale yellowish; bases of clypeus and palpi brown; proboscis brown with pale labellum. Arista with 7 dorsal and 4 ventral branches in addition to the fork. One strong oral; cheeks narrow, brown below eye becoming grayish yellow pos­teriorly. Mesonotum dull reddish brown with a pattern of pale tan to whitish pollinose spots, streaks and blotches. Generally there is a poorly defined pale area on an­terior % between dorsocentral rows, within which there are 3 medium large brown blotches; from this a thin irregular median line continues to scutellum. About 6 pale marks laterad of dorsocentral rows. Acrostichal hairs in 4 rows be­tween anterior dorsocentrals, irregularly 6-rowed in front; one or more bristles in dorsocentral rows enlarged, an especially prominent one just anterior to edge of transverse suture. Scutellum dark brown with 5 gray pollinose areas: one at base on each side and 3 apical ones which may be partly fused. Basal scutellars divergent. Pleura mottled with dark brown, tan and gray pollinose areas, not organized into a describable pattern. Anterior sternopleural 0.7 length posterior; middle bristle thinner but of about same length as anterior one. Halteres with brownish dis­ coloration on front surfaces. Legs brown, especially coxae and femora; tibiae paler with a tendency toward basal and subapical darker bands; tarsi pale. Hind metatarsus with 2 large, 1 smaller, stout black bristles among the pale ones basally below. Abdomen with pattern of dark dull brown apical bands, expanded in the mid­ dle and at lateral margins, the large paramedian basal areas thus formed being grayish pollinose, sometimes almost metallic. Anal plates and ovipositor of fe­ male pale. Wings as in Figure 7. Vein 2L with spur veins as in schildi and quadrum. Costal index 3.1-3.3; 4th vein index 1.3-1.4; 5x index about 0.7. Body length (pinned female) 3.2 mm., wing, 4.0 mm.; males smaller. Other characteristics, relationship, and distribution. Male clasper with 7-9 primary teeth followed by a cluster of pale hairs going around onto lower side, and with 4-5 long bristles in secondary position; genital arch with a single strong bristle 'just above attachment of clasper, and 2 bristles on heel area; lower tip of anal plate with a cluster of small bristles. Ovipositor slender, pointed, with about 17 primary teeth, 6 secondary teeth, and 4-5 pale bristles near tip. Spermatheca, as observed in cleared preparation of pinned specimen, dark, shaped about as in Figure 17. Distribution and types.-Holotype male, 9 paratypes of both sexes, plus 7 ad­ditional specimens, from Hacienda Santa Maria de Ostuma, about 11 klm. north of Matagalpa, Nicaragua, June 1954, W. B. Heed collector. Two specimens (USNM) labelled Panama, Volcan Chiriqui Prov., 9-XII-1952, F. S. Blanton. The University of Texas Publication Relationship.-Belongs to the calloptera group of the subgenus Drosophila. Drosophila maracaya Wheeler, new species. External characters of imagines. ~ , 'i' . Similar to calloptera, but with an entirely different wing pattern (Fig. 8), the 5th vein with a spur vein. Arista with 6 dorsal and 4 ventral branches in addition to the fork. Front, antennae, face and clypeus whitish yellow, ocellar area, orbits and bases of verticals a little darker; clypeus brownish at base. Palpi dark tan; cheeks narrow, grayish brown, darker behind. Mesonotum mottled, dark brown with golden brown pollinose blotches and spots, the appearance varying with the viewing angle; in general the anterior Fw. 7. Drosophila kallima. photograph of wing. Frc. 8. Drosophila maracaya. photograph of wing. Studies in the Genetics of Drosophila % is golden with a partial median brown streak, but which appears dark brown to black when seen from behind; posteriorly and laterally there are a number of golden brown spots and blotches. Scutellum broadly brown except on sides where it is more tan. Acrostichal hairs rather sparse, irregularly 6-rowed; no prescutellars; an enlarged bristle near inner edge of transverse suture. Basal scutellars divergent. Pleura dark brown to blackish brown, pollinose. Posterior sternopleural stout and long, the other two equal in size, about 0.6 length pos­terior. Halteres tan. Legs mostly brown, including coxae; tibiae paler, tarsi grayish yellow. Base of hind metatarsus below with 2 bristles darker than the others. Abdomen with dark brown apical bands with median extensions, leaving paired tan basal mark­ings. Male tergite 2 mostly brown, 3, apical band with large median expansion, 4-5. with narrower expansions, the band of 5 thicker; 6 all dark brown; all bands form solid lateral areas. Female as in male, but tergite 6 with small paired basal tan areas, 7 all dark; anal plates and ovipositor tan. Wings as in Figure 8, vein 2L lacking spur veins but 5L with a spur in the large dark spot before posterior crossvein. Third costal section with the small black bristles on the basal 0.4; costal index about 3.4; 4th vein index about 1.2; 5x index about 0.6. Body length (pinned female) up to 3.5 mm., wing, 3.2 mm.; t smaller. Relationship.-Belongs to the calloptera group of the subgenus Drosophila. Distribution and types.-Holotype t , 9 paratypes of both sexes, Rancho Grande, near Maracay, Venezuela, Nov. 1956, M. Wasserman collector. melanderi group Drosophila ordinaria Coquillett Drosophila ordinaria Coquillett, 1904. Proc. Ent. Soc. Wash. 6:190. An examination of the holotype female showed that this rare species belongs to the melanderi species group, and is quite similar in appearance to both melan­deri and magnafumosa. The type, in the U.S. National Museum collection, is from the White Mountains, N. H., and I have examined a second specimen, in the Museum of Comparative Zoology, from St. John's County, Quebec. Sturte­vant (1921:86) lists a specimen from Chester, Mass. The melanderi group now consists of the following species: melanderi Sturte­vant, magnafumosa Stalker and Spencer, ordinaria Coquillett, all from North America, cameraria Haliday (=pallida Zetterstedt) from Europe, and makinoi Okada from Japan. As far as known, all the species are mushroom feeders and are poorly attracted to fruit baits; they also show many morphological similari­ties to the subgenus Hirtodrosophila. polychaeta group Drosophila polycha.eta Patterson and Wheeler Drosophila polychaeta Patterson and Wheeler, 1942. Univ. Texas Puhl. 4213: 102. Our original specimens came from the wharfs at Galveston, Texas. The species has since been reported from various widely scattered localities, including the Netherlands, Liverpool (on ships from west Africa and Malaya), Hawaii, and Guam, and I have seen specimens from Saipan and Guam (Marianas) and The University of Texas Publication Koror (Palau). New American records are: 1, Cativa, Colon Prov., Panama; 6, Cocos Island, Wafer Bay. Cocos is a small island belonging to Costa Rica, nearly midway between the latter and the Galapagos Islands. These 7 specimens are in the U. S. National Museum collection. canalinea group Recent collections have shown that there are perhaps 6-8 species in Central and South America that are related to canalinea Patterson and Mainland. We have not succeeded in culturing most of these, but hybridization tests between the seven stocks which we did have available showed that three species were present in the laboratory. The results of these tests are given in Table 3. The geo­graphic origins of the stocks used are as follows: canalinea: H50.31 La Lima, Honduras H101.7 Palmira, Colombia H188.21 Santa Marta, Colombia canalinioides: H38.6 San Salvador, El Salvador H66.7 San Salvador, El Salvador H181.55 Barro Colorado Island, Canal Zone paracanalinea: H129.19 El Yunque, Puerto Rico As the table shows, intraspecific crosses are fully fertile, and interspecific crosses are sterile with the single exception of canalinea 'i? (50.31) X paracana­linea J (129.19). The cross was made by mass matings of about 10 J, 10 'i?; the first attempt yielded three F, males which were backcrossed to parental females and proved to be fertile. The cross was made again and watched more closely. The cross produced an estimated two dozen larvae fi-om which only seven pupae developed; from these only three F, males emerged, the remaining pupae under­going an internal degeneration. These males were similarly backcrossed to paren­tal females and proved to be fertile. TABLE 3 Results of crosses between members of the canalinea complex Intraspecific Interspecific Cross Cross Cross 'i? 5 'i? 6 'i? J "'~ 38.6 x 66.7 F 38.6 x 50.31 s 50.31 x 38.6 s "O ·a 38.6 x 181.55 F 38.6 x 101.7 s 50.31 x 66.7 s :@o; ;:: "' u 66.7 x 38.6 66.7 x 181.55 181.55 x 38.6 F F F 38.6 66.7 66.7 x 129.19 x 50.31 x 101.7 s s s 50.31 x 181.55 50.31 x 129.19 101.7 x 38.6 s few s 181.55 x 66.7 F 66.7 x 129.19 s 101.7 x 66.7 s "' 181.55 x 50.31 s 101.7 x 181.55 s ~ ;§ "' 50.31 x 101.7 101.7 x 50.31 F F 181.55 x 101.7 181.55 x 129.19 s s 101.7 x 129.19 129.19 x 38.6 s s ;:: "' 188.21 x 101.7 F 129.19 x 181.55 s 129.19 x 66.7 s u 129.1 9 x 50.31 s 129.19 x 101.7 s F = fertile F, produced S = sterile; no F, Studies in the Genetics of Drosophila 12 14 FIGS. 9-20. FIG. 9, D. trifiloides, spermatheca; FIG. 10, D. paraguttata, spermatheca; FIG. 11, D. paraguttata, genital arch and clasper; FIG. 12, Laccodrosophila heedi, part of front tarsus, ventral view, diagrammatic; FIG. 13, D. sticta, head of penis, ventral view; FIG. 14, D. sticta, spermatheca; FIG. 15-16, Pseudiastata sp. from Cuernavaca, Mexico, genital arch and genitalia from below; FIG. 17, D. kallima, spermatheca; FIG. 18, D. canalinioides, spermatheca; FIG. 19, D. canalinioides, female genitalia showing paragenital fringe, semi-diagrammatic; FIG. 20, D. paracanalinea and canalinea, spermatheca. The University of Texas Publication Drosophila canalinea Patterson and Mainland Drosophila canalinea Patterson and Mainland, 1944. Univ. Texas Publ. 4445:50. This species is widely distributed throughout the Neotropical region. We have material from Mexico; El Salvador, Honduras, Nicaragua, Costa Rica, Panama, Colombia, Trinidad, Ecuador, and Brazil. The only collection of canalinea-like flies from the Caribbean islands was on Puerto Rico, but this stock showed nearly complete reproductive isolation from canalinea and is being described as a new species, D. paracanalinea. Drosophila canalinioides Wheeler, new species. External characters of imagines. 5 , <,? • Arista with 5-7 dorsal branches, 3 ventral ones and a terminal fork. Front dull blackish brown with strong pollinose gray to brown areas along orbits. two large spots anteriorly, and on the large ocellar triangle, the latter extended anteriorly to ptilinal suture, gradually narrowing. Proclinate orbital % length posterior reclinate; anterior reclinate tiny. Antennae blackish with pale yellow apices on 2nd and 3rd segments. Carina broad, rounded; face yellowish gray, cly­peus and cheeks black, the latter with grayish pollen; palpi and proboscis black. One prominent oral. Mesonotum dull, with complex, somewhat variable, pattern of dark brown and pale tannish brown areas with many of the bristles and hairs which arise within the pale areas showing dark spots at their bases; the resulting pattern resembles that of canalinea plus scattered repleta-type spots. Acrostichal hairs in 8 rows; no prescutellars. Anterior scutellars convergent. Scutellum black with grayish brown pollen on each basal angle and broadly over apex. Pleura dull black with mottled grayish pruinescence when viewed from certain angles. Halteres pale, darkened at base. Anterior sternopleural about % posterior one, a middle one scarcely differentiated. Legs mostly black, with annulated tibiae and yellow tarsi. Femoral-tibial junc­tion pale; tibiae with basal and apical black areas, paler in the middle, the pale portion faint on 1st leg, larger on 2nd and still larger on 3rd. Male 1st tarsus with a series of about 14 long recurved hairs along inner side, their length nearly equalling tibial diameter. Base of hind tarsus with pale bristles below. Abdominal tergites with broad apical bands, interrupted medianly, the inter­ruptions becoming progressively narrower on posterior tergites; the bands are wide next the interruption, then narrow, then widen again to form solid lateral areas. The basal yellow area on each side with whitish to silvery pruinosity when viewed from most angles. Circumanal tergite of <,? with a prominent "paragenital fringe" of about 24 long slender bristles on each side (Fig. 19); as far as known, a similar sort of fringe occurs on the females of all species of the canalinea group, but has not yetbeen seen on species of any other group. Wings light brown, paler posteriorly; apex of 1st costal section a bit enlarged, black, with 2 bristles; 3rd costal section with the small black bristles on the basal %. Posterior crossvein with a moderately narrow black cloud, the anterior one with a small cloud. Costal index about 2.2; 4th vein index about 1.6; 5x index about 0.9. Studies in the Genetics of Drosophila Body length, live J, about 3.2 mm., wing, 2.6 mm.; female, about 3.8 mm., wing, 2.6 mm. Internal characters of imagines. Ventral receptacle long with many tight coils; spermatheca (Fig. 18) pale tan, elongate but narrowed apically, most of the surface with fine wrinkles except basally with numerous fine sulci. Ovipositor with about 23 primary teeth, longer than usual, and 4 slender secondary bristles. Posterior Malpighian tubes apposed at the tips, without a continuous lumen. Testes orange (young J J) to rusty red (older J J ) ; sperm pump with two long thick, somewhat tangled, posterior diverticula. Clasper with a slightly curved row of about 10 teeth, the lower 2-3 distinctly more pointed; penis shaped nearly as in canalinea. Other characteristics, relationship, and distribution. Eggs.-With 4 filaments, the anterior pair short and thin, the posterior pair thicker and longer, nearly %the egg length. Larvae.-The larvae skip, but infrequently. Puparium.-Dark tan; anterior spiracular horns short, about equalling the distance between their bases, bearing 9-10 branches. Posterior spiracles rather long, pale, parallel. Chromoso'mes.-The chromosomes are described by Clayton and Wasserman (this bulletin) . Distribution.-Our collectors have taken this species rather commonly in El Salvador (San Salvador, Santa Tecla), and on Barro Colorado Island, Canal Zone. The U.S. National Museum collection has specimens from: Costa Rica (San Ma­teo), Panama (Patino Point, San Carlos, Barro Colorado Island, Loma Borracha, Tocumen), and Venezuela ( Carife) . Types.-Holotype J and 9 paratypes ( J, 'i') from stock No. H66.7 from San Salvador, El Salvador, Sept. 1956, W. B. Heed, collector. This stock originated from 6 individuals collected by Dr. Heed from bracket fungus. Drosophila paracanalinea Wheeler, new species. This new species is quite similar in appearance to canalinea, but a side-by-side comparison of living individuals of a stock from Puerto Rico with individuals of canalinea (Honduras stock) shows some fairly reliable differences: canalinea paracanalinea 1. mesonotum dull; ground color dark 1. slightly shining; brownish black brown 2. eyes very dark red 2. brighter red 3. abdominal bands dark brown, the 3. blacker bands, yellow areas not yellow areas pronounced pronounced 4. sternites pale, grayish 4. sternites darker 5. 'i' circumanal tergite pale below, 5. this tergite dark below, with ca. 9 with ca. 7 hairs in paragenital hairs in paragenital fringe fringe Dissections of male and female genitalia show small, possibly not significant differences: clasper of canalinea with usually 9 primary teeth vs. paracanalinea The University of Texas Publication with usually 10; spermatheca (Fig. 20) of paracanalinea generally more irreg­ularly warty over the apical ¥2, and the inner duct usually a little shorter. The chromosomes also differ, and are described by Clayton and Wasserman (this bulletin). Distribution.-Collected by Dr. W. B. Heed in El Yunque, Caribbean National Forest, Puerto Rico, January 1956, and at the Agricultural Experiment Station, Rio Piedras, Puerto Rico, February 1956. Types.-Holotype J , and 9 paratypes ( J , <;> ) , from El Yunque, Puerto Rico. Drosophila annularis Sturtevant Drosophila annulata Williston, not Fallen. Drosophila annularis Sturtevant, 1916. Ann. Ent. Soc. Amer. 9:327. A female labelled Type, from the collection of the American Museum of Natural History, is headless and a portion of the abdomen is missing; it clearly shows, however, the canalinea-like pattern on the mesonotum and legs, and has an especially well-developed paragenital fringe, the hairs being longer and more numerous than on any other specimen we have seen. The apex of the 1st costal section is enlarged and strongly blackened, but the crossveins show no clouding at the present time. On the remaining part of the abdomen one can see that the midline stripe (interruptions) are strongly grayish to silvery pruinose. We con­clude that this species is distinct from any we have collected, and that the various published records of this species from Central and South American localities are to be viewed with suspicion. Drosophila panamensis Malloch Drosophila panamensis Malloch, 1926. Proc. U.S.N.M. 68 (21) :28. This species is probably best placed as an aberrant member of the canalinea group. We have examined the type in the USNM, and a single <;>, AMNH col­lection, also from Barro Colorado Island, Canal Zone. The general appearance is not that of canalinea, having a golden yellow face and front (the latter with a V-shaped bJ:"OWll mark on the type), and a different sort of mesonotal pattern. The female has, however, a paragenital fringe, a rather typical abdominal pat­tern and leg markings. The wing is quite dark, with still darker clouds over the crossveins; the 3rd costal section has the small black bristles reaching all the way to the 3rd vein, and the 4th vein clearly bends toward the 3rd at its apex. fenestrarum group (Subgenus uncertain) Drosophila basdeni Wheeler, new species. =Scaptomyza sp. B, Wheeler, 1952. Univ. Texas Puhl. 5204:197, 208. External characters of imagines. J . Front tannish yellow, the orbits paler, less than half width of head. An­terior reclinate orbital about % proclinate, Y4 posterior reclinate. Antennae pale yellow, 2nd segment with 4 stout bristles, 3rd small; arista with 5 dorsal and 2 ventral branches in addition to the fork. Face whitish yellow, low, a carina scarcely indicated; cheeks whitish, very narrow. Vibrissa thin, 2nd oral about% as long and placed rather far behind 1st. Palpi pale with a strong apical bristle. Mesonotum and scutellum pale tan, rather shiny; acrostichal hairs in four rows; no prescutellars. Apical scutellars cruciate and bent somewhat upright. Two nearly equal humerals; posterior sternopleural large, the other two thin, middle one longer than 1st. Pleura tan, a trifle darker on mesopleura. Legs whol­ly yellowish. Abdominal tergites blackish, distinctly shining; tergites 1-2 paler. Halteres pale. Sternites pale. Wings clear, rather narrow and somewhat pointed. Two prominent bristles at distal costal break; 3rd costal section with the small black bristles on the basal %. Costal index about 3.2; 4th vein index about 2.0; 5x in­dex about 1.9. Anal vein greatly reduced. Body length (pinned specimen) about 2.1 mm.; wing, 1.9 mm. Distribution and types.-Holotype 6 (USNM), E. Lansing, Mich., May 4, 1949, Ryoji Namba collector. Paratype 6, Mohawk Park, Ohio, May 10, 1937, H. D. Stalker collector. Relationship.-This new species belongs to the fenestrarum species group and is the first member of the group to be reported from North America. It is quite distinct from any of the Palaearctic species: fenestrarum Fallen, forcipata Collin, and acuminata Collin. Collin ( 1952) gives the following features of the fenestrarum group: acrosti­chals irregularly 4-6 rowed; facial carina small; middle sternopleural longer than anterior one; arista with 2 ventral branches basal to the fork; male fore metatarsus ventrally with an apical tuft of long pale hairs, and with a less obvi­ous tuft on the 2nd joint; male hypopygia generally rather large. Collin has pointed out that the group occupies a somewhat isolated position in the genus. Hackman ( 1954) comments that many features, including those of the genitalia, suggest a relationship with some of the species of Scaptomyza, and is of the opin­ion that a new subgenus should be erected for the group. Little is known of the food habits of the species of this group, but since Basden ( 1954) reports taking both fenestrarum and forcipata by sweeping over watercress (Nasturtium mi­crophyllum) in Scotland, a position intermediate between Drosophila and Scap­tomyza is again indicated. The holotype specimen of basdeni was compared with the Palaearctic species by Mr. E. B. Basden of Edinburgh who has graciously permitted the use of his notes. He states that basdeni is most similar to acuminata, especially in the form of the genital arch and the lack of large claspers. D. acuminata also shows, ac­cording to Basden, the following obvious differences: tergites with more obvious apical bands; cheeks pale brownish; palpi blackened; frons broader; mesonotum tannish brown but darker posteriorly and on scutellum; much of the pleura darkened ; wings broader; 3rd costal section with stronger bristles on the basal l/i; etc. In both fenestrarum and forcipata the thoracic and abdominal color is subject to considerable variability. Subgenus Drosophila (unclassified species) Drosophila carsoni Wheeler, new species. External characters of imagines. () . Front dull reddish brown, the orbits and enlarged ocellar triangle darker brown, well-marked, posterior orbits around verticals much paler. Middle orbital small, ~ posterior, proclinate strong, about% length of posterior reclinate. An­tennae brown, 3rd segment darker. Arista with 4 dorsal and 2 ventral thin branches in addition to the terminal fork; there are up to 7 thin lateral branches, more prominent than is usual. Face tan; carina of moderate size, a bit flattened on top. Cheeks, palpi and proboscis pale tan, clypeus darker, especially medianly. Palpi of .:; quite bristly, with numerous thin long hairs and bristles, thickest around apex. 1st oral strong, 2nd thinner, about Yz length 1st. Cheeks rather broad, especially broad and bristly behind. Inner and outer verticals strong; a 3rd vertical, jiist behind outer one and bent inwardly, small. Mesonotum subshining brown to dark brown, lighter far anteriorly and on humeri; pleurae diffusely browned, paler over sutural area between stern<>-and mesopleura. Acrostichal hairs 6-rowed, a bit irregular; usually a few enlarged hairs anteriorly in dorsocentral row. No prescutellars; anterior scutellars diver­gent. Three humerals, upper one small, middle one large, lower one strong, about % length middle one. Two stout sternopleurals, anterior one about % length posterior. Legs pale tan, rather long and slender. Bristles of 1st femur prominent: about 4 on lower side, 5 on outer side of which the apical one is strongest and placed further dorsad. Preapicals on all tibiae; a strong apical on 2nd, no apicals evident on 1st or 3rd tibiae. Halteres pale. Abdominal tergites subshining brown to blackish brown, sometimes paler; two basal segments paler, remaining ones often somewhat paler in midline. Wings clear; two strong bristles at apex of 1st costal section; 3rd section with the small black bristles on the basal %. Costal index about 3.5; 4th vein index about 1.4; 5x index about 0.9-1.0. Body length to 3.5 mm., wing about 3.5 mm. Female: Usually paler than male, abdominal tergites often more tan with diffuse brownish apical bands with median interruptions. Palpi less bristly. Body length to 4.0 mm., wing about 4.0 mm. Other characteristics, distribution, and types. Eggs.-With 4 very thin filaments about equal to egg length. Chromosomes.-The metaphase and salivary gland chromosomes are described by Clayton and Wasserman (this bulletin). Distribution.-About 25 individuals have been captured, from 10 states as fol­lows: Maine, Vermont, New York, Ohio, Tennessee, Missouri, Wisconsin, South Dakota, Colorado, and New Mexico. Male genitalia of specimens from Wisconsin and Colorado have been compared; they agree in all respects. Types.-Holotype male, Mellen, Wisconsin, July 1952, H. L. Carson col­lector; 9 paratypes from: Mellen, Wisconsin, Steelville, Missouri, Espanola, New Mexico, and Pagosa Springs, Colorado. Drosophila sticta Wheeler, new species. External characters of imagines. J, 'i?. A pale tan to yellowish tan species with unmarked wings and a spotted abdominal pattern. Head yellowish tan, including front, face, cheeks, palpi, an­tennae, and proboscis. Proclinate orbital about % posterior reclinate, middle or­ Studies in the Genetics of Drosophila bital minute. Arista with 4--5 dorsal and 2 ventral branches in addition to the fork. Carina broad, rather bulbous, not sulcate. One strong oral, the 2nd about% its length, thin. Orbits and ocellar triangle only slightly differentiated. Mesonotum tan, thinly pollinose, not shiny. Acrostichal hairs in about 6 rows, sometimes more irregular; no prescutellars. Basal scutellars widely divergent. Two humerals, nearly equal. Anterior sternopleural thin, about % length pos­terior one; a middle one present, small. Pleura yellowish tan; halteres pale. Legs pale without unusual bristling. Abdomen tan, subshining, with small dark brown markings apically on ter­gites, rather irregularly formed into a series of paired spots (Fig. 21); there is variation in the shape of the larger paramedian spots and considerable variation FIG. 21. Drosophila sticta, abdominal pattern. Male, left; female, right. in the extent to which the various lateral markings are broken up into spots. Fe­male with sub-anal spines-a pair of small sclerotized spines just below lower base of anal plates and above base of ovipositor. Body length (live flies) , ~, 2.5 mm., wing, 2.2 mm.; males smaller. · Other characteristics, relationship, and distribution. Eggs.-With 4 slender filaments, tending to be irregularly bent and twisted at tips; posterior pair about% egg length, anterior pair a little shorter. · Puparia.-Anterior spiracles with 25-30 long white branches; stalk of anterior spiracle about V5 length of puparium; posterior spiracles pale, rather long, diver• gent or parallel. . Internal features.-Posterior Malpighian tubes apposed at their tips but with­out a continuous lumen. Ventral receptacle spirally coiled, of medium length,; spermatheca (Fig. 14) dark, round, with a strong ridge at base; ovipositbr wi't:4 blunt tip. Testis pale yellow; sperm pump with 2 short blunt diverticula. Cl~spe~ (forceps) with 8-9 teeth, plus strong marginal bristles; head of penis (Fig., 13); with 3 strong spines distally, 2 basally, and 3 smaller ones on each side. · Distribution.-D. sticta has been collected in Nicaragua (E!. Recreo), El Salva­dor (Santa Tecla, Laguna Alegria), Honduras (Lance.tilla), ~nd Colombia (Pal­mira, Rionegro). Collectors at Rionegro, Colombia wel;'e H. L. Carson, M. Wasser­man, and H. Hoenigsberg; all other localities were collected by Dr. W. B. Heed. Types.-Holotype male, 9 paratypes of both sexes, from stock No. H51.15 from Lancetilla, Honduras, collected in April 1954. Relationship.-Belongs to the subgenus Drosophila, but cannot be placed satis­factorily in any of the established species groups. Dr. Frota-Pessoa has examined the male genitalia and reports that the ring of the hypandrium is present but it is of a different shape than in any of the species groups possessing such a ring (Malogolowkin, 1953, lists these groups: quinaria, tripunctata, cardini, guarani, calloptera, macroptera, rubrifrons, testacea, and pallidipennis). In other features this new species does not agree too well with any of these groups. Its position in the subgenus thus remains undecided. Chromosomes.-The chromosomes are discussed in detail by Clayton and Was­serman (this bulletin) . Drosophila paraguttata Peter Thompson, new species. External characters of imagines. & , 'i' . A medium-sized, dark tan species with a pair of light spots on the meso­notum, known only from Jamaica, B.W.I. Arista with 4 dorsal and 2 ventral branches in addition to the fork; front and orbits uniformly dark tan, ocellar area darker. Proclinate orbital about equalling length of posterior reclinate; anterior reclinate about half length proclinate. Antennae somewhat darker than front; face, cheeks, and clypeus grayish tan, palpi darker. Carina broadened below, cuneate; one strong oral, the 2nd about %its length. Pseudotracheae of labellum 8/8 ( & ), 9/9 ( 'i' ). Mesonotum dark tan, faintly striped, with a pair of light spots just anterior to and medial to transverse suture, and with less conspicuous light spots at bases of posterior supra-alars and medial to humerus. Scutellum uniformly dark tan. Acrostichal hairs in 8 rows; no prescutellars; anterior scutellars convergent. Anterior sternopleural thin, about 0.8 length posterior. Legs pale gray. Wings faintly brownish, most obviously so in marginal and submarginal cells; crossveins unclouded. Third costal section with the small black bristles on the basal%; 2 bristles of equal size at apex of 1st section. Costal index about 2.0; 4th vein index about 1.8; 5x index about 1.3. Abdominal tergites pale grayish tan at base and diffusely banded at apex, the apical bands of the basal tergites interrupted in midline. Body length, & , 2.9 mm., wing, 2.8 mm.; 'i' 3.8 mm., wing, 3.0 mm. The fore­going description was prepared from living material. Internal characters of imagines. Ventral receptacle with 12--15 tight coils; spermatheca (Fig. 10) small. Testes pale yellow with about 5 coils; ejaculatory bulb without diverticula. Geni­tal arch and clasper as in Figure 11; clasper large, bearing a primary row of 12 teeth, 2 large marginal bristles, and a line of 6 pale bristles from upper side to below margin. Other characteristics, relationship, and distribution. Eggs.-With 4 filaments about 1.5 times the egg length. Puparia.-Amber; posterior spiracles pale, slightly divergent; anterior spiracle with 10 branches, the horn plus branches about 1/7 length of body. Chromosomes.-The metaphase chromosomes, described by Clayton and Was­serman (this bulletin), consist of 5 pairs of rods and one pair of dots. Distribution and types.-Holotype male and 9 paratypes of both sexes, from stock No. H136.34 which originated from flies collected by Dr. W. B. Heed near Bath, Jamaica, B.W.I., February 1956. Relationship.-Belongs to the subgenus Drosophila, possibly close to the drey­fusi group. Drosophila castanea Patterson and Mainland Drosophila castanea Patterson and Mainland, 1944. Univ. Texas Pub. 4445:51. This species has now been collected over a wide area, from Mexico to Colombia and Venezuela. lntraspecific crosses have been made between 13 geographic strains: Mexico (2), El Salvador (3), Costa Rica (5), Colombia (2), and Vene­zuela ( 1). Of the 24 crosses attempted, including reciprocals, only one failed to produce fertile F1 resulting in an F2 • Figure 22 illustrates many of these crosses; solid lines represent fertile crosses, and the dotted line represents the cross: Me­dellin, Colombia s> X Huatusco, Mexico CS • Two castanea females were received in the laboratory from Medellin and when they failed to establish stocks each was mated to males from the Huatusco culture. One female died shortly there­after, but the other produced many F1 flies. These failed to produce an F2 on in­breeding and were then backcrossed to the Huatusco parent stock. The backcross FIG. 22. Crosses between geographic strains of Drosophila castanea. Localities are: 1, Huatusco, Mexico; 2, Atlixco, Mexico; 3, El Salvador. (3 stocks); 4, Costa Rica (5 stocks); 5, Medellin, Colombia; 6, Palmira, Colombia; 7, Merida, Venezuela. Solid lines show crosses producing fertile F1; the dotted line shows the cross which produced sterile F,. The University of Texas Publication was also sterile, indicating that both F, males and females were sterile. As is shown in the figure, however, castanea from Palmira, Colombia, crossed readily to several other stocks, producing fertile offspring. Drosophila suturalis Wheeler, new species. External characters of imagines. o , ~ .This new species simulates Zapriothrica dispar (Schiner) but it appears to be a case of convergent evolution rather than phylogenetic kinship. Front broad, dull tan to orange-brown, the ocellar area and elongated orbits darker brown, the latter a bit shiny. Head bristles rather short and stout, ocellars arising from outside the ocellar area on a level with the anterior ocellus. Proclinate or­bital far forward, 0.9 length posterior reclinate, the middle orbital hairlike, mid­way between the other two. Antennae, face, cheeks, clypeus, palpi and proboscis pale tan; arista with short branches, about 8 dorsal and 5 ventral, progressively shorter toward apex. Carina broad, rather low, rounded, the foveae deep. One prominent oral; cheeks broad, nearly half eye-height; eyes rather small, dark red with long thick pile. Mesonotum long and narrow, dark blackish brown; acrostichal hairs in 4 rows; no prescutellars. Posterior dorsocentrals in usual position, anterior pair far forward, at transverse suture, about % length posterior ones. Scutellum colored as mesonotum, basal bristles convergent to parallel. Pleura pale tan, con­trasting with the dark mesonotum; sternopleurals hairlike, short, the anterior one 0.8 length posterior. Halteres pale. Legs pale tan; tarsi long and slender; empodium and pulvilli small. Abdomen dark blackish brown, becoming tan laterally; male genital arch and anal plates tan, the latter noticeably elongated ventrally; female anal plates pale, ovipositor dark tan with exceptionally strong teeth apically. · Wings hyaline; 1st costal section with several stout short bristles, the longest one at apex; costa beyond the break weakly pectinate. Costal index about 2.6; ~th vein index about 1.5; 5x index about 1.2. Body length (pinned female) about 3.0 mm., wing, 2.4 mm.; o smaller. Distribution and types.-Known only from 45 specimens taken from a single Monstera flower, Turrialba, Costa Rica, July 29, 1956; collectors were W. B. Heed, H. L. Carson, and M. Wasserman who reported that the flies moved very fast for Drosophilids. We were not able to establish a culture, but a few eggs were observed, and possessed one pair of very short filaments. Holotype male, and 9 paratypes of both sexes, from Turrialba, Costa Rica. Relationship.-Of uncertain affinity, possibly related to the subgenus Phlori­dosa. Drosophila tibialis Wheeler, new species. External characters of imagines. o, ~. This species simulates Zapriothrica dispar (Schiner) and D. suturalis, described above, but is probably not closely related to either. Front orange-tan, o<:;.ellar triangle and elongated orbits gray pollinose; head bristles strong; procli­nate orbital slightly shorter than posterior reclinate, middle orbital % length proclinate and only slightly nearer it than posterior one. Antennae and face tan; arista with 3 dorsal and 1 ventral branches in addition to the often irregular terminal fork. Carina high, narrow and long, rounded; one strong oral. Cheek narrow, a bit brownish; clypeus pale brown, darker on sides; palpi tan; probos­cis pale with darkened labellum. Eyes dark red with thin pile. Mesonotum long and narrow, dark brown pollinose, a little lighter above humeri; dorsocentrals in the usual position. Acrostichal hairs 6-rowed; no pre­scutellars. Scutellum darker than mesonotum, basal bristles divergent. Pleura nearly as dark as mesonotum, the sutures all paler; anterior sternopleural half length posterior, a middle one not developed. Halteres brownish. Legs, including coxae, pale tan except for hind tibiae which are dark brown to black, but vari­able in intensity. Front femur thick, the inner side with a row of stubby bristles, the distal 4-5 of which arise from small tubercles. Abdomen uniformly dark blackish brown, pollinose, though some individuals show narrow basal tan areas on the tergites. Wings weakly smoky throughout; 3rd costal section with the small black bristles on the basal ¥3. Costal index about 3. 7; 4th vein index about 1.2; 5x index 0.9. Body length (pinned female) about 3.2 mm., wing, 2.5 mm.; (; a trifle smaller. The eggs have two pairs of thin filaments. Distribution and types.-Holotype male, 9 paratypes and 16 other specimens, Turrialba, Costa Rica, Oct. 1955, W. B. Heed collector, from flowers of Dimero­costus (Zingiberaceae); an additional 18 specimens, Cerro La Campana, Pana­ma, Aug. 1956, from flowers of Costus (same family). Drosophila reticulata Wheeler, new species. External characters of imagines. (; , c;? • The wing of this very beautiful fly is shown in Figure 23, and is suffi­ciently unusual that a new genus is suggested. Many other features, however, are not so bizarre, and we doubt if this species is more than subgenerically distinct. Arista with 6 dorsal and 2 ventral branches in addition to the fork; front broad, dull yellowish white, strongly sloping so that it and the face are contin­uous; head bristles normal; proclinate orbital % length posterior reclinate, the middle orbital minute. Antennae tan, 3rd joint elongate; face whitish yellow, carina evenly rounded. One prominent oral; cheek of medium width, brown below eye, creamy yellow posteriorly; clypeus pale, rather large. Palpi elongate and pointed, black at base, white at apex, the separation between the two colors nearly longitudinal. Eyes elongate oval with long white pile. Mesonotum rather tan to brown with shining bluish reflection; acrostichal hairs 4-rowed, sparse and thin, difficult to see; no prescutellars. Basal scutellars divergent; dorsocentrals normal. Pleura whitish with 5 brown spots, variable in intensity: just below and behind humerus, on lower middle of mesopleura, ,oh' upper sternopleura, below wing base, and over posterior spiracle. Halteres pale. Legs, including coxae, pale yellow but the "knees" darkened, and front femur with a small brown mark on inner side just beyond middle. Abdomen of female with broad apical bands expanded in the middle to base of previous tergite where they form narrow basal bands; remaining areas pale The University of Texas Publication FIG. 2-3. Drosophila reticulata, photograph of wing. yellowish; laterally the apical bands gradually disappear but the basal ones be­come a little larger. In the male the apical bands of the basal tergites continue to the sides, the basal bands are smaller, and tergite 6 has no basal band. Wings glassy and wrinkled, with the pattern shown in Figure 23; the darker spots in submarginal and posterior cells produced as bullae. Apex of 1st costal section with two bristles; 3rd section with the small black bristles on the basal %-¥2. Costal index about 2.8; 4th vein index about 1.7; 5x index about 1.2. Body length (pinned female) about 3.0 mm., wing, 3.3 mm.; J smaller. Internal characters of imagines. Anterior Malpighian tubes with the common stalk Y3 their total length; pos­terior tubes with their tips apposed, but without a continuous lumen. Ventral receptacle short, with 2-3 tight coils basally, remainder uncoiled. Spermathecae with very small chitinized centers, the surrounding glandular portion unusually large and frothy in appearance. Parovaria long and elliptical. Eggs with 2 pairs of filaments, the anterior pair % egg length, posterior pair % egg length; in the ovaries examined there was but a single developed egg in each ovary. Distribution and types.-Holotype male, and 3 paratypes, Medellin (30 klm. northwest), Colombia, 8000 ft., Nov. 1955, W. B. Heed collector. Other para­types as follows: 3, Bucaramanga, Colombia; 2, Rionegro, Colombia; 4, Rancho Grande, near Maracay, Venezuela. Eight specimens from Ecuador differ slightly in wing pattern are not being considered paratypes. GENUS CHYMOMYZA Chymomyza bicoloripes (Malloch) Drosophila bicoloripes Malloch, 192,6. P. U. S. N. M. 68 (2,1): 31. The generic combination used here has never been made officially although it has been used in an article on the moldy rot of rubber trees in Costa Rica (J. B. Carpenter. 1954. Plant Disease Reporter 38(5): 334-337), the determination of the flies being credited informally to Dr. W. W. Wirth. Carpenter reports that the species has been found constantly associated with active moldy rot lesions and saprophytic colonies of the mold Ceratostomella fimbriata, and it was suspected that the flies were primarily responsible for the spread of the mold. There has been some confusion concerning the type locality. Dr. Paul H. Arnaud, U.S.D.A., has been very helpful in settling this question. The type ( 5 ) bears the labels: "Las Cascadas, Canal Zone, Panama"; "A.H. Jennings Collec­tor"; and "Type No. 28467, USNM." Dr. Arnaud states that "I am inclined to believe that somehow Malloch incorrectly stated the type locality as Higuito, San Mateo, Costa Rica (P. Schild) both with the original description and in the type book." Dr. Arnaud reports that the USNM collection possesses, in addition to the type (which we have seen), 30 specimens from La Francia, Costa Rica, 11 from Turri­alba, Costa Rica, and 4 from Panama. Our own field collectors have taken this species on Barro Colorado Island, Canal Zone, "off fresh cut log," and at Villa­vicenico, Colombia, in August and September. Malloch's species is clearly a Chymomyza although it is unusual in having a strong black cloud over the posterior crossvein and in lacking a row of femoral spines. Chymomyza maculipennis Hendel (1936. Ann. Naturhist. Mus. Wien. 47:97) from the Amazon region of Brazil is, judging from the description, most likely the same species. However, he describes the pleura as lighter than the meso­notum, and we are unable to interpret satisfactorily his description of the 2nd vein as "winkelig gebrochen." Chymomyza mexicana Wheeler Chrmomrza mexicana Wheeler, 1949. Univ. Texas Publ. 4920: 162. The description was based upon a male from Puebla, Mexico. We have now seen five additional specimens as follows : 4, Santa Maria de Ostuma, Nicaragua, June 1954 (W. B. Heed); 1, Volcan Boqueron, El Salvador, 4500 feet, July 1954 (W. B. Heed). The present specimens do not agree exactly with the description, especially the pigmentation pattern of the front, but we believe that they are con­specific. GENUS CLADOCHAETA Cladochaeta nebulosa Coquillett Cladochaeta nebulosa Coquillett, 1900. P. U. S. N. M. 22:264. This species is apparently quite widespread on both the mainland and the islands of the Caribbean region. We have seen 18 specimens from the USNM collection, including the type from Puerto Rico, from Mexico (Vera Cruz, Tam­pico), Panama (Ft. Clayton, Darien Pr.), and Puerto Rico (many localities). Our collectors have saved more than 50 specimens, from Mexico (San Luis Po­tosi); El Salvador (San Salvador, Santa Tecla, Laguna de Zapotitan); Nicaragua (El Recreo); Colombia (Medellin, Bucaramanga) ; Trinidad (Sangre Grande); Venezuela (Merida); St. Lucia; Jamaica; Haiti (Kenscoff) ; and Cuba (Santiago de Cuba). It has also been recorded from Florida and from the Virgin Islands (St. Croix) . We have never been successful in culturing this species in the laboratory. GENUS NEOTANYGASTRELLA N eotanygastrella nigricosta (Malloch), new comb. Drosophila nigricosta Malloch, 1926. Proc. U.S. Nat. Mus. 68 (21 ):30. I have examined the type female from Costa Rica (U.S.N.M.) and a second female from El Recreo, Nicaragua (June 1954, W. B. Heed). There can be no doubt about the generic reference, although the anterior reclinate orbital is a little larger than usual, the face less bulbous below, and the legs all yellow. The large postverticals and absence of femoral spines serve to separate it from Chymo­mvza which it superficially resembles. The very dark costal and m'.1rginal cells are quite distinctive. Neotanygastrella antillea Wheeler, new species. External characters of imagines. t, 'i'. Similar in general appearance to leuco-poda and boliviensis (see Frota­Pessoa and Wheeler, 1951) , differing most obviously as follows: 2nd antenna! joint black. 3rd whitish yellow; palpi yellow; face with a shiny brownish black longitudinal stripe bordered by white on each side, the broad whitish areas be­ginning beneath the antennae and continuing down around the cheeks which gradually become black posteriorly. Front velvety black, strongly whitish pruinose posteriorly when viewed from certain angles; ocelli on a raised prominence, placed rather far forward, nearly equidistant between postverticals and ptilinal suture. Anterior reclinate orbital small, about %length proclinate and placed slightly to the side of and anterior to the latter. Arista with 4 dorsal and 2 ventral branches in addition to the fork. Clypeus dark, small; proboscis dark apically. Mesonotum dark brown to blackish, rather shiny but with thin gray pollen when seen from the side. Scutellum black, velvety, with yellowish apex. Pleura and prosternum dark brown. First legs with white coxae, trochanters, and 4 apical tarsal segments, and with blac;k femora, tibiae and metatarsi. Coxae of 2nd legs also black, remainder of legs yellow. Abdomen uniformly dark brown to black; 1st tergite and part of 2nd yellowed. Anal plates of female yellow, those of male rather small, yellow. Halteres whitish. Wings clear. Costal index about 1.4; 4th vein index about 3.8; 5x index about 1.8. Body length (pinned specimen), 'i', about 2.5 mm.; wing, 2.0 mm.; male slightly smaller. Distribution and types.-Holotype male and 6 paratypes of both sexes from Jamaica, British West Indies, collected February 1956, by Dr. W. B. Heed. Ap­proximately 50 additional specimens were taken by Dr. Heed in the Montego Bay area of Jamaica in July 195 7. Neotanygastrella ornata Wheeler, new species. External characters of imagines. t , 'i' . Similar to tricoloripes Duda but with mesopleura velvety black and strongly contrasting with the pale tan sternopleura, and with more extensive . darkening on the mesonotum. Arista with 4 dorsal and 2 ventral branches in addi­tion to the fork. Front velvety black, whitish pruinose behind when seen from most angles. Antennae pale tan. Face bulbous below, with a broad black median stripe, narrowly yellow along sides; clypeus black, palpi yellow, cheeks yellow becoming black behind. One strong oral. Postverticals strong; anterior reclinate orbital ¥2 length proclinate and placed lateral of and slightly anterior to the latter. Mesonotum basically tan with a broad blackish central stripe between the dorsocentral lines, widening anteriorly to reach humeri; it is also black above and in front of the wing base thus leaving an irregular, winding yellow area from humerus (and fore coxa) upwards and then back to dorsocentrals. Acrostichals irregularly 8-rowed; no prescutellars. Basal scutellars parallel. Scutellum velvety black with white tip. Postscutellum dark brown, this color continued laterally to around haltere base; haltere pale. Legs yellow except: fore coxae whitish, fore femur, tibia and metatarsus black, the 4 apical joints white. Abdomen black, a bit velvety, the basal two tergites paler. Wings clear; 3rd costal section with the small black bristles on the basal%. Costal index about 1.3; 4th vein index about 3.6; 5x index about 2.4. Body length (pinned specimen), c;i, 2.0 mm.; wing, 2.0 mm.; males a little smaller. Distribution and types.-Holotype male and one paratype female, from Me­rida, Venezuela, collected in October 1956, by Dr. Marvin Wasserman. N eotanygastrella tricoloripes Duda Neotanygastrella tricoloripes Duda, 1925. Ann. Mus. Nat. Hung. 22:224. Duda's specimens came from Costa Rica; our own collections have shown that this species is widely distributed: Honduras, Costa Rica, Panama, Colombia, Trinidad, Peru, Brazil, and Puerto Rico (El Yunque). Judging from the 70+ specimens in our collection, we are unable to find any consistent differences from N . brasiliensis (Frota-Pessoa), but a careful comparison of male genitalia has not yet been made. Neotanygastrella chymomyzoides Duda Neotanygastrella chyrrromyzoides Duda, 1927. Arch. Naturg. 91A 11-12 (1925):71. The species was described from specimens from Peru and Bolivia; I have ex­amined one specimen (American Museum of Natural History) labelled "Coty­pus" from "Bolivia-Mapiri." We have preserved about 180 specimemi of this species and have noticed that while most of the specimens show th~,frpical color pattern of the front legs (metatarsus black, apical joints pale) , about 30 of them have all of the fore tarsi dark. The significance of this is not yet knowp..ln the following distributional list, starred countries are those from which dark-legged specimens were taken: El Salvador, Nicaragua, Honduras, Costa Rica"* Pana­ma,* Colombia,* Venezuela, Trinidad, Brazil,* E~uador,* Penh* and ·~;t.' Lucia (B. W.L). GENUS DETTOPSOMYIA Dettopsomyia formosa Lamb Dettopsomyia formosa Lamb, 1914. Trans. Linn. Soc. London 16:350. This very small, pretty species is apparently quite widespread. Lamb's ma­terial came from the Seychelles, and it is also known from Hawaii, the Palau and Solomon Islands, Yap, and the Philippines. We can now report it from Cen­tral America where Dr. Heed collected about 70 specimens as follows: El Sal­vador (Santa Tecla, Laguna de Zapotitan); Honduras (La Lima). Collections were made in March and May, 1954. Live specimens from El Salvador were kept in the laboratory for a short while; the eggs had 4 short filaments, each about Y3 the egg length. GENUS CLASTOPTEROMYIA Clastopteromyia superba (Sturtevant) Drosophila superba Sturtevant, 1916. Ann. Ent. Soc. Am. 9:342. I have examined the type from Guatemala (USNM collection); it agrees quite well with the 11 specimens taken by our collectors. Localities are: El Sal­vador (San Salvador, Santa Tecla, Laguna de Zapotitan); Honduras (Lance­tilla); Costa Rica (Turrialba, La Lola); Colombia (El Recuerdo). We have also examined 3 specimens (USNM) from the Panama Canal Zone. The description states that only a single pair of dorsocentrals is present; on our specimens when the anterior dorsocentrals are missing, one can still detect the empty socket, indicating that two pairs are normally present. Clastopteromyia longipennis Malloch Clastopteromyia longipennis Malloch, 1926. Proc. U. S. Nat. Mus. 68:34. The type, from Costa Rica, shows that the very dark brown prosternum, which contrasts strongly with the pale legs, is a very distinctive feature. We have 10 specimens from: El Salvador (San Salvador), Honduras (Lancetilla), Costa Rica (San Isidro de General), and Canal Zone (Barro Colorado Is.). One individual, from Trinidad, appears to be an undescribed species very closely related to longipennis. Clastopteromyia guttipennis (Duda) Diathoneura guttipennis Duda, 1925. Ann. Mus. Nat. Hung. 22: 171 ; 1927. Arch. Naturg. 91A 11 (1925) :91­Clastopleromyia guttipennis, Frota-Pessoa, 1947. Sum. Brasil. Biol. 1:36. Duda described the species from Z ~ ~ , 1 ~ from Costa Rica and later re­corded 1 ~ , 1 ~ from Peru. We have seen a single specimen from Almirante, Bocas del Toro, Panama (USNM) which we identify as guttipennis although it shows these small differences: marginal cell of wing with an additional white spot, rather small, nearly in line with the posterior crossvein (thus between the two prominent white stripes of the wing); halteres somewhat darkened and palpi definitely brownish; face and orbits withm1t noticeably whitish areas along edge. Clastopteromyia bomplandi (Malloch) Diathoneura bomplandi Malloch, 1934. Dipt. Patag. Chile 6:438. Clastopteromyia bomplandi, Frota-Pessoa, 1947. Sum. Brasil Biol. 1: 18. We have a single specimen, from San Salvador, El Salvador, which extends the known distribution considerably. Malloch's type and paratype came from Bompland, Argentina, and Frota-Pessoa reports it from Brazil. The species is quite similar to taeniatipennis Duda from Costa Rica, but the latter has con­siderable darkening along the leading edge of the marginal cell; and the other dark areas are much more prominent than in bomplandi. Clastopteromyia aberrans Wheeler, new species. The species described here differs from the typical members of the genus in (1) the absence of posterior scutellar bristles and (2) the presence of only two rows of acrostichal hairs. In other respects it appears to be fairly closely related to other members of this genus. The highly marked wings are rather similar to those of guttipennis and borgmeieri. iS, ,about 2.0 mm.; wing, 2.0 mm. Distribution and types.-Holotype male, Cerro La Campana, Panama (about 60 klm. SW Panama City), collected August 1956, by W. B. Heed, H. L. Carson, and M. Wasserman. Two paratypes, same data; 8 paratypes, Barro Colorado Is., Canal Zone. We have, in addition, about 35 specimens from: Nicaragua, Hon­duras, Costa Rica, Colombia, Venezuela, Trinidad, and Brazil. GENUS PARALIODROSOPHILA Paraliodrosophila antennata Wheeler, new species. External characters of imagines. ~ , <;> . Arista with 4 dorsal, 1 ventral branch in addition to the fork. Front dark brown to black, the large shiny frontal triangle gradually tapering to a point at the ptilinal suture; orbits also shiny, rest of front velvety brown. Second antenna! joint whitish yellow, strongly contrasting with the dark front, and with the brown 3rd joint. Postverticals rather small; proclinate and posterior reclinate or­bitals equal in size, the middle one about VJ as long. Face dark tan, the carina of moderate size, rather short; oral margin dark brown as is the area around the vibrissae; cheeks white posteriorly. One strong oral. Clypeus black, dull; palpi small, light brown. Mesonotum dark brownish black, shining; acrostichal hairs in 6 rows, absent posteriorly from the level of posterior dorsocentrals. No prescutellars; basal scutel­lars convergent. Scutellum rather rounded, more pollinose than mesonotuin. Anterior dorsocentral short, about VJ posterior. Pleura pale whitish yellow except for a narrow area above, which is velvety black beginning near base of 1st coxa and going to wing base. Basal joints of halteres brownish, the knob tan. Legs wholly yellow. Abdomen with large dark bands, semi-shining, with creamy yellow areas; on both male and female tergites 1-3 are paler medianly, 4-5 are dark in the middle, leaving basal yellow areas on each side, that of 5 extended all the way laterallv; remaining tergites dark, but all dark bands cease before reaching lateral edge, the edges thus all yellow. Anal plate of 'i' yellow; ovipositor pale, truncate, with dark teeth. Wings hyaline, a little darkened below distal costal break. Small black bristles of 3rd costal section poorly differentiated, but occur on about the basal 0.6. Costal index 1.4; 4th vein index about 2.5; 5x index about 3.0. Body length (pinned specimen) , <;>, up to 2.2 mm.; wing, about 2.4 min~ ; males smaller. Distribution and types.-Holotype male and 9 paratypes, from near Bath, Jamaica, B. W. I., collected February 1956, by Dr. W. B. Heed. There are six ad­ditional specimens from the same locality. GENUS AMIOTA The type species is leucostoma Loew, designated by Coquillett (1910. Proc. U.S. Nat. Mus. 37 (No. 1719): 505) and not humeralis as I have stated earlier (Wheeler, 1952. Univ. Texas Puhl. 5204: 166). Amiota humeralis Loew Amiota humeralis Loew, 1862. Berlin Ent. Zeit. 6:229 (Cent. 11, No. 93). An examination of the holotype female, in the collection of the Museum of Comparative Zoology at Harvard, shows that my earlier concept of this species was in error (Wheeler, op. cit., p. 168). The specimen is in fair condition, but, aside from size, shows only one outstanding feature that may serve to recognize it: the front is noticeably golden pruinose when viewed from most angles. The only other species known to me with this character is setigera Malloch, of which we have 1 J from Rutledge, Tenn. and 1 J from Lithium, Mo. Of the 9 females from Lithium which we consider are the females of setigera, some show the prui­nosity and others have it less developed. There is a possibility, then, that the male of humeralis is setigera, but for the present there seems to be no way to verify this. Amiota steganoptera Malloch Amiota steganoptera Malloch, 1926. Proc. U.S. Nat. Mus. 68:31. This species seems not to have been recorded since its description, but we find that it has a very extensive distribution. It is easily recognized by the presence of thorn-like "warts" on the 3rd costal section, quite like those characteristic of Leucophenga and Stegana. The type, from Higuito, San Mateo, Costa Rica (USNM) has been examined. We have seen 30 additional specimens as follows: 1, Saucier, Miss.; 1, Kushla, Ala.; 1, Blacksburg, Va.; Mexico; El Salvador; Nicaragua; Honduras; Costa Rica; Panama; Brazil; and Puerto Rico (Rio Grande). It should be mentioned that there are at least three undescibed species closely related to steganoptera occur­ring in Brazil. GENUS PsEUDIASTATA Sabrosky ( 1951) summarizes all that is known about this rare genus. All of the species so far recorded are quite similar to the type species, nebulosa, but there are obvious differences in male genitalia. Among the specimens loaned to us from the National Museum collection were four such individuals, and we borrowed another from the American Museum of Natural History. The latter, from Guate­mala, is a female, and cannot be determined further. Two are males, and have been dissected and examined. One, from Cano Saddle, Gatun Lake, Panama, is pseudococcivora Sabrosky; the other male, from Cuernavaca, Mor. ,Mexico, has a genitalial arrangement not figured by Sabrosky. This genitalia is shown in Figures 15-16, but the species is not being named at the present time. The remaining two specimens are females, and have an entirely different wing pattern; further, the 3rd vein is bristled, a character not previously known in the Drosophilidae. These are described below as a new species, P. armata. Pseudiastata armata Wheeler, new species. With the general appearance of nebulosa, but with clearly different wing pat­tern (Fig. 25). Many stout prescutellar bristles, the middle pair quite long and reaching to scutellar apex, the rest (about 6) only half this long. Fringe of calyp- FIG. 25. Pseudiastata armata, wing. ter especially long. Ovipositor with numerous pale hairs. All femora rather thick, the 3rd ones especially so, nearly globose. Third vein bristled, both above and below, from the bifurcation of 2L and 3L to just beyond the anterior crossvein, with about 6 bristles visible dorsally on the best-preserved wing, and 3 ventrally: 1 at the bifurcation, 1 just before anterior crossvein, and 1 about midway between the other two. Second wing of this specimen missing. On female 2, the wings are bent downward in such a way that the ventral surfaces cannot be studied, and the upper sides are considerably rubbed. However, on one wing there are 6 empty sockets dorsally plus 4 small bristles, the distal one just a little beyond the level of the posterior crossvein. It is apparent, therefore, that the actual distribution of these bristles is variable. Distribution and types.-Holotype ~ , USNM collection, labelled "Mojinga Swamp; Ft. Sherman, C.Z." (Canal Zone, Panama); "6-17-52"; "F. S. Blanton Collector." Paratype ~ , samelocality, collected 5-13-52. GENUS LAcconROSOPHILA Duda erected the genus for two species from Bolivia, and it seems not to have been recorded since. Since he did not designate a type species I am selecting his first species, L. fiavipes Duda, as the type species. Laccodrosophila fiavipes Duda Laccodrosophila flavipes Duda, 1927. Arch. Naturg. 91A 11 (1925) :38. Duda described the male only; we have a female (AMNH collection) with the following labels: "Minza Chica; V. Tungurahua; 3750 M.; Ecuad.; 2--14 IV, 1939; F. M. Brown." The legs are all yellow; front and mesonotum black; pleura brownish black, subshining; antennae, face and palpi yellow. Postverticals large. Ocellars ap­parently divergent, placed outside the ocellar triangle, in line with anterior ocel­lus; 3 orbitals extra thin, nearly equidistant from one another. Halteres yellowish white. Empodium and pulvilli unusually large. Front tarsi appearing to consist of but 4 joints (see Duda's figures 6 and 8) but as seen under higher magnification (120X) it can be observed that the apparent "metatarsus" consists of the fused 1st and 2nd joints, each of which bears a pair of stout black spines apically. Two pairs of dorsocentrals, thin. Scutellum with marginal hairs, arising from the lower edge and upturned; both basal and apical scutellars arise from short black tubercles. Prescutellars present. Duda described the male as having the 3 basal tergites black, 4th black at basal angles, otherwise reddish yellow, as is the 5th and 6th. On this female the tergites are yellow with narrow apical bands, broader at the angle. Anal plates yellow, long and flat; ovipositor long and hairy, of unusual shape and partly encased within a tubular sheath. Wings pale, the posterior fringe long. First vein, dorsally, with 1-2 black bristly hairs just before its union with the costa. Laccodrosophila atra Duda Laccodrosophila atra Duda, 19Z7. Arch. Naturg. 91A 11 (19Z5) :40. Duda saw 4 J J , 3 <;> <;> from Bolivia; we have a female which we tentatively identify with this spcies, from the vicinty of Bogota, Colombia (Nov. 1955, W. B. Heed), "off pink Digitalis flower." In this species the face is black as are the palpi and proboscis; abdomen shining black; arista with only a single dorsal branch plus the large fork. The scutellar margin is haired as in the former species, and the scutellars also arise from small tubercles. The legs are thicker, mostly black with yellowish tarsi. First tarsal joint of leg 1 with a very stout spine plus 7-10 ;smaller ones, the 2nd joint with a single smaller spine. Laccodrosophila flavescens Wheeler, new species . .External characters of imagines. J , '? . Quite similar to fiavipes, differing primarily in the shape and color of the face and cheeks, and the much more yellowed abdomen. Face narrow with deep foveae separated by a strong ridge, the latter blackened, the foveae pale; the dark ridge is continuous with a dark structure on anterior surface of proboscis. Cheeks proad, the bristled area dark, the remainder tan ( <;> ) , shiny next the eye, becoming striated next to the bristled area; lower posterior corner dark reddish brown ( '? ) , strongly rugulose, the creases parallel to lower cheek margin. In males, dark areas are more black, the non-striated portion of the cheek much darker than in females. Postverticals % length ocellars, latter long. Orbitals on the best preserved specimen flattened against head, proclinates mildly convergent, middle reclinates somewhat diverging, posterior reclinates inclined outward over the eye. bn.e pair of strong orals. Abdomert"inostlf' yellow, wholly yellow oh males, only the genital arch brownish; in females tergites 4-5 a little darker laterally, the apex shiny brown, intiuding sides of sheath of ovipositor. Legs yellow, both sexes with 1st tarsal spines as iri fiavipes, Wings br0ad, hyaline with yellow veins; apex of 1st vein a bit thickened and discolored at its union with costa, without hairs; costa somewhat pectinate, with small sparse black spines. Costa! index about 1.3; 4th vein index about 2.0; 5x index about 0.9. Body length (pinned female with extended ovipositor) about 4.5 mm.; wing, 4.0 mm.; i , 3.5 mm., wing, 3.7 mm. Distribution and types.-Holotype i , Z i , 1 9 paratypes, USNM collection, labelled: Pedregal, near Pobre Pena, Mexico, 14 Sept. 1942, W. F. Foshag; 1 9, USNM, L. Patzcuaro, Mich., Mexico, 29 Aug. 1945, W.F. Foshag. Laccodrosophila heedi Wheeler, new species. External characters of imagines. i , 9 . A shiny black species with broad cheek, unusually low costal index, and with the apex of 1st vein strongly blackened. Front black, rather shiny, with nu­merous small hairs; posterior reclinate orbital about% length proclinate, middle orbital Yz length posterior. Antennae and palpi dark tan; arista short, with one basal dorsal branch and one more distal ventral branch in addition to the fork. Face deeply sunken, without interfoveal ridge; one strong oral; oral margin, clypeus and cheeks black, the latter very broad, mostly shining, but with a sha­greened quality on under side. Mesonotum and scutellum black; acrostichals typical for this genus; pre­scutellars strong; scutellars tuberculate; scutellar margin typically haired. Pleura shining black; mesopleura with scattered small hairs on upper part (absent on fiavipes, fiavescens, and atra); legs mostly shining black including coxae. Tarsi of fore legs bearing three stout black spines (Fig. 12), fore tarsi black, other tarsi yellow except for blackened apical joint. Empodia and pulvilli large, pale. Abdomen shining black; 9 anal plate long, flat, yellow; i anal plate not well observed but apparently pale. Wings hyaline, veins pale. Apex of 1st costal sec­tion, and apex of 1st vein enlarged and blackened. Body length (pinned female with retracted ovipositor) 3.0 mm., wing, 2.5 mm. Male smaller. Costal index about 0.55, the 2nd costal section being only about half length 3rd; 4th vein index about 2.6; 5x index 2.0. Distribution and types.-Holotype 9 , paratype i , from orchid flowers, orchid garden, Medellin, Colombia, Nov. 16, 1955, W. B. Heed collector. REFERENCES Basden, E. B. 1954. The distribution and biology of Drosophilidae (Diptera) in Scotland, includ­ing a new species of Drosophila. Trans. Royal Soc. Edinburgh 62(3) :603-654. Collin, J. E. 1952. Notes on some Drosophilidae (Dipt.), including five additional British species, two of them new to science. The Ent. Mon. Mag. 88: 197-199. Frota-Pessoa, 0. 1954. Revision of the tripunctata group of Drosophila with description of fifteen new species (Drosophilidae, Diptera). Arquivos do Museu Paranaense, Curitiba, 10:253-330. Frota-Pessoa, 0., and M. R. Wheeler. 1951. A revision of the genus Neotanygastrella Duda. Rev. Brasil. Biol. 11 : 145-151. Hackman, W. 1954. Die Drosophila-Arten Finnlands. Notulae Ent. 34: 130-139. Malogolowkin, C. 1953. Sohre a genitalia dos Drosofilideos. IV. A genitalia masculina no sub­genero Drosphila (Diptera, Drosophilidae). Rev. Brasil. Biol. 13:245-264. Okada, T. 1956. Systematic study of Drosophilidae and allied Families of Japan. Tokyo, G:hodo Co., Ltd. 183 pp. Patterson, J. T., and Th. Dobzhansky. 1945. Incipient reproductive isolation between two sub­species of Drosophila pallidipennis. Genetics 30:429-438. Sabrosky, C. W. 1951. Two new species of Pseudiastata (Dipt., Drosophilidae) pre ravine San Salvador, Dec. 19, 1953 5 c;> c;> ravine 2000 ft. Jan. 20, 1954 1 c;> ravine Mar. 2. 1954 1 c;> ravine Sept. 27, 1955 1 c;> ravine origin of parthenogenetic stock Honduras: Lancetilla, Apr. 4, 1954 1 c;> orchard on m1ngosteenas fruit 100 ft. (Garcinia sp.) . Nicaragua : Santa Maria de June 30, 1954 c;> coffee finca banana-baited trap Ostuma, 4000 ft. July 1, 1954 c;> coffee finca banana-baited trap Costa R;rn: T,1rri1lba, Oct., 1955 1 c;> 2000 ft. Panama Canal Zone: Barro Colorado Nov. 1, 1955 ram only known male to elate Island, 200 ft. 1 i forest Trinidad : Port of Spain, 100 ft. Sangre Grande D ec. 21, 1955 Dec. 26, 1955 Dec. 28, 1955 1 c;> 1 c;> 1 c;> Queen's Savannah on fallen nowers of E rythrina sp. Cacao finca Cacao sweeping over breadfruit finc Holotype female and at the Instituto Tropical de Investigaciones Cientificas, where the collections were made, it has a depth of from 20 to 60 feet and an average width of about 10 feet. The walls are nearly perpendicular and at intervals are quite densely covered with such vegetation as ferns, begonias, Selaginella, bromeliads (Pit­cairnea) and bloodworts (Xiphidium). Sweeping over this vegetation with a long-handled net yielded more than 100 species of drosophilids in an eleven month period. CYTOLOGY AND REPRODUCTION The single strain of D. mangabeirai now extant in the laboratory was estab­lished from a single wild female individual captured at San Salvador, El Salvador on September 27, 1955. Since this time the strain has been reared on standard culture media by routine methods in both the Texas and St. Louis laboratories. When the strain was first subjected to close scrutiny approximately one year after it was established, no male individuals could be found. In a systematic check of the sub-strain in the St. Louis laboratory, 2,365 hatching individuals were screened; all were normal females except for three females with slightly rotated genitalia. A simultaneous check of the strain at The University of Texas laboratory also showed only females and all specimens which had been previously pinned from this strain were likewise found to be females. There is thus no evidence that males have ever occurred in this strain. Cytological studies on the brain and salivary glands of larvae were made using the conventional aceto-orcein technique. Brain metaphases were clearly observed in smears from 14 different larvae; each showed the same configuration, namely, the diploid condition of two pairs of V-shaped chromosomes and one pair of rods (similar to the willistoni group). Furthermore, Dr. H. D. Stalker kindly examined the wing cells of 671 individuals without finding any which showed a cell size suggestive of triploidy or tetraploidy. Salivary gland chromosome preparations were examined and chromosome conditions recorded in a total of 50 individual larvae chosen at random from the stock. Each larva showed five salivary gland chromosome arms. Two of these arms tend to come out of the chromocenter attached to the same heterochromatic mass (Fig. 2, bottom, center). Without exception, in every individual examined, these two arms show the identical heterozygous configurations shown in Figure ~ There is a nearly terminal overlapping inversion configuration in one of the two arms (Fig. 2, left) and a simple, just barely subterminal inversion loop in the other (Fig. 2, right). The other three chromosome arms were homozygous for gene arrangement in every case. The X chromosome cannot be identified. To summarize, this strain apparently consists exclusively of parthenogeneti­cally-produced diploid females, each of which is heterozygous for the same struc­tural karyotype. In order to determine the hatchability of the unfertilized eggs of this species, newly-emerged flies were collected and aged for four days. Ten females were selected at random and each female placed without etherization into an indi­vidual culture vial. Each female was changed to a fresh vial every day for the succeeding 21 days. All females were still vigorous and healthy when they were discarded at the end of the three-week period of observation. Eggs were counted FIG. 2. Heterozygous configurations of salivary glai:id chromosomes in D. mangabeirai. daily. Larvae were counted daily as they hatched and were removed from the vial with a needle. They were either destroyed or transferred to a fresh yeasted vial. Table 2 shows the hatchability of eggs of each of the ten females. The number of transferred larvae which survived to adults is given in Table 3 for each of the 26 vials so prepared. Survival between larva and adult was greater when 12 or fewer larvae were transfer~ed (average survival: 86.6%) than when 13 or more were transferred (average survival: 72.8%). Some of the larval mortality observed is thus apparently due to larval crowding under the conditions of the experiment. Even though D. mangabeirai is a small species (approximately the size of D. melmwgaster), the generation time from egg to adult at 25.5° C. is long, that is, approximately 18 days under optimal conditions. Maturation of females takes about four days so that the minimum generation time is approximately 22 days. As in many other species, however, slight crowding or underfeeding of larvae results in very great extension of the length of larval life. TABLE 2 Hatchability of unfertilized eggs of Drosophila mangabeirai Malogolowkin. Each female was changed to a new vial for 21 successive days. Eggs were counted daily. Female Total number Number of larvae Per cent number of eggs laid hatching hatchability 29 49.2 13 30 8 12 59 26.7 14 135 79 59.5 15 123 84 68.3 16 119 79 66.4 17 149 101 67.8 18 99 63 63.6 19 63 33 52.4 20 96 49 51.0 21 20 5 25.0 893 530 59.4 TABLE 3 Survival of Drosophila mangabeirai from larva to adult. Newly hatched larvae were counted and transferred to fresh vials. The number of adults emerging from each vial were recorded. No. of larvae No. of adults Per cent Vial No. transferred emergmg survival 1 4 3 75 .0 2 6 6 100.00 3 9 7 77.8 4 12 12 100.00 5 8 6 75.0 6 18 15 83.3 7 13 8 61.5 8 17 11 64.7 9 19 14 73.7 10 15 11 73.3 11 17 10 58.8 12 15 14 93.3 13 10 7 70.0 14 9 9 100.0 15 12 10 83.8 16 8 7 87.5 17 19 17 89.5 18 15 10 66.7 19 17 13 76.5 20 19 11 57.9 21 12 11 91.7 22 11 9 81 .8 23 12 10 83.8 24 12 11 91.7 25 12 10 83.8 26 12 11 91.7 Total 333 263 79.0 CONCLUSION Whether thelytokous parthenogenesis as found in the El Salvador strain of D. mangabeirai is characteristic of wild populations of this species or not must remain a matter of conjecture at the present time. Although rare, the very exist­ence of a male in the collections suggests that the species has at least a residual bisexuality. Nevertheless, the rate of parthenogenesis in this strain is so high that culture of it in large numbers by routine laboratory methods is easily accom­plished. Such an efficient method of parthenogenetic reproduction could hardly be a matter of chance occurrence in a single strain. Rather, it is probable that parthenogenesis has somehow been brought to its present level of efficiency by natural selection. The type of parthenogenesis, that is, the manner in which diploidy is main­tained in the absence of syngamy, is not yet known. From the genetic point of view it is important to know if the method involves meiotic reduction and fusion of two haploid nuclei ( automixis) or whether the definitive nucleus of the egg is derived directly from a diploid cell of the mother without intervention of meiosis ( apomixis). Further cytological or genetic evidence will be necessary before either of the above alternatives can be demonstrated. At present, however, the indications are as follows. That automixis may occur is suggested by the fact that Stalker ( 1954) has conclusively demonstrated this process in Drosophila parthenogenetica; it probably also forms the basis for parthenogenesis in the related species D. poly­morpha. Automixis also apparently occurs in the parthenogenetic fly, Lonchop­tera dubia (Stalker, 1956). This latter case is of special interest in that the cyto­logically-observed automictic mechanism apparently involves the fusion of the central two of the four pronuclei produced at meiosis. Such a process would serve to reconstitute exactly the original heterozygosity of the mother, thus maintain­ing intact the fixed heterozygous karyotype characteristic of each of the choromo­somal races in L. dubia. Such a mechanism could also be operative in D. manga­beirai and reproduce the observed fixed heterokaryotype. On the other hand, apomixis cannot be excluded; indeed, certain facts suggest it as a possibility. In the first place, if meiosis is essentially normal, then non­ disjunction of X chromosomes should produce an occasional male. No males have been observed. Secondly, if automixis, involving fusion of haploid products of meiosis is occurring, then occasional triploids due to fusion of three nucl~i would be expected. No triploids have been found. Finally, the maintenance of the fixed heterokaryotype appears to be complete. This suggests apomixis because under automixis, regularity could be assured only if fusion of central nuclei in the egg is likewise highly regular. If automixis of the type which occurs in D. parthenogenetica has served as the starting place for the evolution of parthenogenesis in D. mangabeirai, then natural selection must have eliminated a number of the inefficient features of this system. Further study of mangabeirai may thus not only provide clues to the evolutionary origin of parthenogenesis in the Diptera but might also provide fundamental information on the manner in which nuclear and cellular dynamics themselves may respond to natural selection. SUMMARY Pertinent facts on the taxonomy, relationships, ecology and distribution of the sophophoran species Drosophila mangabeirai Malogolowkin are presented. The only known strain of this species is unique in the genus Drosophila in that it reproduces wholly by diploid parthenogenesis. About 60% of the eggs laid by virgin females hatch. No males have been produced; all individuals examined have been diploid females and all are heterozygous for the same two inversion configurations. Parthenogenetic reproduction, as it now exists in this strain is efficient and it is considered likely that the system has evolved in natural popu­lations and is not a chance occurrence in a single strain. Existing data do not permit a definitive statement as to whether the type of parthenogenesis in this strain is automictic or apomictic. REFERENCES Malogolowkin, C. 1951. Drosofiiideos colhidos na Bahia, com descricao de uma especie nova (Diptera) . Rev. Brasil. Biol. 11(4) : 431-434. Patterson, J. T ., and W . S. Stone. 1952. Evolution in the Genus Drosophila. New York. The Macmillan Co. 610 pp. Stalker, H. D. 1954. Parthenogenesis in Drosophila. Genetics 39 ( 1): 4-34. Stalker, H . D. 1956. On the evolution of parthenogenesis in Lonchoptera (Diptera) . Evolution 10(4): 345-359. Sturtevant, A. H. 1921. The North American Species of Drosophila. Carnegie Institution of Washington Publication No. 301. 150 pp. IX. A Preliminary Note on the Cardini Group of Drosophila in the Lesser Antilles WILLIAM B. HEED1 Townsend and Wheeler ( 1955) described a new species, Drosophila dunni, in the cardini group from Puerto Rico. More recently the author has found a north­west, south-east gradation in abdominal pattern of dunni-like forms on three islands of the Lesser Antilles. Collections were made in January 1956 on Bar­bados, St. Lucia and St. Kitts, British West Indies. The abdominal markings of dunni consist mostly of light paramedian mark­ings on the 3rd and 4th tergites; these markings become slightly heavier and more fused in the St. Kitts form, and they become still darker and broader in the form from St. Lucia. The abdomen of the Barbados form is most distinct of all, being mostly brownish black with a dorsal tan pattern. Superficial study of other characters, such as size, color of mesonotum, number of primary and secondary teeth on the forceps of the male genitalia, and extent of heavy bristles on the 3rd costal section of the wing show a disjunct relation in that the Puerto Rico and St. Lucia forms are more similar while the St. Kitts and Barbados forms are, in turn, more similar to each other. Hybridization tests were made with dunni from Puerto Rico and the dark form from Barbados. When females from Puerto Rico were crossed to males from Barbados, hybrids intermediate in color were produced (Table 1). The hybrids failed to produce progeny after being tested for two weeks. The males were then dissected and examined. The testes were smaller than is typical for either paren­tal form and neither active nor inactive sperm were seen. The hybrid females were not tested for fertility. The reciprocal cross (Barbados <;> X Puerto Rico o) did not produce hybrids. On the basis of the crosses and the clearly distinct abdominal pattern, the Bar­bados form is being described as a new species, D. nigrodunni, by Heed and TABLE 1 Hybridization tests of D. dunni from Puerto Rico (P) and D. nigrodunni from Barbados (B). Cross No. mated Date Date Date Total 0 0 0 mated lumped discarded F, 0 0 P x B 8 8 5/ 11 l 8 9 5/ 11 ~ 5/ 21 6/ 13 12 9 5/ 11 47 32 15 B x P 11 8 5/11 9 11 5/ 11 f 5/ 21 6/ 1* 12 15 5/ 11 J 9 20 5/ 20 6/ 13* P x P 6 8 5/ 11 6/ 13 5 7 5/ 14 6/ 13 12 12 5/ 14 6/ 13 B X B 10 8 5/ 14 6/ 13 8 6 5/ 14 6/ 13 22 17 5/ 17 6/ 13 0 0 267+ 150+ 91 + 46+ 2 19+ 0 0 140 74 46 22 2 8 0 0 127 76 45 24 0 11 • Females dead at this time. 1 Zoological Laboratory, Univ. Pennsylvania, Philadelphia. Wheeler (this bulletin). The results of the crosses are given in Table 1. It may be seen from the control test that nigrodunni is difficult to maintain on banana medium. This is also true of the forms from St. Lucia and St. Kitts and further testing is impractical at this time. The islands adjacent to the Lesser Antilles were also collected. Trinidad was collected in December, and Puerto Rico in February, 1956. No dunni-like forms were taken in Trinidad. This island shows true continental affinities in that five members of the cardini group, all represented on the mainland, have been taken here. They are cardini, polymorpha, cardinoides, neomorpha, and a form very similar to parthenogenetica (differs in having a shiny black abdominal pattern typical of most of the other cardini group species, and a slightly higher secondary bristle count on the palpi. Drosophila neomorpha is being described as new in this bulletin (article by Heed and Wheeler). In Puerto Rico the only species of the cardini group other than dunni was D. cardini, which was also collected on St. Lucia and has been reported from Do­minica (Sturtevant, 1921). One other species of the Lesser Antilles should be mentioned: Drosophila similis, described by Williston from the island of St. Vincent. The author has examined a type specimen from the American Museum of Natural History and has come to the conclusion that this specimen belongs to the dunni-like forms. The cardini group shows an extremely interesting distributional pattern especially in the West Indies and the adjacent mainland. Part of this pattern is briefly outlined above. Future collecting and subsequent analysis following up Stalker's important contribution (1953) to the genetic affinities of the species then known in the cardini group will produce valuable information about the evolution of the group. REFERENCES Stalker, H. D. 1953. Taxonomy and hybridization in the cardini group of Drosophila. Ann. Ent. Soc.Amer.46:343-358. Sturtevant, A. H. 1921. The North American species of Drosophila. Carneg. Inst. Wash. Puhl. 301. 150 pp. Townsend, J. I., and M . R. Wheeler. 1955. Notes on Puerto Rican Drosophilidae, including descriptions of two new species of Drosophila. Jour. Agric. Univ. Puerto Rico 39:57-64. X. Chromosomal Studies of Several Species of Drosophila 1 FRANCES E. CLAYTONAND MARVIN WASSERMAN INTRODUCTION This report includes cytological analyses of a number of new species of Dro­sophila, as well as of additional strains of species which have been reported upon previously by the authors and other investigators. The preparation of the salivary gland chromosomes and larval brain gan­glion slides has been by our usual techniques. The larval ganglion is stained for approximately 15 minutes with aceto-orcein after which it is squashed in 50 per cent acetic acid, ringed with a paraffin-vaseline mixture, and studied as a temporary preparation. The salivary glands are pretreated in 1 N hydrochloric acid for about one minute, stained in aceto-orcein and mounted in 50 per cent acetic acid. Identifications of the species were made by Drs. M. R. Wheeler, W . B. Heed, and the junior author. RESULTS AND DISCUSSION The data resulting from this investigation are given in Table 1, and the chromosomal configurations of some of the strains are illustrated in Plate 1. In Table 1 the stock number and the geographic origin of each culture are given; starred species are those being described by various authors elsewhere in this bulletin. Many of the strains examined show no differences from previous ac­counts and will not be discussed further. Several species, however, have un­usual cytological interest and are described below. D. paranaensis. Fig. 5-7. We have found three types of metaphases among our material. Type (a) was first reported by Dreyfus and de Barros ( 1949) ; type (c) was described by Clayton and Ward (1954). Type (b), reported here, is intermediate between the other two, consisting of three pairs of rods, one pair of large V's, one pair of small V's with the Ya short rod. Crosses between the three types give interesting results (see Wasserman and Wilson, this bulletin) ; for example, F1 males from type (a) crossed to type (b) are sterile; type (a) crossed to type (b) and type (b) crossed with type (c) give variable results. D. fragilis. The stock from La Palma, El Salvador shows three pairs of rods, one pair of J's, and one pair of dots; the stock was about to be lost and only a single larva was dissected. Additional dots may have been present, as reported earlier (Clayton and Ward, 1954), but no other larvae were available to check on this point. D. setula. Fig. 10-11. Metaphase plates of this species have been unusually difficult to interpret and the two stocks we have used seem to disagree with each other. Stock No. H187.9 appears to have four pairs of rods, one pair of V's, and one pair of dots, while stock No. H81.15 has the same V and dot chromosomes but shows only three pairs of rods. However, due to the poor quality of the preparations, these interpretations are still tentative. 1 Dept. Zoology, Univ. Arkansas, Fayetteville. ....... TABLE l to 0) Species Stock No. Locality Meta phase x y Salivary Hirtodrosophila pictiventris H 15.10 San Vincente, El Salvador 1V, 1J, lR, 1D R 0 5A, 1D longala (?) 2268.27 Cuernavaca, Morelos, Mexico 5R, 1D R 0 5A, 1D grisea 2359.6 Rustler Park, Arizona 5R, 1D R 0 (? ) 5A, ID Dorsilopha busckii H 57.27 Santa Maria de Ostuma, Nicaragua 2V, 1R R J (?) 5A, 1D Sophophora saltans group elliptica (?) H 62.51 Lo Palma, El Salvador 2V, 1R "l ;:"?­ willistoni group bocainensis (?) fumipennis suc1nea H 50.4 H 50.4B H 57.29 H 57.23 La Lima, Honduras La Lima, Honduras Santa Maria de Ostuma, Nicaragua Santa Maria de Ostuma, Nicaragua 2V, 1R 1V, 1J, 1R 2V, 1R 2V, 1R v v J v 5A 5A 5A, 1D (?) 5A, 1D ~ t:! ;::i..... ~ ~ bromeliae group bromeliae H 51.24 Lancetilla, Honduras 2V, 1R, 1D ..... ..... ~ bromelioides (?) Drosophila H 57.54 Santa M aria de Ostuma,.Nica ragua 4V v J .Q.. tripunctata group albirostris H 25 .2 San Sa lvador, El Salvador 5R, JD H H 5A, 1D ~ H bipunctata crocina facialba • H 29. 10 H 41.25 H 25.30 H 29. 1b H 26.2a San Salvador, El Salvador Volcan Boqueron, El Salvado r San Salvador. El Salvador San Salvador, El Salvador Volcan Santa Ana, El Salvador 5R, lD 5R, lD 5R, 1D 5R, lD 5R, 1D R R R R R R R R R R 5A, 1D 5A, 1D 5A, 1D 5A,1D ~ "ti i:: ""' ........... () fragilis medionotata (?) H 62.1 H 21.5 H 27.1 La Palma, El Salvador Lago de Coatepeque, El Salvador Lago Pichichuela, El Salvador 3R, 1J, 1D 5R, 1D 5R, 1D R R R R 5A, 1D 5A, 1D ~ ......... . 0 ;::i H 29.18 San Salvador, El Salvador 5R, 1D R R 5A, 1D H 50.10 La Lima, Honduras 5R, 1D H. R 5A, 1D mediopuncta ta mediostriata H 62.10 H 29.1a La Palma, El Salvador San Salvador, El Salvador lV, 1J, 2R, 1D 5R,1D R R R R 5A,1D 5A, 1D H 35.15 San Salvador, El Salvador 5R, 1D R R 5A, 1D H 50.41 La Lima, Honduras 5R, 1D R R 5A, 1D H 51.6 Lancetilla, Honduras 5R, 1D R R 5A, 1D metzii H 56.95 El Reci·eo, Nicaragua 5R, 1D R R 5A, 1D setula• H187.9 Santa M arta, Colombia 4·R, 1V, 1D trapeza • H 81.15 H 29.16 Barro Colorado Is., Canal Zone San Salvador, El Salvador 3R, 1 V, 1D (?) 5R, 1D R R 5A, 1D TABLE 1-Continued Species Stock No. Locality Metaphase x y Salivary trifiloides • H 50.10b La Lima, Honduras 5R, 1D R R tristriata• H109.22 Trinidad, B. W. I. 5R, 1D 5A, 1D repleta group aldrichi (? ) aureata• H 52.12 H180.42 Puerto Triunfo, El Salvador San Jose, Costa Rica 5R, 1D 5R, 1D R R R J 5A, 1D 5A, 1D fulvimacul oides • inartensis • Jnercatoru1n paranaensis type a type b H163.31 H188.1 2 1781.8 H113.5 H194.44 H186.33 Turrialba, Costa Rica Santa Marta, Colombia San Pedro, Coah., Mexico Trinidad, B. V\T. I. Villavi cencio, Colombia Santa Marta, Colombia 5R, 1D 5R, 1D 3R,2V 3R, 1V, 1D 3R, 1V, 1D 3R,2V R R R R R R J v R R R R 5A, 1D 5A, 1D 5A, 1D 5A,1D 5A, 1D VJ..... i::: ~-.~ "' H H 73.6 75.10 Turrialba, Costa Rica Heredia, Costa Rica 3R, 2V 3R, 2V R R R R 5A, 1D 5A, 1D -·;:l type c H 56.94 H 34.20/ 32 El Recreo, Nicaragua San Salvador, El Salvador 3R,2V 3R, 2V R R R R 5A, 1D 5A,1 D ..... ;:i-.. ~ peninsularis repleta H101.1 2 2327.3 H 17.8 Palmira, Colombia Rio Piedras, Puerto Rico Santa Tecla, El Salvador 3R,2V 5R, 1D 5R, 1D R R R R v R 5A, 1D 5A, 1D 5A, 1D cw ~ ;:l ~ H 25.32E H 34.30 San Salvador, El Salvador San Salvador, El Salvador 5R, tD 5R, 1D R R R R 5A, 1D 5A, 1D .....-· (") "' immigrans group 0- immigrans macroptera group submacroptera (?) H 57.21 2257.lb 2257.20 2261.1 6b 2262.4b Santa Maria de Ostuma, Nica ragua Chapulhuacan, Hid., Mexico Chapulhuacan, Hid., Mexico Tehuacan, Puebla, Mexico Huatusco, Vera Cruz, Mexico 3R, 1V 2V, 11, 1D 2V, 11, 1D 2V, 11, 1D 2V, 11, 1D R v 4A, 1D 5A 5A 5A 5A tl-; 0 "' .g ;:i-..-· 2263.3b Tezuitlan, Puebla, Mexico 2V, 11, 1D 5A 1S"' guarani group guaramunu (?) limbinervis 2263.17 H 26.29 Tezuitlan, P uebla, Mexico Volcan Santa Ana, El Salvador 4R, !V 5R, 1D R R 5A(?) 5A, 1D H 42.26 Volcan Boqueron, El Salvador 5R, 1D R R 5A, 1D pallidipennis group pallidipennis centralis H 29.27 6 San Salvador, El Salvador 4R, tV, 1D 5A, 1D x H 34.24'? H 62.58 La Palma, El Salvador 4R, 1V, 1D 5A, 1D calloplera group atrata H203.23 Caripe, Venezuela 5R, 1D R R ...... to '1 ....... TABLE 1-Continued to Species Stock No. Locality Meta phase x y Salivary ornatipennis 23 78.2 St. Vicente, Cuba 3R, 1V, 3D 5A, 1D dreyfusi group briegeri H182.2 H 80.5 Cerro La Campana, Panama Barro Colorado Is., Canal Zone 3V, JD 3V, 1D v v v v 5A, 1D 5A, 1D camargoi H 51.12 Lancetilla, Honduras 3V, 1J v J 5A, 1D cardini group cardini H 15.20 San Vincente, El Salvador 6R R R 5A, 1D H 26.6 Volcan Santa Ana, El Salvador 6R R R 5A, 1D '"'-3 cardinoides canalinea group canalinea canalinioides • H 29.20 H 57.26 H 27.9 H 50.31 H 25.24 H 66.7 San Salvador, El Salvador Santa Maria de Ostuma, Nicaragua Lago Pichichuela, El Salvador La Lima, Honduras San Salvador, El Salvador San Salvador, El Salvador 6R 6R 2V, lR, 1D 2V, 1R, 1D 6R 6R R R R R R R R R 5A, 1D 5A, 1D (?) 4A, 1D (?) 5A, 1D 5A, 1D 5A, 1D ;::i­(I) ~ ;::s;:: . (I) "'I "'-..... · paracanalinea• Unassigned species casta_nea H129.19 H 29.7 H 25 .13 H M.1 3 El Yunque, Puerto Rico San Salvador, El Salvador San Salvador, El Salvador San Salvador, El Salvado1· 1R, 1V, 1J, 1D 1R,2V, 1D 1R, 2V, 1D lR, 2V, 1D v v J J 5A,1D 5A, JD 5A, 1D 5A, 1D ~ .Q.. '"'-3 ~ H 73.8 Turrialba, Costa Rica rn.,2V, 1D 5A, 1D ~ H180.21 San Jose, Costa Ri ca 1R, 2V, 1D 5A, 1D "ij H209.9 H209.39 1802.8 Merida, Venezuela Merida, Venezuela Atlixco, Puebla, Mexico 1R, 2V, 1D 1R, 2V, 1D lR, 2V, 1D v J 5A, JD 5A,1D 5A, 1D ~ \:)"...... t:; · fulvalineata 2075.9 Cliff, New M exico 5R, 1V R v 5A, JD ~-· fulvalineata (?) aracea* 2J58. 13 H 17.1 Patagonia, Arizona Santa Tecla, El Salvador 3R, 2V 1V, 1J, 1R v J 6A, 1D 5A, 1D 0 ;::s H 46.28 Santa Tecla, El Salvador lV, 11, 1R 5A, JD H 57.69 Santa Maria de Ostuma, Nica ragua 1V, 1J, 1R 5A, 1D carsoni • 2351.9 Espanola, New M exico 2R, 2J, 1V, 1D 8A, JD sticta • H 51.15 Lancetilla, Honduras 5R,1D R R 5A, 1D H 57.61 H192.32 Santa Maria de Ostuma, Nicaragua Rio Nei;-rn. Colombia 5R, 1D varies R R 5A, JD paragu tta ta• H136.34 Jamaica, B. W. I. 5R, 1D R R 5A, 1D • New species described elsewhere in this bulletin. Studies in the Genetics of Drosophila 129 ~~ '7u~ I. STICTA-o ~~ .. ~,\~ 4. STICTA-d ~k ~I'~ 7 PARANAENSIS-c ~~!~ "" 10. SETULA-o -~~ ­ - - /1 if PLATE I. ~~ .. 4fi~ 2. STICTA-b ~:~~ 5. PARANAENSIS-o '\ ·~ )>'~·..··­ 8.ORNATIPENNIS ~" Aj\= 11.SETULA-b ~"? H~ ~~ ., .-rn~ 3. STICTA-c ,, ~,,~ 6. PARANAENSIS-b ~~\b ..\\ 9. BRIEGER! I I ~H~ -~" 12. CARSONI ~~ '. ~i\ r.,,. : 15. PARACANALINEA 13. FULVALINEATA-o 14. FULVALINEATA-b D. submacroptera. The metaphase reported for this species was one pair of large V's, two pairs of small V's, one pair of rods and one pair of dots. All of the strains we have examined differ from this configuration, possessing one pair of large V's, one pair of smaller V's, one pair of J's, and one pair of dots. D. guaramunu. The stock studied (Tezuitlan, Mexico) is probably a new species related to guaramunu; its chromosomes differ from the original report in several respects. D. atrata. As was pointed out by Clayton and Ward ( 1954) the metaphase of this species was reported previously by Dobzhansky and Pavan (1943) under the name of calloptera, where it was described as five pairs of rods and one pair of dots. Our preparations agree, with one pair of long rods, four pairs of slightly shorter rods (one pair with satellites), and a pair of dots. D. ornatipennis. Fig. 8. Our determination, based upon a stock from Cuba, differs from the earlier reports. Metz (see discussion in Clayton and Ward, 1954) reported one pair of large V's, three pairs of rods, and one pair of microchromo­somes. We find one pair of large V's, three pairs of rods (one long pair and two shorter pairs), and three pairs of dots. This is a haploid complement of seven, previously reported for two other species, trispina and fragilis. D. briegeri. Fig. 9. Pavan and Breuer (1954) described the metaphase in the species description as follows: three pairs of V's, one pair of dots. They state that the large V's (sex chromosomes) are heteropycnotic, with one arm and half of the other arm positively heteropycnotic in prophase. Our preparations showed approximately the same thing. Salivary chromosomes show some banding pat­terns recognizable on the basic repleta map. We consider that one pair of smaller V's represents a 2-5 fusion, and the other pair of smaller V's represents a 3-4 fusion, using the repleta configuration as the standard. D. camargoi. The original description of this species gives the following meta­phase configuration: one pair of long J's, one pair of rods, and two pairs of small V's. Our determination, using a stock from Lancetilla, Honduras, differs slightly, since we find three pairs of V's, rather than two, and no rods. D. canalinea. Our preparations agree with the metaphase reported in the origi­nal species description, but disagree with that recorded in Patterson and Stone (1952: 159) where it is evident than an error was made, listing the chromosome complement as 1R, 1V, 1D rather than 1R, 2V, 1D. Of the two pairs of V's, one pair is much larger than the other. D. paracanalinea. Fig. 15. The stock from Puerto Rico shows one pair of large V's, one pair of large J's with satellites, one pair of rods and one pair of dots. Salivary preparations show five arms and a dot, and certain portions are recogniz­able in terms of the repleta chromosomes. An X-4 fusion forms one of the meta­centrics, and the other is uncertain, using the repleta configuration as the stand­ard. D. castanea. Wasserman (1954) reported that many portions of the salivary chromosomes showed recognizable repleta patterns. The new strains agree with those examined previously in having a 2-4 fusion and an X-3 fusion, using the repleta chromosomes as a standard. D. fulvalineata. Fig. 13-14. The stock from New Mexico shows the same meta­phase as that reported previously but we see only five arms and the dot in salivary Studies in the Genetics of Drosophila preparations rather than six arms. The stock from Arizona, however, has an en­tirely different metaphase, and is possibly an undescribed species. The metaphase of this strain consists of one pair of large V's, one pair of long rods, one pair of shorter rods, one pair of chromosomes which are probably rods, and one pair of small V's. The X is a large V while the Y is a large J-chromosome. Salivary prepa­rations show six arms and a dot, with two arms rather short. D. carsoni. Fig. 12. Metaphase preparations show one pair of long rods with satellites, one pair of shorter rods, two pairs of J's, one pair of V's (with a satellite seen on one V of the pair in several individuals), and one pair of large dots. The X and Y could not be determined. Salivary preparations showed eight arms and the dot; two arms were about half the length of the others, and one chromosome had a mass of heterochromatin in the middle, this chromosome usually being sep­arated from the chromocenter. D. sticta. Fig. 1-4. The metaphase configurations found in stock No. H192.32 from Colombia show an unusual type of variability. The basic chromosomes are apparently five pairs of long rods and one pair of dots. All preparations consist­ently showed the five long rods, but the remaining chromosomes were either (1) one pair of dots, or (2) one pair of dots and a short rod, or (3) one dot and one short rod, or (4) two short rods and no dots. Nine larvae, chosen at random from the culture, gave the following distribution: 1 'i' , 2? one pair dots, no rods 1 'i' no dots, one pair rods 1 t One dot, one rod 1 t , 3? one pair dots, one rod Thus the "supernumerary" rod may be absent, present once or twice, and the dot may be absent, present once or twice. There is no apparent relation between rods or dots and sex. The X and Y chromosomes could not be identified with certainty. REFERENCES Clayton, F. E. and C. L. Ward. 1954. Chromosomal studies of several species of Drosophilidae. Univ. Texas Puhl. 5422:98-105. Dobzhansky, Th. and C. Pavan. 1943. Chromosome complements of some South American species of Drosophila. P. N. A. S. 29:368-375. Dreyfus, A. and R. de Barros. 1949. Sex-ratio chez certains hybrides interspecifiques de Dro­sophila et son interpretation par !'analyse des chromosomes salavaires. Suppl. La Ricerca Scient. Anno. 19: 1-13. Patterson, J. T. and W. S. Stone. 1952. Evolution in the genus Drosophila. Macmillan Com­pany, New York. 610 pages. Pavan, C. and M . E. Breuer. 1954. Two new species of "Drosophila" of the "dreyfusi" group (Diptera). Rev. Brasil. Biol. 14:459~3. Wasserman, M . 1954. Cytological studies of the repleta group. Univ. Texas Puhl. 5422: 130-152: Wasserman, M. and F. Wilson. Further studies on the repleta group. This bulletin. XI. Furthn Studies on the Repleta Group MARVIN WASSERMAN AND FLORENCE D. WILSON This is a continuation of the work started by the senior author in 1954 on the repleta group of the genus Drosophila. At that time an examination was made of twenty species, mainly from the United States and Mexico. During the past two years the Genetics Foundation of the University of Texas has been carrying out a series of collecting trips to Central and South America and the Caribbean islands, aided by a grant from the National Science Foundation. This investiga­tion is based primarily on cultures derived from these collections. Drs. Marshall R. Wheeler and William B. Heed have made most of the species determinations; the field collecting has been carried out mainly under the leadership of Dr. Heed assisted by Dr. H. L. Carson and the senior author. We are also indebted to Dr; W. S. Stone for suggestions and encouragement, to Mrs. W. B. Mather for the use of some of her unpublished data, and to Miss Gail Kaufman for her aid in the preparation of the manuscript. The repleta group is one of the largest known species groups in the genus Drosophila. Most of the species are either cytologically homozygous or have but few gene sequences. The differences from species to species are also usually slight, and cytological changes from form to form can be readily determined (Wharton, 1942, 1943; Warters, 1944; Dreyfus and de Barros, 1948, 1949; de Barros, 1950; Ward and Stone, 1952; Wasserman, 1954; Stone, 1955) . . · In addition there is a wealth of information on isolating mechanisms among the species in the group. Patterson and Stone (1952: 412) summarized the crosses attempted between members. The group has been subdivided into three or four subgroups; Wharton ( 1944), Wheeler (1949), and Patterson and Stone (1952) recognize the melanopalpa, hydei, '~nd mulleri subgroups. In addition, Wharton (1944) proposed the merca­torum subgroup for D. mercatorum, and D. peninsularis. Patterson and Stone (1952) also recognize the mercatoru~ subgroup and have included in it D. para­n,aensis; they agree with Wheeler, however, in placing peninsularis in the mulleri subgroup. There is also a considerable number of species which have not as yet been assigned to any subgroup, in some cases because not enough information is available, and at other times because they seem to be intermediate in many of their characteristics . . The present investigation deals primarily with the cytological analyses of several members of the repleta group, namely, D. repleta, the fulvirhacula com­plex, the mercatorum subgroup, and the mulleri subgroup. Some data on the hybridization of certain species are1.presented and two new species are described. MATERIALS AND METHODS The 59 geographic strains of the eight species studied are listed in Table 1. Mass matings (20 ct; , 20 '.?) were made to obtain F1 larvae for cytological exam­ination. Several of the strains were also identified by crosses to known laboratory Studies in .the Genetics of Drosophila stocks. If no progeny had been produced from the matings after four weeks, the cross was considered sterile. Cytological examinations were made on the salivary gland chromosomes of the pure strains and on those hybrids which were obtained from the mass matings. In most cases eight slides were made of each stock to test for homozygosity. The observed gene sequence or sequences found in each strain were compared to that found in Drosophila repleta, using the repleta map of Wharton ( 1942) . The symbols used to represent the different inversions have been modified somewhat from the pattern established in the earlier report on the repleta group (Wasserman, 1954). The use of a single alphabet letter (a, b, c, etc.) remains as before, but we are substituting the symbols a2, b2, c2, etc. for the inversions pre­viously recorded as aa, bb, cc, etc., and, due to the great number of inversions in chromosome 2, we are also employing the symbols a3, b3, c3, rather than the cumbersome forms aaa, etc. I. MELANOPALPA SUBGROUP Wharton (1942) and Ward and Stone (1952) have made cytological studies on the melanopalpa subgroup. They have found that D. repleta, D. limensis, D. canapalpa, D. melanopalpa and D. neorepleta form a close-knit group. Among the strains tested they found that only 6 inversions have taken place in the cyto­logical divergence of these 5 species. Ward and Stone (1952) have also demon­strated that melanopalpa, canapalpa and neorepleta share some of these inver­sions. Thus a Mexican melanopalpa strain was homozygous for a gene sequence in the fifth chromosome which was lacking in an Arizona strain of melanopalpa. The strain of canapalpa used was homozygous for this sequence. Similarly, neo­repleta and the Arizona strain of melanopalpa were homozygous for a gene se­quence in the second chromosome. The Mexican strain of melanopalpa was heterozygous for this sequence. Wasserman (1954) examined the salivary gland chromosomes of D. nigro­spiracula. This species had been placed in the melanopalpa subgroup on morpho­logical grounds. Its cytology, however, places it closer to the mulleri and hydei subgroups. Drosophila repleta Wollaston, 18~J; ;>:: ' .! The species D. repleta has been examined a number of times! (Wharton, 1942, 1943; Ward and Stone, 1952; and Wasserman, 1954). It has ·hev;er been known to vary in its gene sequence. The stl:l· fulvimaculoides H29.15 El Salvador j2, j2, }c2, z2, 12, as, b3, cs b .... + + + 5· H57.56 Santa Maria de Ostuma, Nicaragua i2, j2, k2, z2, 12, a3, b3, cs b ;:i + + + H73.23 Turrialba, Costa Rica i2, j2, _k2, z2, 12, as, bS, c 13 b + + + H158.4 Turrialba, Costa Rica i2, j2, k2, z2, 12, a3, b3, c3 + b + + H161.1 Turrialba, Costa Rica i2, j2, k2, z2, 12, as, bS, cs + b + + H163.11 Turrialba, Costa Rica i2, j2, k2, z2, 12, a3, b3, c3 b + + + H163.31 Turrialba, Costa Rica i2, j2, k2, z2, 12, as, b3, cs b + + + mercatorum 141 2.9b Sao Paulo, Brazil d, t, e, u, v b, f, h, nl+ + + + H191.30 Bucaramanga, Colombia i cl, t, e, u, v b, £, h, "g" + + + _J_ H191.46 Bucaramanga, Colombia i d, t, e, u, v b, £, h, "g" I H62.60 2370.5 2393.1 La Palma, El Salvador Oahu, Hawaii Cocha Bamba, Bolivia + + + d, t, e, u, v d, t, e, u, v d, t, e, u, v b, f, h b, f, h b, f, h + + + + + + mcrcatorum 2395.12 Cuzco, Peru + d, t, e, u, v b, f, h + + n111ll eri paranaensis nigricruria martensis H62.13 H194.44 H 34. 20132 H73.6 H 56.94 H163.16 H101.1 2 H75.10 H186.33 H113.5 2268.7 1796.2 1797.1 4 2264.1 4 2263.1 5 2266.1 5 2261.7 H 62.29 H75.11 H163.29 H206.19 H1 88. 12 H 208.1 La Palma, El Salvador Villavice ncio, Colombia El Salvador Turrialba, Costa Rica El Recreo, Nicaragua Turrialba, Costa Ri ca Palmira, Colombia Heredia, Costa Rica Sierra Nevada de Santa Ma rta, Colombia Trinidad Cuernavaca, Morelos, Mexico Morelia, Michoacan, Mexico Mexico City, Mexico San Andres Tuxtla, Veracru z, M exico Tezuitlan, M exico Sierra Madre del Sur, Mex ico Tehuacan, Puebla, M exico La Palma, El Salvador H eredia, Costa Rica Turrialba, Costa Rica Costa Rica A Caripe, Venezuela Santa Marta, Colombia Barquesimeto, Venezuela + + + + + + + + + + a, b, c, k a, b, c, k a, b, c, k a, b, c, k a, b, c, k a, b, c, k a, b, c, k a, b, c, k a, b, c, k a, b, c, k a, b, c, k a, b, c, k a, b, c, j a, b, c, j d, t, e d, t, e d, t, e d, t, e d, t, e d, t, e d, t, e d, t, e d, t, e d, t, e, m 2 a, b, t2, u z, v2, w 2, x2:s2/y2 a, b, t2, u z, v2, ,v2, x2 :s2/y 2 a, b, t 2, u2, v 2, w 2, x 2:s2/y2/ + a, b, t2, u 2, v2, w 2, x 2:s2 a, b, t 2 , u 2 , v2, w z, x2 a, b, t 2 , u z, v2 , vv 2 , x2 :s2/s2y::! a, b, t 2 , u 2 , vz, w 2 , x2 a. b, t 2, u z, v2, w 2, x2 a, b, t 2 , u 2 , v2, w 2, x 2 a, b, t 2 , u 2 , v2, w 2, x 2 a, b, t 2, u 2, v z, w 2, x2 a~ b, t 2 , u 2 , v 2 , w z, x2 a, b, c, d2, e2, f 2, g2I+ a, b, c, d2, e2, f2 , g2I+ b, f + + b, f + + b, f + + b, f, i + + b, f, i + + b, f, i + + b, f, i, gl+ + + b, f, i, gl+ + + b, f, i bl++ b, f, ii+ , gl+ bl++ b c, d f b c, d f b c, d f b + f b c, d f b c, d f b + f b + f b + f b + f b + f b + f a, b, k + + a, b, k + + Cr.! ...... !::: ~-· ~ "' s· ...... ;:i-.. ~ 0 ~ ;:i ~ ......c:; · "' 0-t:i-; 0 "' .g ;:i-..-· ...... ~ "aldrichi" H52.12 H103.2 Puerto T riunfo , El Salvador Sevilla, Colombia a, b,c a, b, c a, b, c, f, g a, b, c, f, g a, b, c a, b, c + + + + -w U'l it and the strain from Mexico, and Patterson (1952) described the Brazilian form as a new subspecies, D. fulvimacula fiavorepleta. Recently a number of strains were collected in Central and South America by W. B. Heed, H. L. Carson and the senior author. These strains have now been tested, and the results show that two species are present. The pertinent data are presented below. The two forms are very similar morphologically, and, without breeding tests, they might quite easily be considered one species. The diagnostic characters are unfortunately either cytological or those which living specimens exhibit best. · All of the original specimens of fulvimacula except the holotype have been lost. Therefore the specimens available for study consist of the holotype and those flies which have been obtained since 1947. However, the authors believe that they have probably identified the holotype of fulvimacula with one of the two species present in the laboratory. We give below a complete redescription of D. fulvi­macula, as we interpret the species, and a description of Drosophila fulvimacu­loides, a new species. Drosophila fulvimacula Patterson and Mainland, 1944. External characters of imagines. J, <;> . Arista with about 8 branches; antennae brownish yellow, 3rd joint darker; 2 prominent bristles on the 2nd joint. Front brownish yellow; orbits lighter with brown areas ventrally; ocellar triangle slightly darker. There may be very faint brownish yellow spots at the bases of the posterior orbitals and inner verticals. Middle orbital about .55 to .625 the other two. Carina flat, slightly broadened ventrally. Face light yellow, tending toward white. Cheeks yellowish white, their greatest width about Vi> greatest diameter of eye. Eyes scarlet-ver­milion (Pl. 2-J12), with dense, short black pile. Acrostical hairs in 8 rows; no prescutellars, but hairs of all the acrostical rows immediately anterior to the scutellar suture enlarged. Anterior scutellars con­vergent. Sterno-index about 0.76. Mesonotum pollinose brownish yellow, hairs and bristles arising from light yellowish brown spots which tend to fuse giving two indistinct stripes between the dorsocentral rows in the darker forms. Sterno­pleurae yellowish; mesopleurae and pteropleurae slightly darker. Legs yellowish, with a slight indication of darker basal bands on the tibiae. Apical bristles on the first and second tibiae, preapicals on all three. Abdomen yellowish with slight greenish cast, each segment with a dark apical band, interrupted medially, which fades at the angle of the tergite, and with anterior projections at the interruption and sometimes at the angle of the tergite. In dead pinned specimens the lateral margin of the tergite also darkens. Wings clear with a yellowish cast, veins brown. Costal index about 3.0; 4th vein index about 1.6; 5x index about 0.9; 4c index about 0.5. Apex of first costal section with two prominent bristles. Third costal section with heavy bristles on its basal %to 1h. Length of body about 4 mm; wing about 3.75 mm. Internal characters of imagines. Testes pale yellow (Pl. 9-H1), with ab9ut 51!2 outer coils and 5 thin, lighter colored, inner coils. Ventral receptacle with about 100 coils, irregularly coiled. Spermathecae not chitinized, bell-shaped. Other characteristics, relationship, and distribution. Eggs,-With 4 filaments, the anterior pair about 4/ 9 length posterior pair. Puparia.-Yellowish brown (Pl. 9-L7), each anterior spiracle with about 16 branches. Horn index about 2.0 (including the spiracle branches). Chromosomes.-Metaphase plate shows 5 pairs of rods and one pair of dots (Clayton and Ward, 1954) . Relationship.-Belongs to the melanopalpa subgroup of the repleta group of the subgenus Drosophila. Distribution.-The holotype is from Sedeno Canon, near Jalapa, Mexico. Other known localities, listed in Table 1, are El Salvador, Honduras, Panama Canal Zone, Colombia, and Brazil. Drosophila fulvimaculoides Wasserman and Wilson, new species. External characters of imagines. i!; , <;> . Arista with about 8 branches; antennae yellowish brown, the 3rd joint darker; two prominent bristles on the second joint. Front brown; orbits pollinose brownish yellow with brown areas ventrally; ocellar triangle brown. The pos­terior orbitals and the inner verticals arise from brown spots. Middle orbital about % length of anterior and Yz posterior orbital. Carina slightly keeled dorsally, flattened and slightly widened ventrally. Face yellowish gray. Cheeks yellowish gray, their greatest width about 116 greatest diameter of the eye. Eyes red (Pl. 2-Kt 1), with dense, short black pile. Acrostical hairs in 8 rows; no prescutellars, but hairs of all the acrostical rows immediately anterior to the scutellar suture enlarged. Anterior scutellars con­vergent. Sterno-index about 0.84. Mesonotum pollinose brownish yellow, hairs and bristles arising from brown spots which tend to fuse into two stripes between the dorsocentral rows. Pleurae yellowish, with indistinct brown stripes from the base of the wing to the humerus, from the haltere to the base of the fore coxa, and along the margin between the sternopleura and the mesa-and pteropleurae. Legs yellowish, with a dark band at the apices of the femora and the bases of the tibiae. Apical bristles on the first and second tibiae, preapicals on all three. Abdomen yellowish with a slight greenish cast, each segment with a black apical band, interrupted medially, with anterior extensions at either side of the interruption, at the angle of the tergite, and at the lateral margin. Wings clear with a slight yellowish cast, veins brown. Costal index about 3.0; 4th vein index about 1.4; 5x index about 1.05; 4c index about 0.5. Apex of the first costal section with two prominent bristles. Third costal section with heavy bristles on the basal Yz . Length of body, about4mm; wing, about3.75 mm. Internal characters of imagines. Testes pale yellow (Pl. 9-H1), with 3 inner and 5 outer coils. Ventral re­ceptacle with about 90 coils, irregularly coiled. Spermathecae not ~hitinized, bell-shaped. Other characteristics, relationship, and distribution. Eggs.-With 4 filaments, the anterior pair %length posterior pair. Puparia.-Yellowish brown (Pl. 11-K12), each anterior spiracle with about 16 branches. Horn index about 1.67 (including spiracle branches). Chromosomes.-Metaphase plate shows 5 pairs of rods and one pair of dots. The Y chromosome is J-shaped (Clayton and Wasserman, this bulletin). Relationship.-Belongs to the melanopalpa subgroup of the repleta group of the subgenus Drosophila. It is quite similar to D. fulvimacula; among the strains present at the Texas laboratory, fulvimaculoides can be distinguished from fulvi­macula by its darker, more distinct pattern on the abdomen and mesonotum, the banding on the pleurae, and the coloring on the front and orbits. Distribution.-The type locality is Turrialba, Costa Rica. The holotype and 9 paratypes are in the collection of the University of Texas. It has also been col­lected at San Salvador, El Salvador, and at Santa Maria de Ostuma, Nicaragua (see Table 1) . Matings Mass matings were carried out among the various strains of fulvimacula and fulvimaculoides. The results are shown in Table 2. In all interspecific crosses where fulvimaculoides was the female, no F1 progeny were obtained. In the reciprocal crosses there was a certain number of F1 males and females. No F2 was obtained. The F1 males proved to be sterile; in four dissected males the in­ternal genitalia appeared normal but the testes were not as highly pigmented as those of either parental male, nor were any motile sperm observed. Some F1 females from the cross Brazilian fulvimacula (1975.5) to Costa Rican fulvimacu­loides (H163.31 ) were backcrossed to H163.31 males; these females proved to be fertile. T ABLE 2 Crosses between strains of the ful vimacula complex. Intraspecific Crosses Interspecific crosses '? (; Results '? (; Results H181.8 X H186.37 F, H163.31 X 1808.37 no progeny reciprocal F, H163.31 X H181.8 no progeny H181.8 X H194.28 F, H163.31 x H186.37 no progeny reciprocal F, H163.31 X H194.28 no progeny ;3"' H181.8 x 1975.5 F, H163.31 X 1975.5 no progeny u reciprocal F, 1808.37 X H163.31 no progeny s"' H186.37 x2H194.28 F, H181.8 X H163.31 no F, - ·:;: reciprocal F, H186.37 X H163.31 no F, ;3 186.37 x 1975.5 F, H194.28 X H163.31 no F, reciprocal F, 1975.5 X H163.31 sterile (; (; H194.28 X 1975.5 F, fertile'? '? reciprocal F, H186.37 X 1808.37 F, Q) "' '"d H163.31 X H161.1 F,·s reciprocal Fo ;3 u H1 63.31 X H163.11 F, reciprocal F, "' a ·:;: H163.31 X H 158.4 F, ....., reciprocal F, ;::s ..... Cytology The cytology of the salivary gland chromosomes of the two species are as follows: X Chromosome: The X chromosome is identical to that of repleta. 2 Chromosome: The changes which have taken place in the 2 chromosome, using the repleta 2 as the standard, are shown in Figure 1. 3 Chromosome: This chromosome differs from repleta in that inversion 3b is present. 4 and 5 Chromosomes: These chromosomes, like the X, are identical to those of repleta. 6 Chromosomq: This is the dot chromosome; no comparisons were made among the various dot chromosomes of the repleta group. Discussion The species fulvimacula had been placed in the melanopalpa subgroup by Wheeler (1949) on morphological grounds. Cytological evidence indicates that both fulvimacula and fulvimaculoides have diverged from the other members of the repleta group from the 3b ancestor and not directly from repleta. This, how­ever, does not invalidate its placement in the melanopalpa subgroup. The original 3b sequence appears to be the focal point from which the melanopalpa subgroup, the mercatorum subgroup, the species peninsularis, and the Xa, Xb, Xe, 2a, 2b, 3b ancestor of the hydei and mulleri subgroups diverge from each other. Cytologi­cally, the fulvimacula complex is quite different from the melanopalpa complex (melanopalpa, repleta, canapalpa, limensis, and neorepleta). However, it is cyto­logically equally or more distinct from any other subgroup. Both fulvimacula and fulvimaculoides are quite.homozygous as to their gene sequences; however, one exception, the fulvimacula strain from Villavicencio, Colombia (H194.28), is quite interesting, being heterozygous for the gene se­quence 2l2. Aside from this arrangement, which no other fulvimacula strain has, it is like the other strains. All of the fulvimaculoides strains are homozygous for this arrangement. In addition, they are homozygous for 2a3 which overlaps 2l2 and therefore must have occurred after 21 2 • This is similar to what had been found by Ward and Stone (1952) among melanopalpa, canapalpa and neore­pleta, and within the mulleri subgroup (Wasserman, 1954). The authors believe that the precursor of the two closely related species was heterozygous for this in­version in some areas. Most of the populations which gave rise to fulvimacula did not reGeive or lost this arrangement. As far as is known, all of the populations which have evolved into fulvimaculoides have maintained this arrangement in the homozygous condition and have added 2a3• Cytologically these forms have also proven interesting. The inversion 2z2 has breakage points which are very similar to those of 2a. Inversions 2h2 of fulvi­macula and 2c3 of fulvimaculoides have arisen independently. However, they both apparently have D3a as one of the breakage points. The other breakage points are quite distinct although fairly close to each other, one being C5b, the other C7-. Our data do not give us information as to the sequence of origin of 2i2, 2j2, and 2k2• We postulate these three inversions with only four breakage points as a N .x C\J N N (\J C\J Q) (.) c: Q) ::J CT Q) VJ ti ....,. I t 0 (.'.) 0 (.!) w t (\) C\J 0 l{) 0 0 <:t LL t 0 (.!) w 0 .2 > Frc. 1. Evolution of the 2 chromosome in the fulvimacula complex. The steps between (a) and (b) are unknown at present. Studies in the Genetics of Drosophila working model to explain the chromosomes as we interpret them. Our breakage points are, of course, too inaccurate to definitely assert that the chromosome has broken at the same place more than once. However, there is definitely a tendency to have certain areas involved in rearrangements. II. MERCATORUM SUBGROUP The mercatorum subgroup is composed of three described forms: the species[). paranaensis, and the two subspecies D. mercatorum mercatorum and D. m. ptira­repleta. D. paranaensis is known from Mexico to Brazil; the subspecies merca­torum has a distribution from Hawaii and the United States, south through Mexico and Central America to Peru and Bolivia. D. m. pararepleta is known only from Brazil. Crosses between paranaensis and mercatorum have yielded a variety of resl,llt$. In all cases, paranaensis females crossed to mercatorum males yield only female F1 • When pararepleta males are used, these F1 females are fertile (Dreyfus and de Barros, 1948 and 1949). When mercatorum males are used, the F1 females are sterile (de Barros, according to Patterson and Stone, 1952). The reciprocal crosses produce both male and female F 1 ; mercatorum female parents produce sterile males and females, while pararepleta female parents produce fertile F1 fem. w The University of Texas Publication · crosses, many F1 flies were obtained. However, only in the crosses to the Villa­vicencio and the Santa Marta, Colombia (H186.33) strains were F2 flies re­covered. · Some of the F1 females obtained from the cross between the two standards, Mexican females to Trinidad males, were backcrossed to Mexican males. These females proved fertile. Apparently the F1 males of this cross, and probably the other crosses as well, were sterile. Mather has unsuccessfully attempted to cross a Turrialba, Costa Rica strain (H73.60) with a Nicaraguan strain (H56.94). Both of these yield fully fertile F1 when crossed to the Mexican standard. Cytology Clayton and Wasserman (this bulletin) report on the metaphase chromosomes of a number of strains of paranaensis. There are three karyotypes. Type (a) shows a large pair of metacentric chromosomes, three pairs of rods and one pair of dots. Type (b) has one pair of large metacentrics, three pairs of rods and a pair of small metacentrics. Type ( c) has two pairs of large metacentrics and three pairs of rods. Type (a) was found in Villavicencio, Colombia; Trinidad; and was reported by Dreyfus and de Barros (1949) in Brazil. Type (b) was found in the Santa Marta Mountains of Colombia and from Heredia and Turrialba, Costa Rica. All of the other strains examined had type (c), including the 5 strains from Mexico and Honduras reported by Clayton and Ward ( 1954) . This karyotype information is related to our crosses. Crosses between strains of type (a) and type ( c) produce the sterile males. The crosses involving type (b) produce v~riable results (see Table 4). We are not certain as to the exact mechanisms in;volved in the sterility encountered. But it seems evident that the amount of hettrochromatin present on the dot chromosome affects the fertility of the hybrid males. The salivary gland chromosomes of the new strains have been examined; and may be described as follows: X Chromosome: The X chromosome of the mercatorum group seems to be identical to the repleta X. All stocks, except the two strains from Bucaramanga, Colombia have the repleta sequence. These two, H191.30 and H191.46, are homozygous for a new gene sequence, Xi. Using the repleta map, region C2c to E3c has inverted to form Xi. 2 Chromosome: One new sequence was found among the new strains. The Trinidad strain of paranaensis is homozygous for 2m2, whose breakage points are approximately C5d to Gt-. It includes gene sequence 2e, whose breakage points are nearer to C6--and F3a than C6--and F1c, as reported in 1954. The resulting 2 chromosome of this strain is shown in Figure 2. 3 Cl:zromosome: In 1954 Wasserman reported that a strain of pararepleta from Brazil (1412.913) was heterozygous for 3i and 3g. We have recently examined I ·:" ,. 2d 2 m2 ,·~fr · . I 2e I A--+82a 8:3~~c1-: 83-.-820 Cl--+C5d GI--F3alc6--F3alc6---.C5d Gl--H ...,I .. , . Fr O> 0 t w 0 0 O> t r<> 0 0 O> 0 <.D 0 0 u:r: <.D 0 u <.D t u o> O> 0 0 t 0 J{) I 0 t 0 o> <.D u <.D .!e' u 0 u t 0 t r<> 0 (]) <.D 0 t u o> J{) t 0 u .L 0 0 0 (]) J{) <.D r D u 0 t(]) r<> "" (]) .L I() t t 0 0 I D 0 (]) iii r<> (]) "' 0 (]) 0 .0 I() "" I() u t (]) t 0 0 t u "" .L t 0 " r (]) 00 (]) u"" r<> "' ~ .0 CT .L (]) "" 0 t J{) ~ 0 u (\j' r<> "'0 (\j N" 0 t u u t N_ N> <; 0 0 0 t t N (\j Ci. u ~ t u u u 0 "" N 3 .L 0 r<> ' u (\j 0 J{) "" "' t t u 0 0 I t r<> 0 1ii u I() t 0 u D r<> I .2 (]) (\j "" "' u t 0 t u t "' 0 r<> -'-0 (]) u t D u"' "" r<> (]) ' 0 "" u "' "' t t . t .2 .2 "' 0 r<> 0 r<> "' 1 Fm. 5. A tentative evolution of the 2 chromosome from the repleta sequence to the nigricruria sequences. Studies in the Genetics of Drosophila Discussion The basic chromosome configuration of the species is Xa, Xb, Xe, Xk, 2a, 2b, 2t2, 2u2, 2v2, 2w2, 2x2, 3b, repleta 4, 5f. The Central and South American strains appear to be homozygous for this genome. Figure 6 gives the known cytological composition of the strains. Standard 2 Standard 4 (South and Central America : 2261.7 ', 62 .29 "75.l l ', 163.29 ', 206.19) l St4', 2s2,St2 ~St4.252 (Mexico : ) ' 2264.14 Mexico:) . ( 2263.15 4c,4d,St2 \ M .. 2 2 ( extco.) 4c,4d;St2,2s ,2y 1797.14 I l M . . · . · 2 2 2 ex1co. Mexico.) 4c, 4d, 2s , 2s y ( 2266.15) ( 1796.2 4c 4d'2s2 2y2 2268.7 ' ' ' FIG. 6. Distribution of inversion sequences among the strains of D. nigricruria. The horizontal line denotes that no interme:liate forms have been found between standard 4 strains and the strains homozygous for 4c and 4d. Our data show that the standard 4 chromosome occurs from Venezuela, Costa Rica, Salvador, and into southern Mexico at Tehuacan, Puebla and San Andres Tuxtla, Vera Cruz. Almost immediately north and west, the standard is com­pletely replaced by the 4c, 4d chromosome in the Tezuitlan, Mexico City, Cuerna­vaca, Morelia and Sierra Madre del Sur populations. The map distance between Tehuacan and Tezuitlan is of the order of 100 miles. The other distances are not much greater, Mexico City and Cuernavaca being approximately 125 to 150 miles from Tehuacan. The basic 2 chromosome has a distribution almost identical to that of the basic 4. It also occurs in South and Central America and is either replaced by or co­exists with newer sequences in southern Mexico. However, it was not found in the San Andres Tuxtla strain where standard 4 is present, and it is present in the strain from Tezuitlan where the 4c, 4d chromosome· is found. The Mexico City strain has 4c, 4d, St 2, 2s2 and 2y2• The other three Mexican populations are apparently either homozygous or heterozygous for 2s2 and 2y2• Thus in this geographical area our data indicates a complete change in the chromosome morphology of the species. Six of the seven populations fall within a semicircle whose radius is 150 miles. There is only one population, from Mexico City, where the standard 2 chromosome has been demonstrated to coexist with the newer sequences. None of the populations seems to be heterozygous for the 4 chromosome. This may be due to insufficient sampling. Probably more collections in this area would yield populations heterozygous for the 4 chromosome. The size of our samples has been small; the collection data of Wheeler and Heed and Wasserman show that these strains originated from collections of from 2 to 44 individuals. Another factor is that some of the strains may have become homozygous in the laboratory. The strains numbered 1700 have been in the laboratory for 10 years; those numbered 2200, for 5 years; and the South and Central American strains, from several months to 3 years. It seems evident, however, that in this area both Standard 2 and Standard 4 are being replaced by the newer sequences. The change in the 2 chromosome appears to be gradual, as one would expect. The validity of the abrupt change in the 4 chromosome should be tested more fully. More collections and an analysis of individual flies should be made. It would be enlightening to discover not only the genetic composition of the populations in this area, but also the genetic consti­tution of the individual flies. A change in the genotype would indicate a change in selective forces or sexual or geographical isolation. Our mating tests have shown no lack of fertility between the different chromo­somal types. Matings in mass do not yield data as critical as paired or preferential matings. However, the variation as to how far north the standard 2 chromosome exists compared to the standard 4 chromosome indicates that there is some gene flow through this area. There is no obvious geographical barrier involved. In Mexico, nigricruria is mainly a high altitude pine-oak forest form. This habitat is present at Morelia, Tezuitlan, Sierra Madre del Sur and Cuernavaca. San Andres Tuxtla is also forest, but at 1700 feet compared to 5-8000 feet in the above-mentioned sites. Mexico City has an altitude of about 7000 feet. Tehuacan is on the Mexican Plateau, in a dry cactus locality at about 5000 feet. The Central and South Ameri­can localities are also forested areas. Cytologically it is interesting to note that 2s2 and 2y2 are small independent inversions. They are situated very close to each other, there being only about 10 bands separating them. In the Mexico City strain there are three types of 2 chromosomes present, the basic 2, 2s2 and 2y2 • The Morelia strain has 2s2 and 2y2• A total of 28 chromosomes of the two strains were in combinations where it could be ascertained that 2y2 and 2s2 were not on the same chromosome. The exact relationship of 2y2 and 2s2 could not be interpreted in 18 chromosomes. In no instance could it be demonstrated that 2s2 and 2y2 were on the same chromo­ some. In the strain from Sierra Madre del Sur, all of the 2 chromosomes examined (28) had the 2s2 sequence. Ten had, in addition to 2s2, the 2y2 sequence. We can­not determine when these two rearrangements arose in relationship to each other because they are independent inversions. However, in order to get them occurring both singly on different chromosomes and also together on the same chromo­some, there must have been at least one crossover in this short region of approxi­mately 10 bands in a cell which was heterozygous for both inversions. This is a Studies in the Genetics of Drosophila somewhat unexpected finding in view of the fact that a heterozygous inversion tends to suppress crossingover in the adjacent regions. Two adjacent independent inversions practically eliminate the crossingover in the intercalary uninverted region (see Carson, 1953 and 1955 for discussion and references) . The mulleri complex The mulleri complex is composed of a group of species, all members of the mulleri subgroup, showing a very close cytological relationship to each other (Wasserman, 1954). These include Drosophila arizonensis Patterson and Wheeler, D. mo;avensis Patterson and Crow, D. wheeleri Patterson and Alex­ander. D. aldrichi Patterson and Crow, D. mulleri Sturtevant, D. ritae Patterson and Wheeler, and D. martensis, new species. Two strains of the new species were obtained in South America. The description follows: Drosophila martensis Wasserman and Wilson, new species. External characters of imagines. ~, ~ . Arista with about 7 branches; antennae yellowish, the base of the sec­ond and the third joint dark brown. Front yellowish brown, orbits and ocellar triangle lighter, pollinose; hairs of the anterior orbits, the anterior and posterior orbitals, and anterior verticals with basal blackish spots. Middle orbital about Yz length of anterior and % length of posterior. Second oral about Yz length of first. Carina broadened below, sulcate. Palpi pale yellow, with one long terminal bristle, two long ventral bristles and a number of smaller weaker bristles. Face brownish yellow. Cheeks yellowish gray, their greatest width about V4 greatest diameter of eyes. Eyes reddish (Pl. 3-L11) with short black pile. Acrostical hairs in 8 rows; no prescutellars. Anterior scutellars convergent. Sterno index about 0.82. Middle sternopleural about % length of posterior. Mesonotum gray, bristles arising from brown spots with a slight tendency to fuse giving a superficial appearance of two brown bands between the dorsocentral rows, separated by a thin pollinose stripe. Some fusion of spots outside of dorso­central rows. Scutellurn dark brown; pollinose spots at angle of the scutellum be­tween the bristles and occasionally at base. Pleurae yellowish gray with indistinct fuscous bands going from the base of the wing to the humerus, from the base of the haltere to the fore coxa, and in the region of the sternopleurals. Legs yellowish gray with indistinct black bands on distal ends of fernora; black bands near bases of tibiae. Apical bristles on first and second tibiae, preapicals on all three. Abdominal segments yellowish pollinose, the second to fifth tergites with an in­terrupted black band with f~rward extensions at the interruption, the lateral margins, and the angles of the tergites. The latter extensions widen rapidly at the anterior margin, usually connecting laterally with the lateral extensions en­closing an irregularly shaped yellow area. Dorsally they cut sharply into the yellow region leaving yellow markings as shown in Figure 7. The 6th tergite with an interrupted indistinct black band which fades at the angle. Wings clear, veins brown; apex of 1st costal section darker. Costa! index about 2.8; 4th vein index about 1.9; 5x index about 1.4; 4c index about 2.0. Two well­ The University of Texas Publication developed bristles at the apex of the 1st costal section; 3rd section with heavy bristles on its basal Y , 1 ~ only (2) = number of attempts u ing 20 pairs in mass. ternal genitalia but no sperm were seen in either the spermathecae of the females or the testes of the males. Three F1 females from the aldrichi X Salvador cross were also found to lack sperm in the spermathecae; three males, however, had many sperm, but very few of them were motile. This latter cross is the one that produced some F 2 flies. Morphologically, except for a difference in color, we have not been able to separate the Salvador and Colombian strains from aldrichi. Genetically they appear to be closer to aldrichi than to any other species tested. The F1 females of these crosses were not tested for fertility because aldrichi-wheeleri crosses pro­duce fertile F1 females as shown by Patterson and Alexander (1952). Their ex­tensive tests have never yielded any fertile F1 males. In the Mexican aldrichi X Salvador strain, some F1 male fertility was present. Due to the paucity of our genetic data, it seems best to consider these two strains as "aldrichi" for the pur­pose of this paper. Cytology The salivary gland chromosomes of "aldrichi" are identical to those of aldrichi, mulleri and wheeleri (Wasserman, 1954) . The chromosomes of martensis are as follows: X Chromosome: The martensis X chromosome differs from the mulleri X (Xa, Xb, Xe) by being homozygous for inversion Xj, which has not been found in any other species. The chromosome is shown in Figure 8. 2 Chromosome: The evolution of the 2 chromosome is esesntially as shown in Figure 9. This is the first species of this complex which has been found to be chromosomally polymorphic. Both strains are heterozygous for 2g2• 3 Chromosome: The martensis 3 chromosome has 3b, 3a, and a new sequence, 3k. It is shown in Figure 8. 4 and 5 Chromosomes: These appear to be identical to repleta. The University of Texas Publication x· X• A-C4g D4b-C4g D4b -E3-F5--G2o Flo -F3o Fla-E3-F5--F3o 2o -H Fm. 8. The X and 3 chromosomes of D. martensis. Discussion It was shown in 1954 that the six species, ritae, mulleri, aldrichi, wheeleri, mo;avensis and arizonensis, although each is apparently homozygous for certain gene sequences, share a series of 5 inversions. The data are such that a simple picture of speciation through geographical divergence could not be constructed. However, the inversions were so placed among the six species that the construc­tion of a linear dine of four chromosomal types (called "subspecies") could be used to explain the data obtained without resorting to hybridization between species. One of the points in question in the hypothetical system was whether two populations could allow a free exchange of certain genes or groups of genes (an inversion) and still not allow other groups of genes to pass from one popu­lation to the other. In nigricruria, there is an indication that such a phenomenon is taking place. The so-called barrier for the standard 4 chromosome does not prevent the various types of 2 chromosomes from migrating from population to population. Table 7 shows the gene sequences which are shared among the seven species. In 1954, the proposed linear dine was ritae; mulleri, aldrichi, wheeleri; mo- TABLE 7 Distribution of inversions among the members of the mulleri complex. mulleri aldrichi Inversion ritae wheeleri mojavenis arzzonenszs martensis 2g present present present present absent 3a present present present absent present 2c absent present present present present 2f absent present present present absent 2h absent absent present present absent 3c present present absent absent absent ;avensis; arizonensis. There was no reason to believe that the precursor of this complex necessarily had a linear distribution. However, the chromosome mor­phology of the six species discussed in 1954 could be interpreted with the linear arrangement as the simplest explanation. The discovery of 2c and 3a in martensis complicates the picture. The lack of 2g which is present in the other six species places martensis off to one side, possibly south since it is now known only from South America. The gene sequences 2c and 3a both occur only in mojavensis and mulleri, aldrichi, and wheeleri. We therefore believe that the precursor of martensis probably occurred farther south than the other known species, border­ing on either the precursor of mojavensis or, more likely, the ancestor of mulleri, aldrichi, and wheeleri. The gene sequences 2c and 3a were fixed in the martensis Studies in the Genetics of Drosophila :r: Q) 0 c Q) !l Q) VJ 0 -Q) Q) 0 0 <.D (.) t O> a 0 <.D (.) t 0 u 0 r<> Ci 0 <.D u t 0 Ci 0 C\J CD t 0 r<> 0 t .2 0 0 N m j 0 u 0 r<> (]) t 0 C\I CD 0 0 t o> 0 0 l.D u t 0 u 0 r<> CD t 0 u 0 r<> 15 0 <.D (.) t 0 Ci 0 r0 m t 0 C\J m 0 a t t N O> 0 N 0 <.O u t O> l[) (.) 0 C\J m t 0 r<> l{) u 0 r<> CD t 0 u 0 r<> 0 0 <.D (.) t 0 0 0 r0 m t 0 C\J m 0 0 t Cl 15 0 <.D (.) t o> l[) (.) 0 N m t 0 r<> l[) (.) 0 r<> m t 0 «t m 0 «t CD t ·0 u 0 r<> X arizonensis ~) produces fertile hy­brids of both sexes, while the reciprocal cross produces fertile females but sterile males. On the basis of current knowledge, these forms are geographically (Pat­terson and Wagner, 1943) and presumably ecologically (Spencer, 1941; Wagner, 1949) isolated (Fig. 1) . The mitotic chromosome configuration of both species consists of 5 pairs of rods and a pair of dots (Wharton, 1942) . The two species differ cytologically by inversions in three chromosomes (Wasserman, 1954). The X chromosomes differ by one simple inversion, the 2nd chromosomes by 4 over~ lapping inversions, and the 3rd chromosomes by 2 overlapping inversions. These two species were chosen for study since fertile hybrids are formed between them and since the composition of the populations could be analyzed cytologically by examining the salivary gland chromosomes for normal and various hybrid combinations. 1 Dept. Biology, Johns Hopkins Univ., Baltimore. The University of Texas Publication \ e MOJAVENSIS Fw. 1. Distribution of the known records of Drosophila arizonensis and Drosophila mojavensis. MATERIALS AND METHODS Experimental populations derived from crosses involving D. arizonensis, D. mojavensis, and their F1 hybrids were maintained in population cages. Crosses were made using males and females of both species to determine if hybridizat:on occurs when there is a choice of mates. This type of cross tests the effects of compe­tition between species and hybrids if hybridization occurs, or solely between the two species if hybridization does not take place. Populations were started with males of one species and females of the other to test the effects of selection between possible gene and chromosome combinations where the F1 hybrids are necessarily formed. Finally, an experimental population was initiated with F1 hybrid males and females to determine the effects of competition in a population where the first generation consisted of various F2 hybrids; here backcrosses, if any, were of necessity between hybrid forms. Each of these experimental populations was analyzed by taking egg samples from the cage and examining the salivary gland chromosomes of the developing larvae. Population changes were determined by the change in frequency of the chromosome inversions characteristic of each species. The homozygous chromo­some combinations for e~ch species could be determined by the banding pattern. The experimental populations were bred in population cages similar to those used by Wright and Dobzhansky ( 1946), which in turn were modifications of those devised by Heritier and Teissier ( 1933). All experiments were carried out at 23 ± 1° C. on a banana-agar-malt-yeast culture medium. The stocks used were Studies in the Genetics of Drosophila Drosophila arizonensis from Tucson, Arizona (stock no. 928.5) and Drosophila mojavensis (strain from Chocolate Mountains, California). The flies were raised in regular culture bottles. Virgins from these were aged for six days and introduced into the population cages in desired proportions. Seven experiments were initiated. The crosses involved, the proportion of flies, and the date started are summarized in table 1. TABLE 1 The initial experimental populations Cage no. Started Initial population Number November 14, 1953 mojavensis 2 500 m'?iavensi_s 6 500 anzonenszs 2 500 anzonenszs 0 500 2 November 14, 1953 anzonenszs mojavensis 2 0 1,000 1,000 3 November 14, 1953 moiavensi.s arz zonenszs 2 6 1,000 1,000 4 February 14, 1955 mojavensis m'?iavensi~ arzzonens;.s arizonensis 2 0 2 6 500 500 500 500 5 February 14, 1955 ari zonensis mojavensis 2 6 1,000 1,000 February14, 1955 m'?iavensi.s 2 1,000 an zonenszs 6 1,000 April 2, 1955 F, hybrid (M X A) 2 1,000 F, hybrid (M X A) () 1,000 Samples were taken from the cages once a month in order to determine the composition of the populations in regard to the three chromosomes mentioned above. A fresh food cup was placed in the cage and was removed after 24 hours. Eggs were taken from various areas of the food and placed in a regular culture vial. The process was repeated for five successive days. Chromosomes were ex­amined from 20 larvae of each of these daily subsamples, giving a total of 100 larvae (600 chromosomes) examined for each monthly sample. Since the X chromosomes were being studied, only female larvae were utilized. Cytological Technique The cytological technique differed slightly from the usual method. The larvae were dissected in Drosophila saline solution, the salivary glands were placed in 1N HCl for six minutes, and then transferred to a drop of distilled water directly on the slide. The water was drained off and a drop of stain added. After five min­utes the stain was drained off, a drop of 40 percent acetic acid was added and the salivary glands covered with a coverslip. Spreading of the chromosomes was done by pressing lightly on the coverslip with a needle. The preparation was then ringed with paraffin-lanolin. The stain used was one percent orcein in 50 percent acetic acid. RESULTS Of the seven experiments, no two were exactly alike. Cages four, five, and six were duplicates of cages one, two, and three in regard to the initial composition of the population; but the first analysis was deferred several months in the lat­ter group to test the effects of selection over a longer period of time. Experiments one, two, and three were started November 14, 1953, but the first analysis was not begun until April, 1955. The duplicate experiments were started February 14, 1955, and samples were taken within 30 days. During December of 1954, cage three was found to have a contamination of. Drosophila melanogaster ; and was discontinued before samples were taken. Since the male hybrids of the cross arizonensis <;> X mojavensis g are sterile, no attempt was made to set up a hy­brid cage reciprocal to cage seven. Tables 2-7 summarize the results of the six experiments. The information is arranged to show the number of heterozygotes and homozygotes for the three chromosomes. Homozygous arizonensis is represented by AA, homozygous mo­javensis by MM, and the heterozygous condition by AM. The initial population composition is given as a percentage (considering both males and females) . Otherwise, since exactly 100 individuals were studied per sample, each entry can be read as an absolute number or a percentage. The percentage of arizonensis chromosomes is given in the last three columns. Since three chromosomes were followed, each of which may be homozygous or heterozygous, one particular larva could be placed in one of 27 classes of chromosome combination. These will be referred to as larval classes. The number of individuals in these various larval classes is presented in the bottom half of tables 2-7. In this case, homozygous arizonensis is denoted by A, h()mozygous mojavensis by M, and the heterozygous condition by H. The data are presented graphically in figures 2-7. The percentage of homo­zygous and heterozygous combinations of the three chromosomes is shown for each population. The time ordinate is given in elapsed numbers of days. Cage I (Table 2, Figure 2) Cage 1 was started with males and females of both species in equal numbers. Following the first analysis samples were taken at 30-day intervals, except be­tween the 638th and 698th day. The population went quite well from its start, with an over-abundance of larvae in each food cup. The experiment was started with a total of 2,000 flies, but within three generations the population increased to over twice this number (by visual estimation). Only the mojavensis X chromosome was present in any of the six successive samples. The arizon.ensis X chromosome was eliminated from the population within 518 days. The 2nd and 3rd chromosomes of both species were still present in the population after 698 days from its initiation. The mojavensis 2nd chromosome homozygotes varied in frequency between 16 and 50 percent and were more frequent than arizonensis homozygotes in every sample. The 2nd chromosome heterozygotes were found in higher fre­quency than either homozygote in all samples except the first, where homozygous TABLE 2 Results of cage 1, started with females and males of both species Date Jl -14--53 4--16-55 5-16-55 Elapsed Time in Days 0 518 548 Chromosone X AA AM MM 50 0 50 0 0 JOO 0 0 JOO Chromosome 2 AA AM MM 50 0 50 50 4. 46 6 78 16 Chromosome 3 AA AM MM 50 0 50 560 44 0 40 60 Percent ARIZONENSIS x 2 3 50.0 50.0 50.0 0.0 27.0 22. 0 0.0 45. 0 20.0 V:l ...... !:::: ~-.ti) "' -·;:i 6-15-55 578 0 0 100 JO 51 39 0 32 68 0.0 35.5 16.0 ...... 7-15-55 608 0 0 JOO 8 64 28 0 30 70 0.0 40.0 15.0 ~ 8-14--55 10-13-55 638 698 0 0 0 0 100 100 18 2 44 64 38 34 0 0 24 14 76 86 0.0 0.0 40.0 34.0 12.0 7.0 ~ ti) ;:i ti) ......-· ~ Elapsed Time in Days (X ) A (2) A (3) A A A H A A M A H A A H H A H M A M A A M H A M M H A A H A H H A M H H A H H H H H M H M A H M H H M M M A A M A H M A M M H A M H H M H M M M A M M H M M M -Q.. t:l ""'.j 0"' 0 ~ 518 548 4 6 16 22 28 56 26 12 26 4 ~-· ....... i::i 578 3 7 17 34 12 27 608 2 6 22 42 6 22 638 2 16 10 34 12 26 698 2 JO 54 2 32 .... O'l .... 518 548 578 608 638 DAYS FIG. 2. Cage 1. Started with males and females of both species. T he percentage of homozygous and heterozygous combinations of the three chromosomes. mojavensis was 4 percent greater. The percentage of arizonensis 2nd chromo­somes varied between 27 and 45 percent. Third chromosome arizonensis homozygotes were not found in any sample. Third chromosome heterozygotes declined steadily from 44 to 14 percent, while mojavensis homozygotes increased reciprocally. There was a gradual decrease in the number of arizoru:msis 3rd chromosomes from 22 to 7 percent. A few arizonensis homozygotes would be expected from matings between heterozygotes. Either the matings did not produce them or they were selected against in the samples. Only six of the 27 possible larval classes were represented. These six classes differed from one another in frequency, and individually varied somewhat be­tween samples. The largest class was M-H-M, with over 50 percent of all larvae analyzed in the 548-and 698-day samples. Of the total number of individuals analyzed in cage 1, 77 percent were known hybrids. Cage 2 (Table 3, Figure 3) The initial population of the second experiment consisted of arizonensis females and mojavensis males. Subsequent to the first analysis, samples were taken after intervals of 30, 60, and 60 days. The population went well at first, but its size dropped in the final six months of the experiment accompanied by bacterial and mold infection. At no time did the number of flies present fall below 1,000-2,000. As in cage 1, only mojavensis X chromosomes were present in the samples. Both chromosome arrangements of the 2nd and 3rd chromosomes were still present in the last analysis. The 2nd chromosome arizonensis homozygotes were below eight percent in each sample. The heterozygous state ranged between 26 and 48 percent and was lower in frequency than the mojavensis homozygotes except in the 578-day sample. The percentage of 2nd chromosomes which were arizonensis varied between 15 and 32 percent in the four samples. The 3rd chromosome percentages were similar in the four successive analyses. Homozygous arizonensis 3rd chromosomes were not found in the 400 larvae examined. The heterozygous condition fluctuated in frequency between 16 and 18 percent, and mojavensis homozygotes ranged between 82 and 84 percent. The number of arizonensis 3rd chromosomes was between eight and nine percent. Only six of the 27 possible larval classes were represented, and these were the same classes found to be present in cage 1. The class M-M-M was the largest, consisting of 34 to 58 percent of the larvae analyzed in each sample. Considering the four samples, 53 percent of all analyzed individuals were known hybrids. Cage4 (Table 4, Figure 4) Cage 4 was started with males and females of both species. The first analysis was made 10 days after the flies were introduced into the cage. The larvae of this sample were produced by the initial population. Subsequent samples were taken at 30-day intervals. The adult population increased in size, and extremely large numbers of larvae were present in the food cups. - ~ TABLE 3 Results of cage 2, started with arizonensis females and mojavensis males. ~ Elapsed Percent (1) Time in Chromosone X Chromosome 2 Chromosome 3 ARIZONENSIS Date Days AA AM MM AA AM MM AA AM MM x 2 3 ~ ;::! ...... 11-14-53 0 67 0 33 50 0 50 50 0 50 66.7 50.0 50.0 <::::: (1) 5-16-55 548 0 0 100 1 41 58 0 16 84 0.0 21.5 8.0 "'( ..... . -"' 6-15-55 578 0 0 100 8 48 44 0 18 82 0.0 32.0 9.0 ~ 8-14-55 638 0 0 100 2 44 54 0 16 84 0.0 24.0 8.0 c 10-13-55 698 0 0 100 2 26 72 0 18 82 0.0 15.0 9.0 ~ ~ ~ "tl Elapsed (X) A A A A A A A A A H H H H H H H H H M M M M M M M M M i::: Time in (2) A A A H H H M M M A A A H H H M M M A A A H H H M M M ~ ...... - ~ Days (3) A H M A H M A H M A H M A H M A H M A H M A H M A H M ~ c;· 548 1 7 34 8 50 ;::! 578 8 8 40 10 34 638 2 8 36 8 46 698 2 4 20 16 58 % 100 90 80 l&J 2 70 0 (/) 600 2 50 0 a: 40 :J: 0 30 )( 20 10 0 100 90 l&J 2 80 0 (/) 70 0 2 60 0 a: 50 :I:: (.J 40 0 30 z N 20 10 0 100 90 l&J 80 2 0 70 (/) 0 60 2 0 a: 50 :J: 0 40 0 30 a: it) 20 10 0 I I I I •-•=MM &-& =AM •-•=A A -~ 548 578 638 698 DAYS Fie. 3. Cage Z. Started with arizonensis females and moiauensis males. The percentage of homozygous and heterozygous combinations of the three chromosomes. ....... Ol Ol TABLE 4 Results of cage 4, started with fem ales and m ales of both species Date 2-14-55 2-24-55 3-26-55 4-25-55 5-25-55 6-24-55 7-24-55 8-23-55 Elapsed Time in Days 0 10 40 70 100 130 160 190 Chromosone X AA AM MM 50 0 50 28 0 7'L 24 24 52 5 24 71 1 30 69 0 24 76 0 19 81 0 25 75 Chromosome 2 AA AM MM 50 0 50 28 0 72 26 16 58 7 31 62 5 44 51 9 52 39 16 56 28 15 50 35 Chromosome 3 AA AM MM 50 0 50 28 0 72 24 20 56 8 26 66 6 29 65 0 42 58 2 43 55 4 50 46 Percent ARIZONENSIS x 2 3 50.0 50.0 50.0 28.0 28 .0 28.0 36.0 34.0 34.0 17.0 22.5 21.0 16.0 27.0 20.5 12.0 35 .0 21.0 9.5 44.0 23 .5 12.5 40.0 29.0 ...., ;:i-.. (I) ~ ;::sI:. (I) ..... "'-..... ~ .Q. ~ Elapsed Time in Days lO 40 70 100 130 160 190 (X) A (2) A (3) A 28 24 5 1 A A H A A M A H A A H H A H M A M A A M H A M M H A A 2 I 1 H A H 2 2 2 4 H A M 2 4 3 H H A 1 3 1 1 H H H 12 7 6 6 2 6 H H M 2 8 8 6 4 3 H M A H M H 2 3 5 5 2 6 H M M 6 3 7 3 3 2 M A A M A H 1 3 5 3 M A M 2 2 4 5 M H A 1 3 M H H 4 5 10 19 18 M H M 2 11 20 30 29 19 M M A M M H 4 12 13 16 12 13 M M M 72 46 44 '2 7 15 12 14 ~ "t:) ;::: \J"' .......-· 2....-· 0 ;::s ILi ::E 0 (/) 0 ::E 0 a: :c (.) )( ILi ::E 0 (/) 0 ::E 0 a: :c (.) 0 z "' ILi ::E 0 (/) 0 ::E 0 a: :I: (.) 0 a:. I') % 90 80 70 60 50 40 30 20 10 0 90 80 70 60 50 40 30 20 10 0 90 80 10 60 50 40 30 20 10 0 10 DAYS FIG. 4. Cage 4. Started with males and females of both species. The percentage of homozygous and heterozygous combinations of the three chromosomes. . ·1· .·· • The University of Texas Publication The results of the 10-day sample were the same for all three chromosomes: 28 per cent ariwnensis homozygotes and 72 percent mojavensis homozygotes. Since a few individuals in the 40-day sample were the result of a backcross or F1 hybrid cross, there must have been some F1 hybrids present in the first generation, although they were not detected because of chance or time of sampling. The arizonensis chromosomes were lower in frequency in all samples; but whereas in the autosomes the decrease of arizonensis chromosomes was complete by 70 days and was even reversed thereafter, there was a more prolonged trend toward loss of the arizonensis X chromosomes. The arizonensis X chromosome homozy­gotes dropped rapidly from 50 percent to none by the 130th day, while mojaven­sis homozygotes increased to 75 percent in the last analysis. The X chromosome heterozygotes increased to 24 percent and remained at approximately this level. After the 10-day analysis 2nd chromosome mojavensis homozygotes gradually dropped to 35 percent in the last sample. There was a reduction of arizonensis homozygotes to an even lower level, 16 percent. Second chromosome heterozy­gotes increased to over 50 percent. The results of the 3rd chromosome were similar to those of the 2nd, except that the arizonensis homozygotes were slightly fewer in number and the mojavensis homozygotes more frequent. Of the 27 possible larval classes, only A-A-A and M-M-M were represented in the 10-day sample. Class M-M-M dropped from 72 per cent in the first analysis to 14 percent in the last sample, while A-A-A decreased to none by the 130th day. There was an increase in the hybrid classes M-H-M, M-H-H, and M-M-H. In the last three samples these classes, along with M-M-M, were the ones most abundant. Within 130 days, 85 percent of the larvae sampled were known hy­brids. Thus in cage 4, where a choice of mates was possible, hybrids were formed and eventually became the majority in the population. Cage5 (Table 5, Figure 5) The initial population of the 5th experiment consisted of arizonensis females and mojavensis males. The cage went well, and a large number of larvae were produced in each generation. The cross was obligatory, producing only F1 hybrids (H-H-H) in the first generation. As stated above, the F1 males of this cross are sterile. "· The arizonensis arrangement of the three chromosomes was lower in frequency than mojavensis in most samples. As in cages 1, 2, and 4, the trend in cage 5 was toward the loss of the arizonensis X chromosome arrangement. The arizonensis X chromosome homozygotes dropped to none in the first generation and remained at a low level. The reduction of heterozygotes after the first generation continued to the last sample, in which only one percent of all X chromosomes was arizonen­sis. The second chromosome heterozygotes were found in higher frequency than either of the homozygotes, at approximately a 50-60 percent level starting with the 40-day analysis. The two homozygous conditions fluctuated somewhat, but mojavensis was usually in higher frequency. After the first generation, the two chromosome arrangements of the 3rd chromosome approximated an equilibrium. Third chromosome arizonensis homozygotes were found to be less than six per­ TADLE 5 Results of cage 5, started with arizonensis females and mojauensis males . Date 2-14-55 2-24-55 Elapsed Time in Days 0 10 Chromosone X AA AM MM 67 0 33 0 100 0 Chromosome 2 AA AM MM 50 0 50 0 100 0 Chromosome 3 AA AM MM 50 0 50 0 100 0 Percent ARIZONENSIS x 2 3 66.7 50.0 50.0 50.0 50.0 50.0 V) ..... $:: ~ ~- 3-26-55 4-25-55 5-25-55 6-24-55 4-0 70 100 130 0 0 0 3 46 53 31 24 54 47 69 73 0 13 14 25 49 58 6 1 57 51 29 25 18 0 2 3 5 58 56 47 50 42 42 50 45 23.0 26.5 15.5 15.0 24.5 42.0 44.5 53.5 29.0 30.0 26.5 30.0 .... ;::! ..... ;:r.. ~ 7-24-55 8-23-55 160 190 0 0 13 2 87 98 J1 12 59 50 30 38 2 6 44 42 54· 52 6.5 1.0 4-0.5 37.0 24.0 27.0 ~ ;::! ~ .....-· ~ c- Elapsed Time in Days (X ) A (2) A (3) A A A H A A M A H A A H H A H M A M A A M H A M M H A A H A H H A M H H A H H H H H M H M A H M H H M M M A A M A H M A M M H A M H H M H M M M A M M H M M M t:1..., c "' .g ;:r..-· ..._ ~ 40 17 9 13 8 12 10 16 ·15 70 4 5 2 20 7 10 4 "2 2 14 15 6 9 100 5 1 2 10 7 3 3 4 4 18 24 l 7 11 130 t 2 1 3 2 7 5 1 5 1 12 7 20 21 5 7 160 1 1 6 1 1 3 1 3 5 1 20 31 I 13 12 190 2 8 'I• 20 28 6 14 18 ....... 0) <.O l&.I 2 0 Cf) 0 2 0 a: :c u x l&.I 2 0 (/) 0 2 0 a: :c u 0 z N l&.I 2 0 Cf) 0 2 0 a: :c u 0 a: ff) % 100 90 80 70 60 50 40 30 20 10 0 100 90 80 70 60 50 40 30 20 10 0 100 90 80 70 60 50 40 30 20 10 0 40 70 100 130 160 190 DAYS FIG. 5. Cage 5. Started with arizonensis females and mojavensis males. The percentage of homozygous and heterozygous combinations of the three chromosomes. cent in all samples. After the 40-day analysis, there was a slight increase of mojavensis homozygotes to about 52 percent and a decrease of heterozygotes to approximately 42 percent. Only H-H-H of the larval classes was represented in the first generation due to the obligatory cross. The number of represented classes increased to 16 by the 130th day but then decreased to eight by the last analysis. Six of these eight classes were the ones found to be present in cages 1 and 2. Classes M-H-M and M-H-H increased rapidly and became the most abundant in the last four samples. Over 82 percent of the larvae analyzed in each sample were known hybrids. Cage 6 (Table 6, Figure 6) This cage was started with mojavensis females and arizonensis males. The cage went very poorly at the beginning, although numerous eggs were laid. Egg sam­ples were taken, but the hatch was too low to yield a significant analysis. It was impossible to make an analysis until the 100th day, when many larvae were pro­duced both in the sample and in the cage. At this time the size of the population increased rapidly until it was substantially larger than the initial number of 2,000 flies. There was a certain degree of fluctuation in each of the zygotic chromosome combination percentages, but no trend was observed. The heterozy­gous condition of each of the three chromosomes was highest in frequency in most samples, while mojavensis homozygotes were the lowest. Thus it may be noted that this was the only experiment having the total number of wizonensis chromosomes greater than 50 percent. In the other cages arizonensis X chromo­somes tended toward less or were eliminated. In all samples of this experiment, the X chromosome heterozygotes were the largest class. Many of the 27 possible larval classes were represented. Individuals of 22 larval classes were present in the 160-day sample. No noticeable trend in the change of class frequency was observed. Cage 7 (Table 7, Figure 7) Experiment 7 was started with male and female F1 hybrids from the cross mojavensis <;> X arizonensis t . The cage went poorly for the first 2-3 months, but then the population started to build up slowly. It was impossible to make an analysis until the 104th day. Even at this time the number of larvae in the sample and in the cage was small. The same was true for the 134-day analysis. The 164­ day sample vials had a large amount of mold present, and not enough larvae were produced to make an analysis. By the 194th day the cage was going very well, with many larvae present in the food cups and sample vials. The X chromosome behavior was similar to that in cages 4 and 5. The homozy­ gous arizonensis combination dropped to none, th~ heterozygous condition de­ creased to 13 percent, and the mojavensis homozygotes increased to 87 percent by the 194th day. The 2nd chromosome heterozygotes were far more frequent than the two homozygotes. The mojavensis homozygotes decreased to 25 per­ cent, while ariwnensis homozygotes were even less frequent, 9 percent in the ..... ~ T ABLE 6 Results of cage 6, started with mojavensis females and arizonensis males. Date 2--14--55 2--24--55 5-25-55 6-24-55 7-24--55 8-23-55 Elapsed Time in Days 0 10 100 130 160 190 Chromosone X AA AM MM 33 0 67 0 100 0 44 46 10 30 47 23 30 42 28 35 49 16 Chromosome 2 AA AM MM 50 0 50 0 100 0 52 42 6 35 47 18 45 48 7 38 53 9 Chromosome 3 AA AM MM 50 0 50 0 100 0 48 40 12 37 48 15 43 47 10 47 49 4 P ercent ARIZONENSIS x 2 3 33.3 50.0 50.0 50.0 50.0 50.0 67.0 73.0 68.0 53.5 58.5 61.0 51.0 69.0 66.5 59.5 64.5 71.5 "-3;:::-.. II> ~ ;::!-.<:::: II>.., "'-...... ~ .Q_ ~ " ~ Elapsed Time in Days 100 130 160 190 (X) A (2) A (3) A 22 11 12 10 A A H 6 10 7 11 A A M A H A 6 7 7 10 A H H 8 1 3 5 A H M 1 1 A M A 2 1 A M H 1 A M M H A A 12 10 10 H A H 6 7 8 7 H A M 6 1 H H A 6 5 13 H H H 12 5 11 13 H H M 6 13 4 2 H M A 3 1 H M H 2 3 2 1 H M M 2 4 1 M A A 6 2 1 M A H 4 4 M A M 1 1 M H A 3 4 2 M H H 2 9 11 7 M H M 4 5 3 M M A 1 1 M M H 4 4 M M M "ti !:: \:!" .......(:) • i::i .....c;· ;::! Studies in the Genetics of Drosophila lLI :E 0 !/) 0 :E 0 a: :I: (.) x lLI :E 0 !/) 0 :E 0 a: :i: u c z N lLI :E 0 (/) 0 :E 0 a: :i: u c a: ,., °lo 100 90 80 70 60 50 40 30 20 10 0 100 90 80 70 60 50 40 30 20 10 0 100 90 80 70 60 50 40 30 20 10 0 10 40 70 100 130 160 DAYS FIG. 6. Started with mojavensis females and arizonensis males. The percentage of homozygous and heterozygous combinations of the three chromosomes. ....... '1 .... TABLE 7 Results of cage 7, started with hybrid (moiauensis/ arizonensis) femal es and males. '"'-j ~ (1) Elapsed Time in Chromosone X Chromosome 2 Chromosome 3 Percent ARIZONENSIS c:'] ;:!-· Date 4-2-55 7-15-55 Days 0 104 AA 33 2 AM 0 30 MM 67 68 AA 0 12 AM 100 53 MM 0 35 AA 0 20 AM 100 55 MM 0 25 x 33.3 17.0 2 50.0 38.5 3 50.0 47.5 <:: (1) "j "'-· .... ~ 8-14-55 10-13-55 134 194 0 0 24 13 76 87 15 9 53 66 32 25 23 12 47 61 30 27 12.0 6.5 41.5 42.0 46.5 42.5 -Q.. ~ ~ Elapsed Time in Days (X ) A (2) A (3) A A A H A A M A H A A H H A H M A M A A M H A M M H A A H A H H A M H H A H H H H H M H M A H M H H M M M A A M A H M A M M H A M H H M H M M M A M M H M M M "ti !::: ~-(:; • !:).... 104 134 1 1 4 2 2 1 1 4 4 7 5 8 2 2 4 5 1 1 2 3 3 5 2 4 11 17 15 12 16 4 1 21 14 5 9 s· ;:! 194 1 1 7 4 1 5 1 7 34 19 3 10 7 Studies in the Genetics of Drosophila ILi 2 0 (/) 0 2 0 a: x 0 )( ILi 2 0 (/) 0 2 0 a: x 0 0 z N ILi 2 0 (/) 0 2 0 a: x 0 0 a: ,,, % 100 90 80 70 60 50 40 30 20 10 0 100 90 80 70 60 50 40 30 20 10 0 100 90 80 70 60 50 40 30 20 10 0 104 134 194 DAYS FIG. 7. Cage 7. Started with hybrid ( mojavensis/ arizonensis) males and females. The percent­. age of homozygous and heterozygous combinations of the three chromosomes. The University of Texas Publication last analysis. The relative frequencies for the three zygotic chromosome combi­ nations of the 3rd chromosome were very similar to those of the 2nd chromosome. The arizonensis arrangements of each chromosome were lower in frequency in each sample. Several larval classes were represented. There was an increase in frequency of classes M-H-H and M-H-M and a decrease of class M-M-H. These three classes · were the ones most abundant in the three samples. DISCUSSlON Inversions, which serve as markers in this analysis of the experimental popu­ lations, represent blocks of genes in particular sequences. Mutational differences exist between the two species utilized. A certain m,1mber of these genes are bound within the inversions, since in heterozygotes the free recombination of the dif­ ferent genes is restricted by inversion loops, at least in the vicinity of the breakage points. These gene sequences are the actual entities being followed. Within the experime~tal populations pure species cannot be identified, since the inversions represent only a portion of the total karyotype. By appropriate crosses, various hybrids are formed with different combinations of the gene arrangements. The hybrids and homozygous "species" have genie differences, which are expressed by the competitive ability of the different forms under stringent selection set up in the population cages. The relative frequencies of the gene sequences for each -0f the three chromosomes studied sometimes underwent rapid changes from one . generation to the next, demonstrating that the carriers of different chromosome types have different selective values. The results of the competition between the forms with different arrangements are most easily discussed separately for each of the three chromosomes. X Chromosome On the assumption that a carrier of some particular gene arrangement is better · adapted than one with another sequence, it would be expected that the less adapted one should be lost. In cages 1 and 2, arizonensis X chromosomes were · completely eliminated, and in cages 4, 5, and 7 there was a rapid reduction in their : number. The mojavensis X chromosome homozygotes are generally superior in competitive ability in the environment of the population cages, depending upon the initial population. The percentage of arizonensis X chromosomes increased . in cage 6. The initial cross in cage 6 ( mojavensis ~ X ari:wnensis i ) was reciprocal to that of cage 5 (arizonensis ~ X mojavensis i ) . Both crosses were obligatory, producing only hybrids in the first generation. The reciprocal results in the early analyses of these experiments are explicable if the majority of the matings which produced the second generation were F, females backcrossing to parental males. The backcross was a necessity in cage 5, since the F, males of this initial cross are sterile (Patterson, 1942). In the 40-day analysis (second generation) of cage 5, the observed frequencies did not deviate significantly from the expected ratio of 50 percent heterozygotes and 50 percent mojavensis homozygotes. The eight 1arv¥ f;:lasses observed, in approximately equal frequencies, would be expected due to chromosome segregation in the gamete formation of the F1 females. The backcross was likewise very probable in cage 6. By the time the F1 flies emerged the parental mojavensis females were too old to produce many eggs which would develop. Males of the mulleri subgroup usually do not mate until 6-7 days after eclosion. Thus the majority of the matings might be expected to be F1 females backcrossing to parental ariwnensis males. This would increase the number of arizonensis chromosomes in the second generation and yield reciprocal results to those seen in cage 5. Backcrossing explains the reciprocal results in the early samples of cages 5 and 6, but it is apparent from the results of later analyses that selection strongly favored mojavensis chromosomes in cage 5 and did not in cage 6. Only arizonensis Y chromosomes were present in the cage 6 experiment. An unbalanced genie system in the carriers of arizonensis Y and mojavensis X chromosomes could be selected against, giving a higher percentage of arizonensis X chromosomes than expected from the results of the other cages. Baker (1947) reported that no progeny was produced when this type of male (arizonensis Y, mojavensis X) was crossed to arizonensis, mojavensis, or hybrid females, in pair matings, but progeny were produced in mass matings. He dissected 15 of the males and found that 13 did.not contain motile sperm. In cage 7, where only arizonensis Y chromo­somes are present, the results are similar to those of cages 1, 2, 4, and 5, and not to those of cage 6. It might be necessary to postulate the existence of different genes on the chromosomes of the initial populations of cages 6 and 7 or the pro­duction of rare recombinations in cage 7 to eliminate this discrepancy. Alter­natively, a mojavensis Y might have come in by non-disjunction or error. Second Chromosome In the majority of the samples from all cages, the 2nd chromosome hetero­ zygotes are slightly higher in number than expected on the basis of the Hardy­ Weinberg formula. After 698 days of selection, both 2nd chromosome arrange­ ments were present in cages 1 and 2. This time period corresponds to approxi­ mately 28 generations. The retention of both gene orders can be explained by the theory of balanced polymorphism. When there is heterosis of the heterozygote, selection will not eliminate either chromosome from the population. The popula­ tion will be polymorphic and will reach an equilibrium. The percentage of ari­ zonensis 2nd chromosomes after a few generations reached a level of approxi­ mately 40 percent in cages 1, 4, 5, and 7. In cage 2 it was nearer 20 percent. This cage was going poorly during the sampling period (latter six months), and a great amount of bacteria and mold was present in the food cµps. It is possible that the selective forces were different in this cage and reduced the level of arizonensis 2nd chromosomes. Dobzhansky and Wallace (1953) gave evidence that in popu­ lations of D. pseudoobscura the different inversions in a particular locality are coadapted to give a superior heterozygote. In the present investigation heterosis is not a matter of coadaptation, since distinct species are involved and not merely strains of one species from the same locality. Each species has genes which are selectively retained for adaptation to its particular environment, and certain combinations of the different genes in hybrids establish a well-integrated physio­ logical system to such a degree that heterosis is apparent. The resµlts of these experiments show that the selective advantage of a par­ ticular karyotype depends not only on the physical environment but also on the other karyotypes present in the population. In cage 6, with the arizonensis Y and a high percentage of arizonensis X and 3rd chromosomes and numerous larval classes represented, the frequency of arizonensis 2nd chromosomes was about 55 percent after 190 days. In the other experiments, with fewer arizonensis X and 3rd chromosomes and fewer larval classes represented, the frequency of arizonensis 2nd chromosomes was approximately 40 percent. There was also a difference in the types and frequencies of karyotypes in cage 6 versus the other experiments. Third Chromosome The results of the 3rd chromosome are similar to those of the 2nd chromosome in each experiment, in that the heterozygotes show heterosis. Nearly every sample of each cage had slightly more heterozygotes than are to be expected according to the Hardy-Weinberg formula. Both gene arrangements were present in the final samples of cages 1 and 2, with balanced polymorphic equilibrium apparently reached in cage 2. In cages 1 and 2, where only X chromosome mojavensis homozygotes occur, 3rd chromosome arizonensis homozygotes did not appear in the samples, and the number of arizonensis 3rd chromosomes was between seven and nine percent. There was a high percentage of arizonensis X chromosomes (about 60 percent) in cage 6. The number of arizonensis 3rd chromosomes was also about 60-70 per­cent, and numerous arizonensis homozygotes were present. A direct relation was also found in the other experiments. In each experiment, very few larvae had one of these chromosomes homozygous for arizonensis and the other homozygous for mojavensis. Larval classes A-A-M, A-H-M, A-M-M, M-A-A, M-H-A, and M-M-A were low in frequency in every experiment (tables 2-7). Either the crosses failed to produce these classes, which is improbable, or the larvae were selected against in the samples. It cannot be considered that these larval classes are complete lethals, since most of the classes are represented by a few larvae in some samples. In cages 1 and 2, arizonensis 3rd chromosome homozygotes were not found among the 1,000 larvae examined, although some would be expected from random mating. The samples were not completely free of selection. Al­though extra yeast was added to each sample vial, the conditions were still slightly overcrowded. If the individuals of the above classes have an extremely low se­lective value they could be eliminated by selection in the samples. From these data it seems that a genie unbalance exists in individuals having the X chromo­some homozygous for the gene sequence of one species and the 3rd chromosome homozygous for the gene order of the other species. The equilibrium level of the two 3rd chromosome arrangements thus depends not only on the physical envi­ronment and the respective selective values of the two gene sequences, but also on the frequency of the X chromosome karyotypes. The results of a similar investigation by Bruneau (Unpublished thesis), using species of the virilis group, are comparable to those reported here. Using the same type of experimental cages, conditions, and method of analysis, he obtained similar results with the species Drosophila virilis, D. novamexicana, and D. tex­ana. The frequency of virilis chromosomes was predominant in crosses with either texana or novamexicana. In most cases the heterozygotes showed heterosis. In the population started with males and females of arizonensis and moiavensis, hybrids were formed and replaced the parental types. Hybrids were produced and heterosis was apparent in the same type of population initiated with texana and novamexicana. There seems, however, to be a greater degree of isolation between virilis and either texana or novamexicana, since hybrids were not formed when there was a choice of mates. A trend toward species replacement resulted in these cages, with virilis predominating. In the present report the X chromosome mo;av­ensis homozygotes had a greater selective value, as evidenced by the loss or trend toward loss of the arizonensis X chromosomes. Only in cage 6 did the X chromosome heterozygotes show heterosis. The results of the virilis group crosses indicated that the X chromosome heterozygotes showed. heterosis except in the crosses with a choice of mates and in the population started with novamexicana females and virilis males. Bruneau reports that, for any two of the three species, the reciprocal obligatory populations gave similar results and that little difference existed between these and the F1 initiated populations. As already discussed, the results of the F1 (moiavensis X arizonensis) population were similar to the one started with arizonensis females and mo;avensis males, but reciprocal results were observed in the cross of moiavensis females to arizonensis males. The production of hybrids in nature between populations normally separated by isolating mechanisms frequently leads to an increase in the variation and adaptive versatility of the species involved. For hybridization to occur isolating barriers need to be incomplete. Gene exchange, leading to more variability of a species due to an occasional hybridization with another species without destroy­ing specific identities, is termed introgressive hybridization by Anderson and Hubricht ( 1938). This process has been extensively studied by Anderson ( 1949, 1953). Although little is known of the frequency of introgression, most cases studied have been found primarily among plants. The results of the present in­vestigation, with the production of heterotic hybrids, show that there is a possi­bility that introgression or intergradation might conceivably occur in natural populations of Drosophila. The present investigation, as well as that of Bruneau, shows that occasional crosses between species in nature could produce heterotic combinations. These could survive and continue to contribute genes from one species to another. SUMMARY The two closely related species Drosophila arizonensis and Drosophila moiav­ensis are not known to be sympatric. They have been shown to produce some fertile hybrids in the laboratory. Populations derived from crosses involving these species were maintained in population cages. They were analyzed fre­quently to determine the effects of competition between the species and their interspecific hybrids when formed. Seven populations were initiated as follows: males and females of both species (in duplicate); moiavensis females and arizo­nensis males (in duplicate, but one was discontinued) ; arizonensis females and moiavensis males (in duplicate); and F1 hybrids from the cross of moiavensis females and arizonensis males. The latter three crosses forced the production of hybrids. The populations could be analyzed cytologically since the species dif­fered by inversions in the X, 2nd, and 3rd chromosomes. In the experiments initiated with males and females of both species not only were hybrids formed, but they survived and tended to eliminate the parental species. In all experiments except cage 6, which was started with mojavensis females and arizonensis males, the mojavensis X chromosome homozygotes proved superior in ability to compete. The 2nd chromosome heterozygotes ex­hibited heterosis in each experiment. The level of balanced polymorphic equilib­rium was reached relatively soon and was dependent upon the genetic background of the population. Although arizonensis 3rd chromosomes were highly selected against, heterosis was sufficient to retain the chromosome arrangements of both species. A genie balance system was found to exist between the Y, 3rd and X chromosomes. The mojavensis arrangements of the three chromosomes became more frequent than arizonensis in all populations except that in cage 6. The reciprocal results of this experiment are discussed, and a comparison is made between this study and a similar one by Bruneau. REFERENCES Anderson, E. 1949. lntrogressive Hybridization. New York: Wiley and Sons. 109 pp. ----.1953. Introgressive hybridization. Biol. Rev. 28: 280-307. Anderson, E. and L. Hubricht. 1938. Hybridization in Tradescantia. III. The evidence for in­trogressive hybridization. Amer. Jour. Bot. 25: 396--402. Baker, W. K. 1947. A study of the isolating mechanisms found in Drosophila arizonensis and D. mojavensis. Univ. Texas Puhl. 4720: 126-136. Brncic, D. 1954. Heterosis and the integration of the genotype in geographic populations of Drosophila pseudoobscura. Genetics 39: 77-88. Bruneau, L. H. Unpublished thesis. Cain, A. J. and P. M. Sheppard. 1954. The theory of adaptive polymorphism. Amer. Nat. 88:321­ 327. ' Crow, J. F. 1942. Cross fertility and isolating mechanisms in the Drosophila mulleri group. Univ. Texas Puhl. 4228:54-67. Dobzhansky, T. 1951. Genetics and the Origin of Species. 3rd. Ed. New York: Columbia Univ. Press. 364 pp. Dobzhansky, T. and H. Levene. 1941. Development of heterosis through natural selection in experimental populations of Drosophila pseudoobscura. Amer. Nat. 85:247-264. Dobzhansky, T. and B. Wallace. 1953. The genetics of homeostasis in Drosophila. Proc. Nat. Acad. Sci. 39:162-171. Koopman, K. F. 1950. Natural selection for reproductive isolation between Drosophila pseudo­?~scura and Drosophila persimilis. Evolution 4: 135-148. Levene, H., 0. Pavlovsky, and T. Dobzhansky. 1954. Interaction of the adaptive values in poly­morphic experimental populations of Drosophila pseudoobscura. Evolution 8:335-349. Levine, L. 1955. Genetic background and heterosis in Drosophila pseudoobscura. Genetics 40: 832-849. L'Heritier, Ph. and G. Teissier. 1933. Etude d'une population de Drosophiles en equilibre. C. R. Acad. Sci. Paris 197: 1765-1768. ----: 1934. Une experience de selection naturelle. Courbe d'elimination de gene "Bar" dans une population de Drosophiles en equilibre. C. R. Soc. Biol. 117: 1049-1051. Merrell, D. J. 1951. lnterspecific '~ompetition between Drosophila funebris and Drosophila melanogaster. Amer. Nat. 85: 159-169. Moore, J. A. 1952. Competition between Drosophila melanogaster and Drosophila simulans. Evolution 6:407-420. Patterson, J. T. 1942. lnterspecific hybridization in the genus Drosophila. Univ. Texas Puhl. 4228:7-15. -----. 1947. Sexual isolation in the mulleri subgroup. Univ. Texas Puhl. 4720: 32-40. Patterson, J. T. and W. S. Stone. 1952. Evolution in the Genus Drosophila. New York: Mac­millan Company. 610 pp. Patterson, J. T. and R. P. Wagner. 1943. Geographical distribution of species of the genus Drosophila in the United States and Mexico. Univ. Texas Puhl. 4313:217-281. Spencer, W. P. 1941. Ecological factors and Drosophila speciation. Ohio Jour. of Sci. 41 : 190-200. Stone, W. S. 1942. Heterosis in Drosophila hrdei. Univ. Texas Puhl. 4228: 16-22. Stone, W . S., M. L. Alexander, and F. E. Clayton. 1954. Heterosis studies with species of Droso­phila living in small populations. Univ. Texas Puhl. 5422:272-307. Teissier, G. and Ph. L'Heritier. 1937. L'elimination des forme~ mutantes dans les population de Drosophiles. 70 Congres Soc. Savantes: 297-302. · Wagner, R. P. 1949. Nutritional differences in the mulleri group. Univ. Texas Puhl. 4920:39-41. Wasserman, M. 1954. Cytological studies of the repleta group. Univ. Texas Puhl. 5422: 130-153. Wharton, L. T. 1942. Analysis of the repleta group of Drosophila. Univ. Texas Puhl. 4228:23-52. Wright, S. and T. Dobzhansky. 1946. Genetics of natural populations. XII. Experimental repro­duction of some of the changes caused by natural selection in certain populations of Dro­sophila pseudoobscura. Genetics 31: 125-156. XIII. An Attempt to Detect Hybrid Matings Between D. mulleri and D. aldrichi Under Natural Conditions 1 WILLIAM B. HEED A new isolating mechanism in the genus Drosophila was reported by Patterson ( 1946, 1947) in the form of a prolonged insemination reaction in the vagina of the female resulting from interspecific matings. Patterson examined all pos­sible crosses in the mulleri subgroup and found a correlation in the degree of se­verity of the mass with subsequent success or failure in hybrid production. A reaction mass is usually formed in intraspecific matings also but they are less severe and pass from the vagina in a shorter time. I-iere then is presented a quan­titative method by which one could detect attempted hybrid matings under natural conditions by isolating freshly caught females until all homogamic (in­traspecific) reaction masses had been passed and then dissecting out the repro­ductive parts. Any reaction mass found would presumably be a heterogamic one and would indicate a hybrid mating in nature. This procedure was followed in respect to D. mulleri Sturtevant and D. aldrichi Patterson and Crow where they were trapped in the fall of 1951 at the Aldrich Farm near Austin. The two species are closely related and are sympatric in the south central part of Texas. Both larval types utilize the fruit of the cactus Opun­tia although D. mulleri also inhabits other fruits and vegetables. Sexual isolation is high in the laboratory and the cross of aldrichi females with mulleri males does not produce hybrids. The reciprocal cross will yield a few sterile hybrid males and females (Patterson and Crow, 1940; Crow, 1942). Patterson (1947) identi­fied hybrid males from field collections but only when the proportion of aldrichi males was at least one-third of the total aldrichi-mulleri male population. In respect to the reaction mass, Patterson found that when aldrichi females were exposed to mulleri males for 96 hours and the females dissected, all of the 24 that were inseminated had a reaction mass in the vagina and five had motile sperm in the ventral receptacles. This cross does not yield hybrids. The re~iprocal cross showed that of the 36 females inseminated, 20 had a reaction mass and 18 had motile sperm. This cross does produce hybrids. Wheeler ( 1947) reported the behavior of the insemination reaction resulting from intraspecific matings from a survey of the genus. METHODS The only species tested was mulleri females for possible matings to aldrichi males. The flies were brought into the laboratory immediately after collection and the female mulleri were isolated for at least 12 hours. The data presented by Patterson showed that reaction masses from homogamic matings pass from the vagina in eight to nine hours. The reproductive tracts were then dissected directly into saline after various hours of isolation and examined under the microscope. Two techniques were used in handling the control material. First the usual method of ether anesthetic was employed. As the experiment progressed it was Zoological Laboratory, Univ. Pennsylvania, Philadelphia. feared that etherization was delaying the passage of the reaction mass. Carbon dioxide was then administered as an anesthetic. The usual mating arrangement for the controls was 20 virgin females and 10 virgin males per large shell vial. The flies were aged about one week before mating. The controls were run on first and second generation individuals originally collected at the Aldrich Farm. RESULTS Table 1 shows that 84 reaction masses were found in 1,973 mulleri females in­seminated in the wild. Table 2 shows that a small proportion of the masses were found up to 47 hours isolation. Most of the masses were of medium to small size in the pouch of the vagina. Two of the masses however differed markedly. One female, dissected after 29 hours isolation, contained a mass which made the whole vagina turgid. The other female, after 24 hours isolation, had a very large re­fractive dark pouch. Of all the dissections containing a reaction mass, four were TABLE 1 Collection data October November December (9 Collect. ) ( 4 Collect. ) ( 4 Collect.) Total mulleri 'i' 6427 1022 387 7836 mulleri ct; 6561 1060 387 8008 aldrichi 'i' 111 187 45 343 aldrichi ct; 193 265 71 529 Percent aldrichi ct; 2.9% 20.0% 15.5% No. mulleri 'i' Dissected 1707 750 338 2795 No. Dissected with V.R. 1220 709 319 2248 No. with Sperm in V.R. 1091 617 265 1973 Percent with Active Sperm 89.4% 87.0% 83.1% No. Reaction Masses 43 23 18 84 Percent Reaction Mass 3.9% 3.7% 6.8% TABLE 2 Proportion of reaction masses in etherized wild D. mulleri. Least Isolation Reaction Mass Mating Time Fiducial Limits Time (Hours) No. Inseminated of Expectation 1Z-14 6/89 -6.7% 2.5-13.8 15-17 10/159-6.3% 3.1-11.1 I 18-20 34/436-7.8% 5.4-10.7 21-2:3 9/139-6.5% 3.0-11.7 I 24-26 11/331-3.3% 1.7-5.7 Unknown 27-29 7/85 -8.2% 3.4-16.0 35-37 1/ 136-0.7% 0.0-4.1 39-41 1/266-0.4% 0.0-2.1 4Z-44 4/237-1.7% 0.5-4.3 45-47 1/57 -1.8% 0.0-9.8 '63 0/32 -0.0% 0.0-11.5 not dissected complete with ventral receptacles. Of the 80 remaining dissections, only two had no sperm in the receptacles. Table 3 shows that a small proportion of homogamic reaction masses remains in the control females even after four days isloation and in approximately the same proportion as that found in the wild flies. The fiducial limits of expectation are taken at the 95 %confidence band. Table 3 shows that etherization does not slow down passage of the reaction mass to any marked degree. The etherization does however retard mating ability considerably. An average of 15 hours for mating was necessary to obtain a satis­factory number of inseminated females. In Table 4 is listed a random sample of T ABLE 3 f'roportion of reaction masses in control homogamic matings of D. mulleri Isolation Reaction Mass Mating Time Fiducial Limits Technique Time (Hours) No. Inseminated Of Expectation Ether 12 hrs. none 14/ 22-63.6% co, co, Ether co, Ether 10 min. 10 min. 15 hrs. 10min. ZO hrs. 1-2 5­6 8-11 13-14 18­20 62/ 85-72.9% 17/ 63-27 .0% 4/ 49­8.1 % 2/ 63­3.2% 5190­5.5% 2.3-19.2 0.4-1 0.7 1.8-12.1 co, Ether co, Ether co, co, co, co, 10min. 22 hrs. 10min. 12 hrs. 10min. 10min. 10min. 10min. 19­20 26-28 26-30 33-37 40 48 87-88 99-102 2/ 53-3.8% 2/ 41-4.8% 3/ 99-3.0% 1/ 48-2.1 % 0/ 37-0.0% 1/50-2.0% 2/51-3.9% 2/68­2.9% 0.5-12.7 0.6-16.5 0.6­8.9 0.1-1 1.6 0.0­9.9 0.5-14.4 0.5-13.4 0.3-9.9 T ABLE 4 Mating success with CO, and ether anesthetized virgin mulleri Age (days) 9 10 9 8 9 CO, M ethod 9 9 10 11 10 10 9 10 10 10 Ether~co, M ethod 8 8 11 10 10 ;\· •, Technique 19<;>, 10$ 19<;>, 10$ 11<;>, 10$ 17<;>, 10$ 20<;>, 10 $ 20<;>, 10 $ 18<;>, 10 $ 15<;>, 10$ 15 'i', 10 s 15<;>, 10$ 19<;>, 10$ 20<;>, 10 $ 20<;>, 10$ 21 'i', 10 s 20<;>, 10$ 20<;>, 10 $ 15<;>, 10 $ 20 'i', 10 s 19'i',10 $ 18<;>, 10 $ Time for Mating 10 minutes 10 minutes 10 minutes 10 minutes 10 minutes 10 minutes 10 minutes 10 minutes 10 minutes 10 minutes 33 hours 13 hours 10 hours 1 hour 1 hour 15 minutes 15 minutes 10 minutes 10 minutes 10 minutes No.Females Inseminate:! 16 13 10 1'6 19 20 15 14 15 12 9 9 12 9 10 7 7 0 6 6 the experiment comparing mating success after virgins were anesthetized with carbon dioxide or ether, aged for about 9-10 days, set up for mating with C02 and etherized just before dissection after a varying number of hours isolation. These data indicate that given over a week to recover and a much longer time to mate, the etherized flies will not mate as readily as individuals taken virgin with C02• Courtship is vigorous with the C02 method but poor with the ether method. In order to observe the behavior of heterogamic reaction masses 379 control mulleri females were tested with aldrichi males in many different trials under varying mating conditions of light, temperature and proportion of males to fe­males. Both the ether and carbon dioxide techniques were employed and in the lat­ter case the aldrichi males courted actively as soon as placed with mulleri females. The females, however, refused to accept the males. Only 16 females of nine dif­ferent trials were found to be inseminated upon dissection. Five females con­tained sperm in the ventral receptacle but no reaction mass (none of the isolation times was more than nine hours; average mating time was 18 hours). Of the re­maining 11 with a reaction mass, four had no sperm in the ventral receptacles. Only one of the masses completely filled the vagina. Four masses were of medium size and six masses were small and usually indistinguishable from homogamic masses. CONCLUSIONS The heterogamic reaction mass is not a suitable criterion to detect hybrid mat­ing attempts in nature for the hybrid-producing cross of mulleri female to al­drichi male under the conditions of the experiment. This is so since a small pro­portion of homogamic masses remain in the female even after four days isolation and since the less severe heterogamic masses cannot be distinguished from homogamic ones. Of the 84 reaction masses from the wild population, two could be safely distinguished as a severe heterogamic type. The probable functions of the homogamic reaction mass are discussed by Pat­terson (1946) and Wheeler (1947). One of the functions stated is to prepare the reproductive tract for the fertilization mechanism. It is believed that even though some homogamic masses do remain for a considerable period after mating, they are of such a nature as not to interfere with normal egg passage and fertilization. The superior mating ability of flies handled throughout their life with carbon dioxide instead of ether suggests that this method should be seriously considered as a technique for Drosophila workers. REFERENCES Crow, J. F. 1942. Cross fertility and isolating mechanisms in the Drosophila mulleri group. Univ. Texas Pub. 4228:53-67. Patterson, J. T. 1946. A new type of isolating mechanism in Drosophila. Proc. Nat. Acad, Sci. 32: 202---208. ----.1947a. Sexual isolation in the mulleri subgroup. Univ. Texas Pub. 4720:32-4-0. ----. 1947b. The insemination reaction and its bearing on the problem of speciation in the muller.i sµbgroup. Univ. Texas Pub. 4720:41-17. ----. ,and i. F. Crow. 1940. Hybridization in the mulleri group of Drosophila. Univ. Texas Pub. 4032:251-256. Wheeler, M. R. 1947. The insemination reaction in intraspecific matings of Drosophila. Univ. Texas Pub. 4720:78-115. XIV. Genetic Studies on Drosophila mulleri. I. The Genetic Analysis of a Population 1 WARREN P. SPENCER INTRODUCTION Drosophila mulleri was first recognized as a distinct species and described by Sturtevant ( 1921). More recently Patterson ( 1943) has redescribed the species, including a complex of taxonomic characters which makes it possible to separate it from many other closely related forms. Wharton (1942) described the meta­phase chromosomes as five pairs of rods and a pair of dots, with the X-chromo­somes longer than the autosomes, and the Y-chromosome much shorter than the X. She found that the salivary chromosomes consisted of five long arms and a dot-like element. Patterson and Wagner (1943) published a distribution map of the species and stated: "Drosophila mulleri has been found chiefly in Texas and northeastern Mexico. A few specimens have been collected at single points in Oklahoma, Arkansas, Louisiana and Florida. This species is very common in Texas, especially in the central and southern parts of the state." Wagner (1944) studied the food habits of the species. He found larvae in the rotting fruit of the prickly pear cactus, Opuntia Lindheimeri, collected at several localities in Texas. Nine yeasts were isolated from these fruits, and it was established that D. mulleri would develop to maturity on a cactus juice-agar medium inoculated with each of the yeasts, but would not grow on sterile medium alone. D. aldrichi, a closely related species, developed to maturity on only six of the yeasts. Wagner stated: "Collections of mulleri have been made in fruit stores, fruit dumps, etc., but in general it may be said that this species restricts itself to cactus provided none of the domesticating influences of man are around to tempt it." The same author (Wagner, 1949) studied the larval development of the five closely related species, mulleri, aldrichi, moiavensis, arizonensis, and buzzatii, on the eight species of yeasts grown on a synthetic basal medium. He found that mulleri and arizonensis showed similar growth on the several yeasts, while the others differed in yeast requirements from these two and from each other. He concluded: "The work reported here gives concrete evidence that significant physiological differences exist between five closely related species of Drosophila which are of a type that conceivably could have been of importance in the evolution of these species." The chief interest in D. mulleri, however, relates to certain possibilities it offers for comparative genetic and cytological studies. This point can best be docu­mented by a brief review of a few facts concerning the taxonomy and cytology of the .genus. Patterson and Wheeler ( 1949) catalogued 605 species described and assigned to the genus Drosophila. While a few of these have since been reas­signed to other genera, the collection and description of new species has added considerably to this total. Of the eight subgenera now recognized from material available for study by far the largest is the subgenus Drosophila, wjth over 180 species, followed by the subgenus Sophophora, with about a third as many species. 1 Dept. Biology, College of Wooster, Wooster, Ohio. The subgenus Drosophila was first divided into 14 species groups by Sturtevant ( 1942). Patterson and Stone ( 1952) have listed additional groups, bringing the total to 22. Each species group contains those forms considered to be most closely related on the basis of comparative taxonomic, cytological and ecological studies. At this stage in the development of our knowledge the most significant contribu­tions to evolution in the genus are likely to be made at the level of intra-and inter­specific studies within the species group. Of the 22 species groups now recognized in the subgenus Drosophila by far the largest in described species is the repleta group. Recent collections of Dr. Wil­liam Heed in Central America indicate that the tripunctata group may eventu­ally rival the repleta group in this respect. A considerable number of the repleta group are readily cultured in the laboratory; the flies are large and vigorous and have a high reproductive potential. Of the 51 described species in this group 39 have been further assigned to subgroups as follows: hydei subgroup, six species; melanopalpa subgroup, 13 species; mercatorum subgroup, two species; mulleri subgroup, 18 species (Patterson and Stone, 1952; Patterson and Alexander, 1952; Wheeler, 1954). The cytological picture in this large and successful species group is of unusual interest and significance in relation to an analysis of the mechanism of evolution in the genus. In a number of species of both the Sophophora and the Drosophila subgenera numerous intraspecific inversions have been found in natural popu­lations. In some species the selective advantage of flies heterozygous for certain of these inversions has been demonstrated both in natural populations and in popu­lation cage experiments (Dobzhansky, 1947; Dobzhansky and Levene, 1948). It has been postulated that the selective advantage of inversion heterozygotes may have served in the past and may continue to serve as a potent factor in evolution. However, in this species group no evidence has been found of any considerable frequency of intraspecific inversions. Wharton (1942) reported finding no inver­sions in strains of D. repleta from Japan, Turkey, Guatemala, Texas, and Con­necticut when these were crossed. Warters (1944) tested 21 strains of D. meri­diana from ten areas to a standard strain and found no inversions. She re­ported no inversions in cross tests of five strains of D. bi/urea from widely sepa­rated localities. In 26 strains of D. hydei collected at several points in the United States only one inversion was found by this author. This inversion, about one­third the length of the longest autosome, was widely spread in American popu­lation samples and was also found in collections from Hawaii and South Africa. In an extensive cytological study of the repleta group Wasserman ( 1954a) found no intraspecific inversions in three strains of D. nigrospiracula of the melanopalpa subgroup, to which D. repleta also belongs. In the mercatorum sub­group five strains of D. paranaensis varied in respect to two inversions in the third chromosome, and three of the 14 strains of D. mercatorum tested varied in respect to three inversions in the third chromosome. In the mulleri subgroup 39 strains, distributed among 12 species, showed no intraspecific inversions with the exeption of one inversion in three of the seven strains of D. buzzatii. On the basis of these and some additional population samplings it may be concluded that this species group contains very few intraspecific inversions in natural populations. In many of these species such inversions are either absent or extremely rare. This is the situation in populations of Drosophila mulleri thus far tested. Wasserman's major contribution involved the careful comparison of the band­ ing pattern of the salivary chromosomes of 18 species and one subspecies of the repleta group to the salivary maps of D. repleta and D. mulleri. All major inter­ specific inversions were observed and on the basis of these observations a phylo­ genetic tree of these species was constructed. His observations indicated that the fourth and fifth salivary chromosomes have remained largely unchanged in their banding pattern, with few interspecific inversions in these 18 species. Others of the chromosomes, including the X, were shown to have identical patterns in several of the species. This elegant cytological study lays the foundation for and serves as added stimulus to comparative genetic studies in this species group, in­ cluding the construction of genetic linkage maps of certain key species and their comparison against the background of the comparative cytology. Thus, a fairly extensive genetic map of the X-chromosome of D. hydei is already available (Spencer, 1949). Wasserman reported no interspecific inversions between the X of D. hydei and D. mulleri. A genetic map of the X of mulleri, including a series of gene loci homologous to those already known in hydei would thus make possible a new approach to certain problems in comparative cytogenetics. For several reasons D. mulleri would seem to be the most logical choice as a key representative of the mulleri subgroup. In the first place it is relatively easy to culture this species in the laboratory, in contrast to many of the 18. species as­. signed to this subgroup. Furthermore, collection records indicate that D. mulleri is a very successful species over its main geographical range of Texas and north­ern Mexico, ensuring easily collected and abundant samples for laboratory analysis, in contrast to many of the other species in this subgroup. In respect to interspecific hybridization D. mulleri occupies a favorable posi­ tion in the subgroup. Patterson and Alexander (1952) reported on hybridiza­ tion cross-tests between six closely related species in the mulleri subgroup. Table 1 is adapted from their table on cross-tests. It will be seen by reference to the table that mulleri will produce fertile female hybrids with two of the species and sterile hybrids with the other three species. Mojavensis also produces fertile hy­ brids with two of the species, and in one of these crosses, mojavensis females x arizonensis males, hybrids of both sexes are fertile. But this species forms sterile TABLE 1 P, crosses between six members of the mulleri subgroup (Adapted from Patterson and Alexander, 1952,) * males mojavensis wheeleri aldrichi arizonensis buzzatii mulleri females mulleri ft.f ;st.m ft.f ;st.m st.f;st.m st.m ab.flies mojavensis st.m none ft.f;ft.m none none wheeleri st.f ft.f;st.m st.f ab.f st.f aldrichi st.f ft.f;st.m st.£ none none arizonensis ft.f;st.m none none larvae none buzzatii none larvae none none none *Key to above table. ft.f-fertile females ft.m-fertile males st.f-sterile females st.m-sterile males ab.f-abnormal females ab.flies-abnormal flies Studies in the Genetics of Drosophila hybrids with only two of the remaining three species. W heeleri, aldrichi, and arizonensis produce fertile hybrids with only one other species and buzzatii with none. Thus for experiments on gene transfer from one species to another and in the investigation of genetic factors in F1 sterile hybrids, mulleri and mo;avensis may be given an approximately equal rating and one distinctly above the other four species. No other species in the mulleri subgroup is as favorable as these two for hybridization studies. The Drosophila mulleri Population The author spent the academic year 1954-1955 on research leave at The Uni­versity of Texas and was fortunate to find living quarters within 10 minutes walk of the campus and at the same time in an area which seemed ideal for Dro­sophila collecting. Using a method of Drosophila collecting first used by Dr. Har­rison Stalker ai;id later described by the author (Spencer, 1950) about 50 paper drinking cups containing banana mash with yeast added were hung on the lower branches of trees and bushes, or set in the garage, all within 150 feet of the author's home. All of the traps were set in an oval area 85 feet long by 40 feet wide, with a total area of approximately 3400 square feet. It was estimated that the collecting area was approximately one two-billionth of the area of the state of Texas. No doubt many of the flies taken in the traps were drawn to them from contiguous territory outside this area. Wasserman ( 1954b) made a study of population dynamics in D. mulleri, and discovered that population movement in this species is very rapid. No attempt was made to list the plants in the collecting area. There was a con­siderable variety of plants including bushes with ripe berries, trees, vines, and herbs. A small stream flowed along one side of the area, and beyond this stream was a wooded park. Live oaks grew in the vicinity but notin the collecting area. It should be noted that there were no cactus growing within the collecting area, and the nearest cactus plants were a few specimens in a garden over a hundred yards from the area. It seems almost certain that the large D. mulleri population, together with that of other species reported below, was not being maintained on cactus. The major food source maintaining the population was not determined. The city maintained a regular garbage collecting service so it seemed unlikely that this could be a chief food source. This observation is in no way a contra­diction of Wagner's observations on cactus as the main food plant of D. mulleri in natural habitats, but it seems quite apparent that in those habitats where man is a disturbing influence the species may maintain itself prolifically on other food sources. From November 5, 1954, through December 15, 1954, collections were made twice daily in the morning and evening. On cool or cloudy days collections were also made at noon. Metal cylinders with cheese cloth covering one end were used as collectors. One of these was brought close to the trap; the latter was suddenly picked up and thrust into the lower open end of the cylinder and flies present in the traps or lures flew up toward the lighted upper end, covered with cheese cloth. After collecting from several traps the open end of the collector was plugged with a large cotton plug. These collectors were then taken to the laboratory immediately; the flies were etherized in the collectors and examined. Records The University of Texas Publication were kept of the numbers of specimens of males and females of each species. In the determination of species the author wishes to acknowledge the invaluable help of Dr. J. T. Patterson, who gave unsparingly of his time and expert knowledge of Drosophila species. Table 2 presents a summary of the collection records for the six-week period. While sexes were recorded separately it was thought unnecessary to record them in the table, except for the affinis-algonquin series in which males can easily be TABLE z Numbers of each Drosophila species taken in collecting area at 805 Leonard Ave., Austin, Texas; November 5-December 15, 1954. Species arranged in order of abundance. Species Total Species Total 1. Z-3. 4. 5. 4-5. 6. D. mulleri D. melanogaster-simulans D. affinis males D. algonquin males D . affin.-algon. females D. rnericliana meridiana 7389 4853 1546 153 635 1Z9 13. 14. 15. 16. 17. 18. D. longicornis D. macrospina D. putrida D. micromelanica D. busckii D. duncani 57 5Z 47 zo 13 3 6. D. meridiana rioensis Z5 19. D. tripunctata 3 7. D. hamatofila 13Z ZO. D. ananassae 1 8. D. aldrichi 1Z3 Z1. D. carbonaria 1 9. D . melanica 107 zz. D. cardini 1 10. D. hydei 101 Z3. D. immigrans 1 11 . D. pseudoobscura 93 Z4. D. repleta 1 rn. D. virilis 64 Z5. D. victoria 1 Total Collection-15,551 Flies. separated, while females of these two species are not distinguishable. As in other collections from this laboratory melanogaster-simulans are listed together. Of the 15,551 specimens of Drosophila collected 7389 were D. mulleri, 47.5% ofthe total. The melanogaster-simulans collection of 4853 specimens constituted 31.2% of the total, and the affinis-algonquin collection of 2331 specimens made up 15.0% of the total. Of the other twenty species, with two subspecies of D. meri­diana, meridiana, hamatofila, and aldrichi represented approximately one per­cent each of the population, and other species were represented by still fewer specimens, with six species, immigrans, cardini, repleta, victoria, carbonaria, and ananassae represented by single specimens. By far the largest coll~ction of Drosophila ever made was that of Patterson ( 1943) and collaborators from the Aldrich plot a few miles from Austin, Texas where collections were made almost continuously from July, 1938 to May, 1941, a total of 141,126 specimens of Drosophila distributed among 31 species. Our collection over a six-week period included 25 species. In spite of the fact that cactus grew abundantly on the Aldrich plot the total of mulleri and aldrichi collected on this plot in the three-year period was 16,203. Our collection showed almost half this number of mulleri for the six-week period. The peak collections for mulleri and aldrichi at the Aldrich plot came in October and November re­spectively for the two years in which autumn collections were made. It seems likely that our collections were made at about the time of the maximum develop­ment of the population. Much more work would have to be done on the ecology of this species together with analysis of population samples from contiguous areas to determine the actual extent of the population from which our collection sample was derived. In fact the D. mulleri populations in the vicinity of Austin, Texas may approach a panmictic state during their peak development in the autumn and early winter. Genetic Analysis of a Population Sample A preliminary account of the data presented here was given in a report by Stone (1955) on genetic and chromosomal variability in Drosophila. In a few of the later collections of D. mulleri the sexes were not recorded separately. In that part of the collection where sexes were recorded there were 3518 males and 3290 females. In order to estimate the kinds and numbers of recessive mutant types carried in the D. mulleri population 1000 pair matings of wild flies were made up in small vials on the standard culture medium used in the Texas laboratory. In order to space the work of analysis these matings were made up in small lots during the six-week collecting period. Because of the failure of some cultures and because certain others had to be discarded during the necessary absence of the author from the laboratory, only 736 of these pair matings gave F1 progeny which were used in the analysis. Undoubtedly many if not most of the females used had already been inseminated before capture. Whether the females used in this study were inseminated by the male provided in the laboratory or by a male in nature, the analysis is based on the assumption that multiple insemination had not occurred. This seems to be a valid assumption in view of the extensive work of Patterson (1947) on the insemination reaction in the mulleri subgroup. He states: "We found, however, that in a species in which a strong reaction occurs, the female usually does not remate for a long time. It was further found, both after homogamic and heterogamic matings, that the females do not im­mediately respond to the vigorous courtship of males." As D. mulleri shows a strong insemination reaction it seems valid to conclude that the data presented below are based on pair matings and not multiple inseminations. Spencer (1947a) has reviewed the different methods of analysis of wild popu­lations for mutants and has pointed out that, when feasible, pair matings of the F1 progeny give the most accurate statistical results. When pair matings produce a large progeny it is possible to analyse the total mutant load carried by a popu­lation sample quite accurately. If, however, many pair matings give no progeny and many others give only a few flies, this method of analysis must be discarded and small mass matings must be used. From the first fifty pairs of wild flies of D. mulleri a small mass mating and three pair matings were made up. Because of the disappointing number of failures from the pair matings the author re­luctantly abandoned the pair mating technique in favor of small F1 mass matings. Fortunately the author had run extensive tests on the mass mating versus pair mating technique in the analysis of population samples of D. hydei, D. immi­grans, and D. melanogaster, and could estimate that about one-third as many mutants would be recovered by the mass mating technique as by rearing three F1 pair matings in these species in which pair matings go very well. The problem of why pair matings do not go well in D. mulleri is an intriguing and exasperating one, and should be given further study. It has been this author's experience that six females of the species, if placed together in a small mass mating, will produce many more offspring than will the same six females if placed separately in six culture vials. If Drosophila mulleri is to be used exten­sively in genetic studies this problem should be solved. This author found that when pairs of D. mulleri were placed in large soda straws pushed into culture medium the flies gave a much larger percentage of successful cultures than when pairs were placed in small glass vials. But he is under the impression that mulleri in mass cultures produce more offspring than in pair matings, and he feels that more study should be given to the problem of how to rear this species successfully in pair matings. One small mass mating from each of the 736 F1 cultures was reared. The F2 offspring were examined by etherizing the flies and looking for new mutant types under the binocular microscope. Routinely the flies emerging from each culture were examined as they first emerged and twice subsequently at about two or three day intervals. With the culture technique used, in which absorbent tissue paper, soaked in a suspension of baker's yeast, had been placed in each vial, large progenies were generally secured and most of the flies had emerged by the time of the last examination of the progeny. Mutant types were scored and given temporary names at the time they were found. Cultures of the more vigorous and interesting types were established, but many of the weak and poorly expressed mutants were immediately discarded because of lack of time for further study. From the 736 F 2 progenies of the mass matings 263 mutant types were re­corded. These were not all mutants at different loci as shown by the analysis below of the numerous recurrences of bright scarlet-like eye colors. From former experience with the mass mating versus pair mating technique of extracting visibles it was estimated that the population sample of 1472 wild flies carried somewhere between 900 and 1400 mutant genes giving visible phenotypes simi­lar to those reported for D. melanogaster in the comprehensive monograph of Bridges and Brehme ( 1944) on the mutants of this species. This gives an average of 0.6 to 1 mutant gene per wild fly tested. The author (Spencer, 1946, 1947b) analysed two population samples, each of 110 flies of D. immigrans, by the pair mating technique and estimated approximately 100 visibles to be present in the sample, approximately 0.5 mutant gene per fly tested. From fairly extensive samples of several populations of D. melanogaster and D. simulans (Spencer, unpublished) 0.7 mutant per fly was estimated to be present. Large samples of the species, D. hydei and D. robusta (Spencer, unpublished), have given some­what lower estimates of visible mutants per fly tested. In the analysis of D. hydei, mutants at the bobbed locus, which can be demonstrated in most wild flies, were omitted from the calculations. Alexander (1949, 1952) analysed population samples of several species of Drosophila, using the F1 pair mating technique, and estimated the frequency of mutant genes for D. hydei to be 2.38 per fly tested; for D. limpiensis, 1.14; for D. americana, 1.69; for D. texana, 1.32; and for D. novamexicana, 0.55 mutants per fly tested. On the basis of the population analysis using the rather crude mass mating technique it appears that this population of D. mulleri carried a visible mutation load falling well within the range of the varying mutation loads carried by populations in several other species. Many of the mutants recovered in such an analysis as the one presented here are obviously semi-lethal types. The flies appear weak and die within a few days of emergence. Such mutants have been discarded although they could be kept Studies in the Genetics of Drosophila in stock by breeding from heterozygotes, using pair matings. Other mutants which appear vigorous enough phenotypically prove to be sterile in one sex or the other. These also were discarded in most cases. A third type of mutant shows many normal overlaps in homozygous cultures. These are also discarded. Any of the above types could be cultured but when one is starting the genetic analysis of a species such types require too much time for culture and can well be dis­carded until the genetics of the species has been worked out, using those visible mutants which have a relatively high viability and fecundity. The Mutants Extracted from the Population In the procedure of extracting mutants from the population sample the F, mass cultures were numbered consecutively from 1 to 736. The mutants found were given a number corresponding to the stock number and a tentative name. A brief description of each mutant was included in the record except for eye colors, where the name was generally considered to be a sufficient tentative de­scription. The names of some mutants were later changed after a more thorough study indicated probable or possible homologies with mutants of D. melanogaster (Bridges and Brehme, 1944). After considering several methods of presenting the data it has been decided to include a check list of the mutants in the consecu­tive order in which they were found, rather than to group them under various phenotypic categories. This procedure has some disadvantages but it does give the reader a clear picture of the variety of mutant types that turn up in the day by day analysis. Perhaps the casual reader may glance through the table and get some concept of the sense of anticipation which the worker develops as he examines culture after culture, knowing that sooner or later new and exotic types never seen before in any species will be found and equally interesting homo­logues of mutants seen in other species, but for the first time revealed against a new genetic background, will appear. Table 3 contains a check list of the mutants discovered, including the stock number, tentative name, and except for eye colors, a brief diagnostic description. The eye colors are recorded as scarlet-like, peach-like, etc. Subsequent analysis showed many cases of identity or allelism in eye colors recorded, but this was not known at the time the mutant was found. There are, of course, numercnis cases of apparently phenotypically identical eye colors occupying different ,focJ., so-called mimic mutations, in D. melanogaster and other species. TABLE 3 Mutants recovered from the F2 progeny of 736 pair matings of wild D. mulleri. Single F, mass: matings were made up from progeny of each pair of wild flies and numbered consecutively. , These stock numbers precede the name of each mutant extracted from the stock. Tenta­tive names in this table were later changed after further study. Most of the mutants were discarded and names used in this table are to be considered descriptive and not permanent symbols. Short diagnostic descriptions follow the names except in case of eye colors. ( 1) 1 bronze; eye color. (2) 1 small bristle; late emerging, bristles reduced in size. (3) 2 erect; post-scutellars erect. (4) 4 rough-coarse; eyes rough; wings coarse; more extreme in male~·:· ..· \ ....,., (5) 5 scarlet-like; eye color. .. , TABLE 3-Continued Mutants recovered from the F, progeny of 736 pair matings of wild D. mulleri. Single F, mass matings were made up from progeny of each pair of wild flies and numbered consecutively. These stock numbers precede the name of each mutant extracted from the stock. Tenta­ tive names in this table were later changed after further study. Most of the mutants were discarded and names used in this table are to be considered descriptive and not permanent symbols. Short diagnostic descriptions follow the names except in case of eye colors. (6) 5 lozenge-like-thick; eyes rough, lozenge shaped; legs thick. (7) 6 roof; wings held at 45 degree angle, roof-like. (8) 7 scarlet-like; eye color. (9) 9 scarlet-like; eye color. (10) 10 peach-like; eye color. (11) 10 light; body color, spots reduced on thorax; only in males when found; sex-linked and carried in P, male. (12) 11 abnormal abdomen; extreme in females; tergites very irregular. (13) 11 missing-scutellars; post-scutellars missing; normal overlaps. (14) 13 wavy-like; wing surface wavy. ( 15) 20 abnormal abdomen; tergites irregular in females; less extreme in males. (16) 22 purple-like; eye color; males sterile. ( 17) 23 rough-small; eyes small and rough; wings often partly unfolded. (18) 30 scarlet-like; eye color. (19) 31 brown-like; eye color; best in young flies. (20) 31 inturned-like; dorso-centrals turn in; hairs on wing margin stand out. (21) 31 wavy-like; wing surface wavy. (22) 34 peach-like; eye color. (23) 34 thin-wing; wing thin textured; crumpled near distal end. (24) 36 rough-small; eyes small and rough; variable in expression. (25) 37 dark; eye color; difficult to classify. (26) 38 light; body color; spots much reduced on thorax; sex-linked. (27) 41 rough-notched; eyes moderately rough, small; wings notched; sometimes abnormal abdomen. (28) 42 peach-like; eye color. (29) 42 spineless; very small bristles; female sterile. (30) 42 cup-wing; wings form small cup, convex above, or are inflated; wings about one-third normal length and at right angles to body. (31) 47 short-narrow; wings short and narrow; body chunky; flies weak. (32) 47 fringe-like; hairs on margin of wing irregular. (33) 51 ragged-roof; hairs on wing margin ragged; wings roof-like. (34) 53 semi-lethal; eyes very small and rough; bristles small and many missing; wings do not unfold; flies soon die. (35) 53 scarlet-like; eye color. (36) 53 silver-like; wings and frons silver; thorax dark. (37) 56 peach-like; eye color. (38) 61 brown-like; eye color; best in young flies. (39) 63 broken; posterior cross-vein broken; variable. (40) 65 light; body color; spots on thorax reduced; probably sex-linked. ( 41) 66 silver-like; wings, thorax and frons silvery; variable expression. (42) 67 purple-like; eye color. (43) 71 blister; wings with blisters; very variable. (44) 71 rough-like; eyes medium rough. (45) 73 scarlet-like; eye color. (46) 76 broken; posterior cross-vein broken; very variable. (47) 77 rotated; male genitalia rotated. (48) 80 peach-like; eye color. (49) 90 scarlet-like; eye color. (50) 91 small bristle; bristles about half normal length. (51) 95 fringe-like; hairs on margin of wing irregular. (52) 97 scarlet-like; eye color. (53) 106 scarlet-like; eye color. (54) 108 roughest; eye facets very irregular; some facets enlarged. (55) 109 peach-like; eye color. (56) 111 scarlet-like; eye color. (57) 115 shaved; many bristles missing; wings nicked; eyes rough. (58) 117 naked-like; hairs very sparse on thorax. (59) 119 rough-like; small rough eyes. (60) 120 inturned-like; dorsocentrals turned in; wing hairs stand out on margin of wing. Studies in the Genetics of Drosophila (61) 120 reinforced; margin of wing with a vein-like thickening at distal end of wing. (62) 122 wavr-like; wing surface in waves. (63) 122 abnormal abdomen; abdominal tergites very irregular. (64) 126 knotted; thick knot at end of fifth vein. (65) 126 pearly-like; body color pearly; bristles silvery. (66) 127 purple-like; eye color. (67) 130 scarlet-like; eye color. (68) 131 dumpy-like; wings truncated, often blistere:l. (69) 132 broken; posterior cross-vein broken; very variable. (70) 134 scarlet-like; eye color. (71) 135 dumpy-like; wings short and truncated; body short; variable but no normal overlaps. (72) 135 radius-incompletus; second longitudinal vein broken near base. (73) 144 cut-net; wing with margin incised and net veins. (7 4) 144 purple-like; eye color. (75) 147 Minute; one male with very small bristles; lost before testing. (76) 148 abnorm~l abdomen; tergites irregular; medium expression. (77) 150 extra; small bits of vein between L 2 and L 3 near wing tip. (78) 153 tiny-like; bristles about one-third normal length. (79) 154 silver-like; body and wings silvery; thoracic spots gone. (80) 156 anterior-cross veinless; anterior cross-vein missing. (81) 158 brown-like; eye color. (82) 160 two-iointed; tarsi with only two joints, the basal and distal. (83) 161 grooveless-like; groove between scutellum and thorax missing; etherizes very rapidly; flies weak. (84) 163 silver-like; body and wings silvery; antennae white. (85) 164 brownish; entire thorax brownish; spots almost obliterated. (86) 170 bent-down; wing margin bent down near tip on outer edge. (87) 174 peach-like; eye color. (88) 175 rough-cut; eyes rough; wing margins cut; wings may be blistered and veins thickened. (89) 175 pearly-like; body color like silver but less extreme. (90) 175 spineless-like; very tiny bristles. (91) 177 small bristle; bristles slightly reduced in size. (92) 178 pearly-like; body color. (93) 181 pearly-like; body color. (94) 183 scarlet-like; eye color, but little darker than other scarlets, and with a cherry tinge. (95) 189 scarlet-like; eye color. (96) 193 brown-like; eye color. (97) 193 silver-like; body color. (98) 199 scarlet-like; eye color. (99) 200 small bristle; bristles about two-thirds normal length. (100) 201 brown-like; eye color. (101) 203 pearly-like; body color. (102) 205 scarlet-like; eye color. (103) 205 silver-like; body color. (104) 208 mahogany-like; eye color. (105) 212 blackish; wings and thorax very dark; flies weak. (106) 214 scarlet-like; eye color. (107) 214 pearly-like; body color. · (108) 214 narrow; wings long and narrow. (109) 219 scarlet-like; eye color. (110) 222 rough-abnormal; rough eye; abnormal abdomen; wings often nicked. (111) 224 grooveless-like; groove between scutellum and thorax missing. (112) 230 serrate-like; wings cut along both margins; eyes small. (113) 232 peach-like; eye color. (114) 237 broken; posterior cross-vein broken; variable. (115) 238 bent wing; wings bent down near tip. (116) 240 scarlet-like; eye color. (117) 245 roughened; eye large and rough. (118) 249 purple-like; eye color. (119) 251 peach-like; eye color. (120) 253 humpy-like; thorax humped; flies small. (121) 256 cross-vein-dome; posterior cross-vein missing; wings broad and convex dorsally. (122) 260 fuzzy-like; hairs on wing margin stand out, but dorso-centrals not turned in. (123) 262 curled; wings curled up strongly; posterior scutellars erect and sharply crossed; flies dark. (124) 262 brown-like; eye color. (125) 263 scarlet-like; eye color. (126) 269 sparse; hairs on wing margin sparse. (127) 272 peach-like; eye color. TABLE 3-Continued Muta~ts recovered from the F, progeny of 736 pair matings of wild D. mulleri. Single F, mass matmgs were made up from progeny of each pair of wild flies and numbered consecutively. These stock numbers precede the name of each mutant extracted from the stock. Tenta­ tive names in this table were later changed after further study. Most of the mutants were discarded and names used in this table are to be considered descriptive and not permanent symbols. Short diagnostic descriptions follow the names except in case of eye colors. (128) 276 rotated abdomen; right side of abdomen rotated down, left side up; both sexes fertile. (129) 276 dachsous; flies short and chunky; wings short; x-veins close. (130) 282 scarlet-like; eye color. (131) 288 broken; posterior cross-vein broken; variable. (132) 289 spread-like; wings held out; flies very weak. (133) 290 tiny-rough; bristles very small; eyes rough; wings nicked; semi-lethal. (134) 295 scarlet-like; eye color. (135) 295 peach-like; eye color. (136) 298 lanceolate; wings long and narrow; pointed at end. (137) 303 dark brown; eye color. (138) 303 Extension; semi-dominant; in heterozygote extends size of dark spots on thorax; in homozygote thorax color an even black or dark brown; thorax humpy; head bristles lie flat. (139) 304 brown-like; eye color. (140) 304 scarlet-like; eye color. (141) 305 scarlet-like; eye color. (142) 305 small; flies small; pigment of thorax light; eyes rough; bristles small; viability very poor. (143) 306 ·cross-veinless; posterior cross-vein missing. ( 144) 307 scarlet-like; eye color. (145) 307 blister-like; large, discolored blisters on wing. (1416) 313 thick-vein; veins thickened; particularly strong in L 5. (147) 321 thickened; posterior cross-veins thick; normal overlaps. (148) 336 serrate-like; wing margin serrated. (149) 339 small bristle; bristles about one-half normal length; flies small. (150) 341 short; fifth longitudinal short. (151) 341 warped-like; wing warped in both axes. (152) 343 peach-like; eye color. (153) 344 chubby-like; flies short; cross-veins close together; legs thick. (154) 346 erupt-like; erupted pustule on posterior margin of eye. (155) 348 peach-like; eye color. (156) 348 polychaete-like; extra bristles on thorax. (157) 348 crease-eye; a distinct furrow across eye. (158) 351 peach-like; eye color. (159) 357 hoary; eye pile white rather than dark as in normal eye. (160) 361 scarlet-like; eye color. (161) 362 scarlet-like; eye color. (162) 364 broken; posterior cross-vein broken; variable. ( 163) 369 broad wing; wings short and broad. ( 164) 370 scarlet-like; eye color. (165) 371 scarlet-like; eye color. (166) 372 thickened; posterior cross-veins thick; normal overlaps. (167) 373 light; body color; spots on thorax reduced; sex-linked. (168) 37'6 peach-like; eye color. (169) 377 abnormal abdomen; abdominal tergites slightly abnormal. (170) 379 rough-like; eyes large and rough textured. {171) 392 broken; posterior cross-vein broken; variable. (f72) 396 short; fifth longitudinal short. \,,",\. {173) 398 serrate-like; wing margins nicked in several places. ( 174) 403 thick-4; end of fourth longitudinal thickened. { 175) 404 delta-like; all longitudinal veins thickened at ends. {176) 404 small bristle; bristles small; flies weak. {177) 408 thick; anterior and posterior cross-veins thick. {178) 413 extra bristles; extra dorsocentral bristles. (179) 414 broken; posterior cross-vein broken; variable. ( 180) 416 bobbed; bristles in females very small; abdominal tergites irregular· sex-linked and sex-limited. ' {181) 417 scarlet-like; eye color. (182) 420 spread wing; wings widely spread and drooping. (183) 433 sepia-like; eye color. (184) 443 silver-like; body color. (185) 445 dash-vein; small piece of vein near wing tip between L 3 and L 4. (186) 449 rough-like; eyes medium rough. (187) 451 inturned-like; dorsocentrals inturned; hairs on wing margin stand out. (188) 451 small bristle; bristles about half normal length. (189) 454 thin-wing; thin textured wing. (190) 456 eyeless; extreme; eyes absent; head greatly reduced in size; flies sluggish but viable and fertile. (191) 460 rough; eyes reduced in size and rough; uniform expression at a given temperature. (192) 463 split-thorax; longitudinal groove at anterior end of thorax. (193) 464 scarlet-like; found in one male; sex-linked vermilion. (194) 468 polychaete; extra bristles on thorax and head; variable but always some extra bristles near mid-line anterior to dorsocentrals; small.bits of extra vein in wing. (195) 470 serrated; many small notches in wing near distal margin; variable but no normal overlaps. (196) 475 dark eye; eye color, but difficult to classify. (197) 482 orange-like; eye color. (198) 483 scarlet-like; eye color. (199) 484 brown; a translucent brown eye color. (200) 490 thickened; posterior cross-veins thick. (201) 496 spineless-like; bristles very small, some missing. (202) 498 curled.Zike; wings turned up, but posterior scutellars normal. (203) 500 pearly-like; body color. (204) 501 crippled; legs twisted and gnarled. (205) 501 net-like; rather extreme netting of veins. (206) 503 nicked-like; wings notched near distal end. (207) 504 brown-like; eye color. (208) 508 mosaic; light areas in eye. (209) 508 scarlet-like; eye color. (210) 510 rough-blister; eyes rough; blisters on wings. (211) 512 scarlet-like; eye color. (212) 516 humped-like; thorax humped and short; posterior scutellars often erect and wings bent down. (213) 519 rough-like; medium rough eye. (214) 521 stubble-like; bristles short and heavy. (215) 523 rough-like; eye rough near posterior border. (216) 524 speck-like; dark speck at wing axil and darkened sutures. (217) 528 small bristle; bristles reduced in size. (218) 529 de-pigmented; pigment lacking along ventral margin of tergites; stronger expression in males. (219) 530 purple-slant; eyes purple; posterior cross-vein oblique; flies weak. (220) 532 abnormal abdomen; extremely abnormal abdominal tergites in females; character less strong in males. (221) 542 stubble-like; bristles short, straight, and heavy; normal overlaps. (222) 557 semi-lethal; wings not unfolded: flies sinall and weak. (223) 568 spineless-like; bristles very small. (224) 576 chubby-like; flies short and thick-set; cross-veins close together. (225) 581 blurred; spots on thorax not distinct; background color dark. (226) 584 garnet-like; eye color found in several males; sex·linked. (227) 591 short-5; fifth longitudinal vein short. (228) 592 delta-like; heavy deltoid thickening at tips of longitudinal veins; wing margin often incised. (229) 603 abnormal abdomen; extreme expression; very abnormal tergites. (230) 606 scarlet-like; eye color. (231) 609 split-thorax; longitudinal groove at anterior end of thorax. (232) 615 gap-vein; gap in fifth longitudinal near base; fourth and fifth veins close near posterior cross-vein. (233) 623 held-out; wings held out at right angle to body, not drooping. (234) 625 inturned-like; dorsocentrals inturned; hairs on wing margin stand out; males sterile. (235) 633 blister-wing; blisters in wings; good expression. (236) 635 rough-like; medium rough eye. (237) 635 extra-bristles; extra dorsocentrals; variable. (238) 641 abnormal abdomen; medium expression. (239) 642 stubble-like; slightly thickened, stubbly bristles. (240) 644 rolled-like; wings thin textured, crumpled an:l rolle:l under at distal outer margin. (241) 647 dark ere; eye color dark. (242) 647 pads-like; wings not unfolded; flies small and weak. (243) 659 small-eye; eyes slightly smaller than normal. The University of Texas Publication TABLE 3-Continued Mutants recovered from the F2 progeny of 736 pair matings of wild D. mulleri. Single F, mass matings were made up from progeny of each pair of wild flies and numbered consecutively. These stock numbers precede the name of each mutant extracted from the stock. Tenta­ tive names in this table were later changed after further study. Most of the mutants were discarded and names used in this table are to be considered descriptive and not permanent symbols. Short diagnostic descriptions follow the names except in case of eye colors. (244) 663 grooveless-like; groove between scutellum and thorax missing; black excrescences on sides of scutellum. (245 ) 680 cherry-like; eye color. (246) 681 grooveless-like; groove between scutellum and thorax missing. (247) 682 javelin-like; bristles straight; not tapering as in normal; often forked near the end. (248) 686 pearly-like; body color. (249) 687 purple-like; eye color. (250) 690 dumpy-like; body short and chunky; wings truncated. (251) 692 rotated genitalia; in males. which are sterile. (252) 694 pads-like; wings not unfolded; semi-lethal. (253) 695 brown-like; eye color; classify in young flies. (254) 697 scarlet-like; eye colar. (255) 706 stubble-like; bristles short and straight; difficult to classify. (256) 708 abnormal-tarsus; tarsal joints short and often fused; flies weak. (257) 715 semi-lethal; rough eyes; grooveless; tumor at base af last leg. (258) 719 taxi-like; wings held at wide angle and elevated; wings slightly darker than normal. (259) 721 dull; eve color. (260) 722 sepia-like; eye color. (261) 723 scarlet-like; eye colar. (262) 726 brown; translucent brawn eye color. (263) 730 peach-like; eye color; best in young flies. Analysis of the Scarlet-like Eye Colors From the standpoint of population structure the most interesting observation on the mutants found was the repeated occurrence of scarlet-like eye colors. A check of Table 3 will show 35 entries for these, all of which appeared identical in phenotype except 183 scarlet-like, which was a little darker than the others, · more of a cherry color. Some of the scarlet-like mutants were found in only a few flies from the mass mating and no stock was established. A few of the others were discarded without testing. No. 464 scarlet-like was found as a single male and it was assumed that it might be sex-linked vermilion, which it turned out to be. Twenty of the scarlet-like stocks were retained for testing. On the assumption that there might be more than one locus involved 18 of these stocks were mated simultaneously to 189 scarlet-like and 205 scarlet-like, chosen at random. When the F1 progeny began to emerge it became apparent that stocks 189 and 205 were indeed different. Nine of the tested stocks turned out to be alleles or identical to 189, and three of the stocks to be alleles or identical to 205. But, in addition, stock 214, which gave only scarlet-like offspring when cross-tested to 189 also gave about half the progeny scarlet-like when tested to 205. Thus both scarlet-like 189 and 205 were carried in the wild parents of 214. It was, of course, impossible to determine whether both mutants were carried in the same wild fly or whether each parent carried a mutant. The fact that none of the offspring of stock 214, after it was established, were wild-type is, of course, corroborative evidence of the point made earlier that many mutants present are not picked up by the mass mating technique. If, in isolating mutants from 214, some homozygous for 189 and others homozygous for 205 had been used to establish stock 214, then one would have expected some wild-type flies in the next generation. Apparently all of the flies used in establishing this stock were homozygous for 189, but some of them were also heterozygous for 205. In any case it was fortunate that 214 was not chosen as a tester stock. Six of the stocks mated to 189 and 205 gave only wild-type offspring with both testers. One of these, 90 scarlet-like, was chosen as a tester and the other five were mated to it. Four of these proved to be alleles or identical to 90, and as was to be expected, 183 turned out not to be an allele. Thus five bright scarlet-like loci were represented in the 21 stocks tested: 464 vermilion, 90, 189, and 205 all apparently phenotypically identical and 183, a little darker than the others. However, very careful examination of stock 90 showed a long light shadow running across the eye in young flies, which was_ not present in 189 and 205. A stock of 90 could be recognized as distinct from the others, but it would be difficult if not impossible to separate flies from a mixture of the stocks, particularly if they varied in age. If one assumes that the same proportion of the three mutant genes, 90, 189, and 205 were present in the 14 scarlet-like types not tested, and that approximately one-third of the mutants present are picked up by the mass mating technique, then 189 scarlet was present in about 50 of the 2944 genes at this locus in the population sample, a gene frequency of 1.7%. The other two mutants, recovered half as frequently, would each have a gene frequency of about 0.8% in the popu­lation sample. The author (Spencer, 1946) recorded gene frequencies of 10% and 4% respectively for the mutants, stubble bristle and brick eye color, in a population sample of D. immigrans, and found a frequency of 4% for the mu­tant, rotund (Spencer, unpublished), in a population of D. melanogaster. The finding of high gene frequencies for certain mutant genes in Drosophila popu­lations is therefore nothing unique. But it is an amazing coincidence that three Jnimic scarlet-like eye colors should have attained a high frequency simultane­ously in this population. Peach-like eye colors were found 17 times. But several of these were phenotypically different; others proved to be sterile in one or both sexes. It is doubtful if any of the peach-like mutants had attained a frequency as high as the scarlets, although they were not tested as carefully. When the scarlet-like mutants had been analysed markers for the four long autosomes had not yet been determined. On the assumption that the three mu­tants, 90, 189, and 205, might represent homologues of cinnabar, scarlet, and cardinal in elements C, D, and E respectively of D. melanogaster, it was de­cided to run immediate tests on whether these three mutants were on the same or separate chromosomes of D. mulleri. If any two were in the same linkage group then a cross of these two should give a second generation ratio of 1,wild­type: 1 scarlet; but if in different linkage groups the second generation ratio should be 9 wild-type: 7 scarlet. Table 4 gives the results of the cross-tests, indi­cating that the three mutant types were in three different linkage groups. Sub­sequent tests to other markers for the chromosomes have established that 90 scarlet-like is cardinal on the second chromosome, homologue of element E, the right end of chromosome III in melanogaster; 189 scarlet-like is scarlet on the fifth chromosome, homologue of element D, the left end of chromosome III in melanogaster; and 205 scarlet-like is cinnabar on the third chromosome, homo­logue of element C, the right end of chromosome II in melanogaster. The University of Texas Publication TABLE 4 F, counts of cross-tests of the three scarlet-like eye colors, stocks 90, 189, and 205, indicating by the 9: 7 ratios that each mutant lies in a separate chromosome F, of stock 90 X stock 189 Actual Count ·Expecte:l Deviation 9 wild-type 314 317 -3 7 scarlet 249 246 +3 Totals 563 563 F, of stock 90 X stock 205 Actual Count Expected Deviation 9 wild-type 7 scarlet 213 169 210 172 +3 -3 Totals 382 382 F, of stock 189 X stock 205 Actual Count Expected Deviation 9 wild-type 325 338 -13 7 scadet 276 2fi3 +13 Totals 601 601 Visibly Different Alleles in the Population In the population sample several other cases were found in which mutants which were phenotypically identical proved to be actually identical when cross~ tested. Thus 67, 127, and 249 purplish proved identical on test and the same mutant was picked up in later generations from the stock of 256 cross-vein-dome, subseque::itly designated elbow. Other cases proved by test to be identical or in­distinguishable alleles were: 484 and 726 brown; 42 and 295 peach; and 10, 38, and 373 light, a sex-linked body color. However, two phenotypically identical mutants, 47 fringe and 95 fringe, were shown not to be alleles, and in fact subse­quent tests showed them to be on chromosomes III and V respectively. For lack of time many cross-tests were not made between certain similar mutants before they were lost or discarded. It seems probable that several other cases of allelism were present among the mutants extracted. Such sporadic cases of the same mutant appearing more than once in the population sample were to be expected on the basis of the population structure revealed by the scarlet-like eye color analysis. But it was rather surprising to find in this population several cases of ,non­identical alleles. Spencer ( 1944) had shown that populations of D. hyd(Ji carr;ied a large series of distinguishable alleles at the bobbed locus, and other p}leno­typically indistinguishable iso-alleles, which could be seriated by the ~se of special genetic tools. One of the primary considerntions which led to the present study was to discover whether populations of another species in the repleta group also contained such a series of bobbed alleles. As the analysis progressed it soon became apparent that D. mulleri contained no such remarkable allelic series. Bobbed, a sex-linked and sex-limited recessive, is one of the mutants which has been found in many species of Drosophila. In the present analysis two distin­guishable alleles were found'. A medium bobbed, with bristles in females.reduced to a bout half normal length,; turned .up in one ofthe F1 pair matings from ~ulture 15. An extreme bobbed, wit):J. females having very tiny bristles and abnormal abdominal tergites, was found in mass culture 416. The finding of two such alleles in an analysis of the same scope would not be surprising in several Dro­sophila species. It seems apparent that the bobbed case in D. hydei is unique and quite different from the situation in D. mulleri. The other two cases seem more remarkable. No. 120 reinforced is an excellent mutant, in which the margin of the wing at the distal end is thickened. While the mutant is variable in expression there are no normal overlaps. No. 592 delta­like is more extreme, with heavy deltoid expansions at the tips of the longi­tudinal veins often accompanied by notching of the wing margin. The hetero­zygote is identical to reinforced. Thus the allele with weaker phenotypic ex­pression is dominant to the other. No. 170 bent-down is a mutant in which the distal outer margin of the wing is bent down; this mutant shows a few normal overlaps. No. 341 warped-like produces curvatures in both axes of the wing, with no normal overlaps. The heterozygote is identical to bent-down. Again the phenotypically weaker allele is dominant. In general, mutations at any one locus are rare. But certain Drosophila species show frequent mutations at some specific locus. Possibly these two loci in D. mulleri are unusually mutable. It would be of interest to test samples of different populations of this species by crossing the flies to 120 reinforced and to 170 bent-down. DISCUSSION The present analysis has given some information on the structure of the D. mulleri population under study. In the first place the mutants recovered indicate in some degree the hidden genetic variability present in the population. Only a fraction of the visibles present in the sample of 736 pairs of flies were recovered. This sample represented less than one-fifth of the D. mulleri captured during the collecting period. At no time during this period was there any indication that the total population was being trapped out; fluctuations in numbers of flies taken from day to day were rather obviously due to temperature and other environ­mental factors. Apparently the total population was much larger than the sample trapped. Furthermore, a glance at Table 3, which represents roughly a chronological series of mutants extracted from smaller sub-samples taken over the collecting period, indicates that the law of diminishing returns was not operating dras­tically. Obviously certain of the later mutants extracted were identical to some of the earlier ones, but many new mutant types were being found toward the close of the analysis. It seems apparent that the visibles found represent only a fraction, though perhaps a sizeable fraction, of the total number of different visibles present. When one considers that the visibles of the type to be observed by the investigator constitute only a small fraction of the total mutants, lethals, semi-lethals, iso-alleles, and physiological mutants with no apparent morpho­logical effects, the tremendous mutant load or perhaps better the tremendous evolutionary potential may be surmised. Some idea of the variability present in the population may be gained from the following item. The author carefully checked all of the mutant autosomal eye color loci recorded in the monograph on the mutants of D. melanogaster (Bridges and Brehme, 1944) and found that over half this number of separate autosomal eye color loci were represented in The University of Texas Publication those mutants found in this analysis and shown to be non-alleles. Many other eye color mutants were lost before being tested. While in some respects this was the most interesting population sample this author has analysed, it was by no means atypical in regard to total mutation load in comparison to samples of populations of D. melanogaster, simulans, ananassae, hydei, immigrans, and robusta formerly analysed. However, the high gene frequencies of mutants at certain loci in the popula­tion sample, particularly the scarlet, cardinal, and cinnabar loci, deserve some consideration in connection with the problem of the possible breeding structure of this and presumably other populations of this species. Elton (1927) first called attention to the importance of fluctuation in numbers in populations as a potent factor in evolution. In non-mathematical terms he states the matter thus: "Many animals periodically undergo rapid increase with practically no checks at all. In fact the struggle for existence sometimes tends to disappear almost entirely. Dur­ing the expansion in numbers from a minimum almost every animal survives, or at any rate a very high proportion of them do so, and an immeasurably larger number survives than when the population remains constant. If therefore a heritable variation were to occur in the small nucleus of animals left at a mini­mum of numbers, it would spread very quickly and automatically, so that a very large proportion of numbers of individuals would possess it when the species had regained its normal numbers. In this way it would be possible for non-adaptive characters to spread in the population, and we should have a partial explanation of the puzzling facts about closely allied species, and of the existence of so many non-adaptive characters in animals... . Finally, what little we know about the regulation of numbers in animals enables us to say that the problem of the origin of species can only be successfully solved by the aid of work on numbers." In a brilliant series of papers Wright (1931, 1932, 1940, 1942, 1948, and many others) has expressed this idea in mathematical terms, and has discussed its re­lation to many other factors in a comprehensive theory of the mechanism of evolution at the incipient level. The phenomenon, which Wright prefers to term random fluctuation, has also been designated genetic drift or the Sewall Wright effect. On the matter of fluctuating population size Wright (1931) states in his first paper dealing with these matters: "If the population fluctuates greatly, the effective N (population number) is much closer to the minimum number than to the maximum number. If there is a great difference between the number of mature males and females, it is closer to the smaller number than to the larger.... The conditions of random sampling of gametes will seldom be closely approached. The number of surviving offspring left by different parents may vary tremendously either through selection or merely accidental causes, a condi­tion which tends to reduce the effective N far below the actual number of parents or even of grandparents." And again (Wright, 1942) he states: "The effective value of N should often be much smaller than its apparent value. It obviously ap­plies only to individuals that reach maturity. If there is cyclic variation in popu­lation size, N is more closely related to the minimum than to the maximum number. It is also reduced if there is excessive variability in the number of mature offspring from different parents." If one were to base his ideas of population number in D. mulleri in the vicinity of Austin, Texas, on collections made during the season when the populations attain a maximum, the late autumn, he might conclude that N was very large and that genetic drift was a negligible factor. This author made no month by month observations on population size in this species. Fortunately Patterson (1943) and his colleagues had already done so. An examination of the month by month collection records of this group at the Aldrich plot shows that mulleri and aldrichi build up to very large populations in the late autumn and early winter, then decline rapidly to extremely low populations in the late winter and spring, to build up slowly again through the summer. Thus for the one year when col­lections were made every month there were 5,715 mulleri and aldrichi taken out of 15,634 Drosophila collected in October and only 61 of these species out of 13,710 Drosophila collected the following May. In the interim the proportionate number of these two species had dropped even lower. Thus they have established that D. mulleri undergoes tremendous fluctuations in population numbers in an annual period in this area. Presumably there is considerable variation in this fluctuation from year to year, depending upon seasonal rainfall, temperature and other ecological factors. In view of these fluctuations in population size it is not surprising that some particular mutant gene, such as scarlet, had attained a frequency of 1.7% in the population, and others, cinnabar and cardinal, a frequency of 0.8%. Actually the variety of mutant types found is more difficult to understand, and it probably points to a tendency for the mulleri populations in the vicinity of Austin to ap­proach a panmictic· state during peak populations, with much migration into and out of a given population. Of course the particular events which led to the high frequencies of the scarlet-like mutants may have occurred many years previ­ously, and a more or less endemic population might have acquired most of the remaining mutation load subsequently. It should be pointed out that Dr. Wright has always maintained that random fluctuation is only one of many factors involved in the mechanism of evolution, and that over long periods selection, in the final analysis, is the major factor in speciation. It may further be emphasized, as he has done, that selection and genetic drift are not necessarily antithetical. They may on occasion act in the same direction in establishing high gene frequencies and combinations of gene frequencies adaptively favorable to the organism. No attempt will be made here to evaluate the evolutionary role of genetic drift in this or other populations of D. mulleri. The facts seem to indicate that it has played a role in establishing certain high gene frequencies, quite possibly non-adaptive or detrimental, in this particular population. It seems likely that the relative roles of various factors differ in speciation in different species groups and sub-groups in this genus. In those species with few intraspecific inversions in the chromosomes possibly random drift may play a larger significant role; Freer recombinations of genes are possible, so that certain mutant types, possibly with some selective advantage, might reach high frequencies and become fixed with­out the accompanying load of deleterious genes originally present in contiguous chromosome segments. Possibly freer crossing-over, resulting in longer chromo­some maps as found in virilis and hydei and subsequently to be reported in mulleri, is a factor in this mechanism. On the other hand the development of many intraspecific inversions in a species may well lead that species along the pathway of enforced chromosome heterozygosity. This latter mechanism is cer­tainly not found extensively in the repleta group, which is at present represented by more species than any other group in the genus. ACKNOWLEDGMENTS This study was made possible by the generous research leave program insti­tuted by President Howard F. Lowry of the College of Wooster and endorsed by the Board of Trustees of that institution, and by the Genetics Foundation and Department of Zoology of The University of Texas, where excellent laboratory facilities and equipment were provided. The author wishes to thank personally Dr. J. T. Patterson for aid in identifying species and Dr. Wilson Stone and many others for the warm hospitality generously extended during his year of research leave at The University of Texas. REFERENCES Alexander, Mary L. 1949. Note on gene variability in natural populations of Drosophila. Univ. Texas Puhl. 4920:63-69. ----. 1952. Gene variability in the americana-texana-novamexicana complex of the virilis group of Drosophila. Univ, Texas Publ. 5204: 73-105. Bridges, C. B., and K. S. Brehme. 1944. The mutants of Drosophila melanogaster. Carnegie Inst. Wash. Publ. 552. 252 pp. Dobzhansky, Th. 1947. Genetics of natural populations. XIV. A response of certain gene ar­rangements in the third chromosome of Drosophila pseudoobscura to natural selection. Genetics 32: 142-160. Dobzhansky, Th., and Howard Levene. 1948. Genetics of natural populations. XVII. Proof of operation of natural selection in wild populations of Drosophila pseudoobscura. Genetics 33: 537-547. Elton, Charles. 1927. Animal Ecology. Sidgwick and Jackson, Ltd. London. 207 pp. Patterson, J. T. 1943. The Drosophilidae of the Southwest. Univ. Texas Puhl. 4313: 7-214. ----. 1947. The insemination reaction and its bearing on the problem of speciation in the mulleri subgroup. Univ. Texas Puhl. 4720:41-77. Patterson, J. T., and Mary L. Alexander. 1952. Drosophila wheeleri, a new member of the mulleri subgroup. Univ. Texas Publ. 5204: 129-136. Patterson, J. T., and W. S. Stone. 1952. Evolution in the Genus Drosophila. New York. The Macmillan Co. 610 pp. Patterson, J. T. and R. P. Wagner. 1943. Geographical distribution of species of the genus Dro­sophila in the United States and Mexico. Univ. Texas Puhl. 4313:217-281. Patterson, J. T., and Marshall R. Wheeler. 1949. Catalogue of described species belonging to the genus Drosophila, with observations on their geographical distribution. Univ. Texas Puhl. 4920:207-233. Spencer, Warren P. 1944. !so-alleles at the bobbed locus in Drosophila hydei populations. Genetics 29:520-536. ----. 1946. High mutant gene frequencies .in a population of Drosophila immigrans. Ohio J. Sci. 46: 143-151. ----. 1947 a. Mutations in wild populations in Drosophila. Advances in Genetics 1: 359­ 402. New York. Academic Press, Inc. -----. 1947b. Genetic drift in a population of Drosophila immigrans. Evolution 1:103-110. -----. 1949. Gene homologies and the mutants of Drosophila hydei. Genetics, Paleontology, and Evolution: 23-44. Princeton, N. J. Princeton Univ. Press. ----. 1950. Collection and laboratory culture. Biology of Drosophila, Chap. VII: 535-590. New York. John Wiley and Sons, Inc. Stone, Wilson S. 1955. Genetic and chromosomal variability in Drosophila. Cold Spring Harbor Symp. on Quant. Biol. 20:256-270. Sturtevant, A. H. 1921. The North American species of Drosophila. Carneg. Inst. Wash. Puhl. 301. 150 pp. -----. 1942. The classification of the genus Drosophila, with descriptions of nine new species. Univ. Texas Puhl. 4213:5--51. Wagner, R. P. 1944. The nutrition of Drosophila rruulleri and D. aldrichi. Growth of the larvae on cactus extract and the microorganisms found in cactus. Univ. Texas Puhl. 4445 : 104-128. -----. 1949. Nutritional differences in the mulleri group. Univ. Texas Puhl. 4920:39-41. Warters, Mary. 1944. Chromosomal aberrations in wild populations of Drosophila. Univ. Texas Puhl. 4445:129-174. Wasserman, Marvin. 1954a. Cytological studies of the repleta group. Univ. Texas Puhl. 5422: 130-152. ----. 1954b. Population studies with Drosophila mulleri. Univ. Texas Puhl. 5422:166-188. Wharton, L. T. 19'42. Analysis of the repleta group of Drosophila. Univ. Texas Puhl. 4228:23-52. Wheeler, Marshall R. 1954. Taxonomic studies of American Drosophilidae. Univ. Texas Puhl. 5422: 47-ti4. Wright, Sewall. 1931. Evolution in Mendelian populations. Genetics 16:97-159. -----. 1932. The roles of mutation, inbreeding, cross-breeding, and selection in evolution. Proc. 6th lnternat. Congress of Genetics 1: 356-366. -----. 1940. Breeding structure of populations in relation to speciation. Amer. Nat. 74: 232-248. -----. 1942. Statistical genetics and evolution. Bulletin Amer. Math. Soc. 48:223-246. -----. 1948. On the roles of directed and random changes in gene frequency in the genetics of populations. Evolution 2:279-294. XV. Genetic Studies on Drosophila mulleri. II. Linkage Maps of the X and Chromosome II with Special Reference to Gene and Chromosome Homologies 1 WARREN P. SPENCER INTRODUCTION While some sporadic observations on gene homologies in the early work on the genetics of Drosophila species had appeared, Dr. A. H. Sturtevant made the first significant contribution in this field in a series of papers on the mutants of Drosophila simulans, culminating in a monograph (Sturtevant, 1929) on the ge­netics of this species. At that time the only case of species hybridization known in the genus Drosophila was the cross of D. simulans to D. melanogaster, which gave sterile hybrids. Sturtevant demonstrated that 14 recessive mutant types in the X of simµlans were actual homologues or alleles of similar mutants in me­lanogaster by making the appropriate crosses and observing the recessive char­acter in the sterile female hybrid. He also established homologies for three re­cessive mutants in chromosome II and five in chromosome III. This work remains the most significant contribution to the study of gene homologies in the genus. It should be noted that Sturtevant was able to predict with a high degree of ac­curacy which mutants would behave as alleles or homologues. Sturtevant and Novitski ( 1941) published a comprehensive report of gene and chromosome homologies in Drosophila species, summarizing the data available up to that time. For anyone interested in the subject this report should serve as necessary background material. They present convincing evidence that the chromosome elements of D. melanogaster, X, left and right limbs of chromosome II, left and right limbs of chromosome III, and the dot-like chromosome IV have their homologous chromosomes and/ or chromosome limbs, as revealed by homo­logous mutant genes, in the species simulans, affinis, algonquin, ananassa.e, azteca, miranda, pseudoobscura, montium and virilis. This author (Spencer, 1949) discussed gene and chromosome homologies in D. hydei, comparing certain mutant genes found in this species with those in me­lanogaster, which still serves as a base of reference for such studies. He has also considered the criteria and pitfalls to be avoided in establishing homologies, particularly in the case of autosomal mutants. In a significant paper dealing with the comparative cytology of the salivary chromosome of species in the repleta group Wasserman ( 1954) reported on the sequence of the banding pattern in these choromsomes. He studied two species in the melanopalpa subgroup, one of which was D. repleta, the type species of the group, two species in the mercatorum subgroup, two species in the hydei sub­ group, including D. hydei, and 12 species in the mulleri subgroup. In this thor­ ough cytological analysis Wasserman made a study of the interspecific inversions which had occurred in the course of evolution and worked out the chromosome phylogeny for the X and second chromosomes of the species studied. He pre­ 1 Dept. Biology, College of Wooster, Wooster, Ohio. sented an interesting discussion of the possible course of explosive evolution in the repl.eta group. As Wasserman points out, in the absence of paleontological evidence, these hypotheses remain highly interesting though not conclusive speculations. Of unusual interest was the fact that Wasserman found that certain chromo­somes and parts of chromosomes had retained their identical pattern of banding in certain species which on morphological and ecological grounds had diverged to a point where they are classified in different subgroups. For instance his analy­sis indicated an identical banding pattern in the X of hydei and mulleri even though they belong to different subgroups. As the author (Spencer, 1949) had ac­cumulated a considerable body of data on the genetic maps of D. hydei, particu­larly the X, and as he had available a large number of mutant types (Spencer, this bulletin) in D. mulleri, it seemed only logical to undertake a comparative genetic study of these two species, which might conceivably supplement the cytological work of Wasserman. The present paper is a preliminary report on the linkage maps and homologous gene loci in D. mulleri as compared to hydei and melanogaster. THE X CHROMOSOME The Mutan.t Loci and Alleles During the course of analyzing the population sample for hidden recessive autosomal mutants and in the subsequent linkage tests on these mutants, several . sex-linked recessives have been found. Of these all are obvious homologues of sex-linked mutants known in melanogaster, with the possible exception of carna­tion eye color and sable body color. In the following section each mutant is given a name, and a symbol corresponding to that used for its homologue in melano­gaster; these are followed by the record of the origin of the mutant and a short description. The mutants are arranged in the order of their loci in the X chromo­some of mulleri. No locus number is assigned in the description because this will undoubtedly change with the discovery of new mutants and the accumulation of linkage data on some of the longer as yet unmapped regions. However, the tem­porary locus number assigned to each mutant may be ascertained by reference to the X chromosome map, Figure 1. vesiculated (vs). Several males in a stock of the sex-linked mutant, light body color. Wings very abnormal, blistered, often shriveled, and divergent; sometimes shows in only one wing, and some normal overlaps. When character is strongly expressed the flies are small and weak. vermilion (v) . One male in F2 mass culture from wild pair 464. Evidently arose as a mutant in the laboratory. Brilliant scarlet-like eye color. In the double recessive, vermilion-brown III, the eye color is white. white-ivory (w1). One male from the F2 of a cross of rudimentary male x forked-light female. This male was light-rudimentary. Eye color very light yel­lowish; vermilion-white-ivory is white in phenotype and easily distinguished from white-ivory alone. light (Ii). Recovered three times from wild pairs 10, 38 and 373. Apparently carried in one of the two flies of each pair. As light in hydei is an allele or pseudo­ FIGURE I. 0.0 vesiculated ­ -- - - - flared 0.0 7.3 vermilion ­ -- --­--­ --­ vermilion 7. 4 ___ scute 15.6 - - - Extra scutellor 16.5 -­-white 19.0 20.2 white - - - - -- - Notch 22.3 - - - lethal 25.4 __ -lethal 31.2 ---singed 31.4 --­ garnet 32.5 --- sable 35.8 :::: - - yellow 38.8 - - light 39.0 44.3 light-­- - - ---tiny wing 47.3 ---scaly 47.4 _ - - dusky 59.4 - - - miniature 59.6 69.0 rudimentary­-- ---cut 77.3 - - -rusty 88.9 - - -russett 94.5 - - -jauntex 112.0 113.9 forked-­- -- _ - -cherry 115. 7 ' - - - bobbed 116.0 I I 126.4 sable ­ - - - - I I I I I I I I I I I 148.6 carnation ­ - - - - I I I I I I I I I 167.8 lozenge ­----bobbed----­ I I FIG. 1. Genetic maps of X-chromosomes of D. mulleri (left) and D. hydei. allele of yellow, the same probably holds for mulleri, although no yellow mutant has been found as yet in this species. Spots of dark pigment on thorax much re­duced in size; heterozygous females intermediate. An obvious homologue of light in hydei and repleta. rudimentary (r). Many males in second chromosome linkage experiment in­volving cardinal-depigmented-polychaete-two joint. Wings often truncated to one-half to two-thirds normal length. Gaps and irregularities in wing-veins. Ap­parently temperature sensitive with some flies showing wings of almost normal length, but these can always be classified easily by the sparse and irregular ar­rangement of hairs on the outer wing margin. Females almost completely sterile. forked (£). From wild stock 253 as several males. Head and thoracic bristles slightly gnarled or twisted. Variable but with no normal overlaps. Normal via­bility and fertility in both sexes. extreme forked (£•). Recovered repeatedly from forked stock. Forked seems to mutate to extreme forked roughly about 1: 1000, but this should be given further study. Apparently extreme forked is stable, not mutating back to the slighter allele. All bristles and hairs extremely gnarled and twisted as in some singed alleles in melanogaster. Flies easily mired but both sexes fertile. carnation (car). Four males in F2 of wild pair 584. Apparently arose in this laboratory stock. Dark, translucent eye color as in mutant of this name in melano­gaster; females somewhat infertile. This, of course, may be a homologue of one of the other sex-linked eye colors in melanogaster. lozenge (lz). Several males in a cross of stock 120 reinforced x stock 372 thickened. Eyes lozenge shaped, with many facets tending to be fused, giving eye surface a very rough appearance in some areas and a glassy texture in others. Abnormal pigment distribution in the eye. Lozenge-vermilion has little pigment in the eye. No terminal tarsal claws; females almost sterile and completely so with lozenge males. bobbed (bb) . From an F, pair mating from wild pair 15. Medium bobbed allele. Bristles reduced in size in females but abdominal tergites normal. Males do not show the character. bobbed2 (bb2). An extreme bobbed allele recovered from F2 of wild pair 416. In females bristles much reduced in size and abdominal tergites abnormal. In addition to the above mutants, all of which have been established in stock and used in linkage studies, a single miniature or dusky male was found, but died without giving offspring. A dusky-like mosaic with one short dark wing and a singed or forked like mosaic failed to give any mutant offspring when flies from them were inbred. They may have been somatic mutations not affecting the germ cells. The Map of the X Chromosome InTable 1 all linkage data on sex-linked genes are summarized and from these data a genetic map of the X chromosome was constructed. Figure 1 shows the genetic maps of the X chromosomes of D. mulleri and D. hydei drawn to the same scale. The map of the hydei Xis identical to that published by Spencer (1949) except for the position of dusky which in the earlier map was placed about four units away from miniature. A large scale miniature-dusky linkage experiment TABLE 1 Summary of all linkage data on the X-chromosome of D. mulleri. . Those items in the table preceded by a number in parenthesis are the items actually used m mapping the chromosome, and the numbers refer to the successive map region, beginning at the left or 0.0 end of the map. Loci Total Flies Crossovers Per-cent carnation-forked 540 162 30.00 carnation-light 3181 1587 49.89 (7) carnation-sable 2906 646 22.20 carnation-vermilion 2106 1084 51.40 forked-light 3900 1889 48.44 forked-lozenge 5209 2261 43.41 (5) forked-rudimentary 635 285 44.88 (6) forked-sable 2003 250 12.48 forked-vermilion 3277 1511 4'6.11 forked-vesiculated 563 276 49.00 light-lozenge 5439 2789 51.09 (4) light-rudimentary 1215 301 24.77 light-sable 1421 651 45.81 light-vermilion 3372 1073 31.82 light-vesiculated 82 24 29.30 (3) light-white 249 60 24. 10 (8) lozenge-sable 3186 1320 41.40 lozenge-vermilion 3951 1976 50.03 rudimentary-vermilion 580 238 41.04 rudimentary-white 249 102 40.96 sable-vermilion 4046 1940 47.90 sable-vesiculated 136 58 42.70 ( 1) vermilion-vesiculated 82 6 7.32 (2) vermilion-white 249 32 12.85 Total Linkage Determinations-48,577. with marker genes on either side showed that dusky lies about .2 of a map unit to the left of miniature. With the author's permission Stone (1955) published a preliminary report of the work on D. mulleri and included a tentative map of the mulleri X based on the fragmentary data then available and compared with the hydei X. This map placed lozenge at the 0.0 locus, followed in order by carnation, forked, vermilion, light, and bobbed. This order was based on a recombination value of about 32% between forked and vermilion in a small experiment. The true recombination value for forked-vermilion is close to 50%. Our table shows 46.11 %, but this is almost certainly low, due to the inclusion of one set of data with an abnormally low value. One experiment in 1865 flies gave a value of 49.17%. With the recent discovery of rudimentary, which linkage tests show to lie be­tween light and forked, it became apparent that the genes earlier mapped at the left end of the X actually lie in reversed order far to the right of light. This im­mediately cleared up what had seemed a puzzling situation. Early in this work the author crossed lozenge males to bobbed females, and from the F2 selected lozenge males and mated them singly each to several lozenge/ bobbed females. If lozenge and bobbed were at opposite ends of the chromosome presumably several of these males would be genetically .bobbed. But of the 12 males which gave progeny apparently none carried bobbed, for no bobbed females appeared among their daughters. It is obvious that lozenge lies close to bobbed, perhaps less than 10 units away. Inspection of crossover values in the table may not be convincing for the plac­ing of vesiculated to the left of vermilion, but in the one small experiment in­cluding vermilion, vesiculated, and light the results showed this to be the position of vesiculated. The present map of the mulleri X is almost 170 units long, and yet it includes two regions where the contiguous genes used in mapping show recombination values of over 40%. Eventually the total map length from vesicu­lated to bobbed should be considerably more than 170 units. Actually the re­combination value for carnation and lozenge remains to be determined and will undoubtedly result in lengthening the present map. Chino (1936) has published the genetic maps of the virilis chromosomes. The X in this species is 170.5 units in length, based on 35 loci with no unmapped regions. longer than 15 units. It seems almost certain that the mulleri map will eventually be longer than that of any species yet investigated. On the hydei map furnished Stone for publication (Stone, 1955) there is one glaring error. The carnation locus, 50 units to the left of flared on that map is a spurious locus, and should be deleted as we have in the present map. On finding a wild hydei male showing an eye color the author assumed that it was sex-linked and set up a curious sequence of matings testing the linkage to mutants all along the X, in no case making a mating which would show that the mutant was a sex­linked recessive or was not. For instance the original stock was established by mating the male to stock hydei females and then remating him to his daughters. Mr. Thomas Gregg, a former student of the author, and doing graduate work at Texas, discovered the error and passed the word along by messenger. The case is doubly embarrassing because, having gotten rid of the left end of the mulleri X by swinging it in to the right end, it would seem that the author had to get rid of a long section at the left end of the hydei X in order to present a consistent story. As the hydei map now stands it will probably not grow much in length be­ tween the two ends now mapped, for there are no long regions as yet unmapped as in the mulleri X. Certain comparisons between the X chromosome maps of the two species will be made following the presentation of data on chromosome II. CHROMOSOME II The Mutant Loci Of the autosomal recessives extracted from the D. mulleri population many were discarded as not suitable for further study; some others were accidentally lost. Of those remaining a few seemed clearly to be homologues of mutants al­ ready known in D. hydei and D. melanogaster. Certain of these were first used in cross-tests to each other and later to other mutants. With over 250 of these cross­ tests in addition to the series of tests on the scarlet-like eye colors (Spencer, this bulletin) five autosomal linkage groups were established, as one would have pre­ dicted from the chromosome number in the species. Table 2 presents the list of mutants localized to each chromosome. Those clearly homologues of mutants in melanogaster, hydei and virilis are listed separately. It should be noted that many of the mutants in the lower list, particularly eye colors, are probable homologues of mutants known in D. melanogaster. We are using here the same numbering of the linkage groups as that used by TABLE 2 List of D. mulleri mutants located in the five autosomes. The homologous chromosome element of D. melanogaster is in parenthesis after each chromosome number of m ulleri. The numbers preceding each mutant are numbers of wild stocks from which they were derived. II (E-3R) 90 cardinal 262 curled 42peach 468 polychaete 460 rough 144 sepiaoid 719 taxi Mutants recognized as homologues in other species III (C-2R) IV (B-2L) V (D-3L) 484 brown 276 dachsous 663 ascute 205 cinnabar 256 elbow 95 fringe V 298 lanceolate 623 held out 120 inturned 644 rolled 682 javelin 470 serrated 276 rotated 189 scarlet 433 sepia VI (F-4) 303 Extension 456 eyeless Mutants not clearly recognized as homologues 61 brown-like 42 cup wing 170 bentdown 158 brown-like 529 depigmented 339 small bristled 37 dark eye 47 fringe III 303 dark brown 224 grooveless 31 brown-like 592 delta-like 160 two joint 482orange-like 26a narrow w. 135 dumpy-like 109 peach-like 341 warped-like 272 peach-like 127 purple-like 730 peach-like 454 thin wing 259 sparse 183 scarlet-like Spencer (1949) for hydei. The same system has been used by Weinberg (1954) for the linkage groups of D. macrospina. It should be noted, however, that Chino's (1936) chromosome III in D. virilis corresponds to the mulleri chromosome V, and chromome V of virilis is homologous to chromosome III of mulleri. Chromo­somes II, IV and VI are the same in mulleri and virilis. For other chromosome homologies consult Sturtevant and Novitski ( 1941). We shall confine further discussion of autosomal linkage to chromosome II, for which the linkage map has been worked out, and report later on the other autosomes on the completion of work now in progress. The work has progressed to a point where it should be possible to identify the linkage groups with the sali­vary chromosomes in D. mulleri and D. hydei. Unfortunately the translocation stocks have not been prepared and tested to date. But the evidence strongly favors linkage group II as corresponding to the longest salivary autosome which has been designated II. Thus Warters (1944) reported finding a heterozygous in­version in salivary chromosome II of D. hydei, widespread in wild stocks, and no other inversions in this species. Certain aberrant results of the author (Spencer, unpublished) on linkage in group II of D. hydei are best explained on the basis of a heterozygous inversion in this chromosome. In describing the mutants of chromosome II the same system will be followed as for the X chromosome, the order being determined by the position of the mu­tant in the chromosome, starting with two joint at the 0.0 end of the chromosome map, Figure 2. This map was constructed on the basis of the linkage data pre­sented in Table 3. It should be noted that all loci have been located on the basis of cross-over values between contiguous genes except for the right end locus, sepiaoid. Only one set of data for sepiaoid cross-overs is available, the cardinal­sepiaoid data. Sepiaoid cannot be worked accurately with rough, a small rough eye. So it was planned to run a sepiaoid-rough crossover experiment on a cardinal background. Cardinal-sepiaoid can easily be distinguished from cardinal in rough Studies in the Genetics of Drosophila FIGURE 2. 0.0 ------­ 17.73 ---­19.23---­ 55.48 _______ two-joint polychaete small bristle taxi curled 73.28---­ 100.16 ---­ 147.53 ---­ 158.57---­ 172. 27 --­ ---peach ---rough ---cardinal ---depigmented ---sepiaoid FIG. Z. Genetic map of chromosome II in D. mulleri. The University of Texas Publication TABLE 3 Surrunary of all linkage data on chromosome II of D. mulleri. Those items in the table preceded by a number in parenthesis are the items actually used in mapping the chromosome, and the numbers refer to the successive map regions, beginning at the left or 0.0 end of the map. Loci Total Flies Crossovers Per-cent cardinal-curled 4135 2031 49.12 (7) cardinal-depigmented 951 105 11.04 cardinal-polychaete 910 473 51.98 (6) cardinal-rough 4135 1959 47.37 (8) cardinal-sepiaoid 861 213 24.74 cardinal-taxi 678 328 48.41 cardinal-two joint 910 470 51.65 curled-depigmented 497 269 54.10 (4) curled-peach 1388 247 17.80 (3) curled-polychaete 4342 1639 37.75 curled-rough 885•6 3088 34.87 curled-small bristle 1292 449 34.75 curled-two joint 4754 2096 44.09 depigmented-peach 954 462 48.42 depigmented-polychaete 497 250 50.30 depigmented-rough 1494 730 48.86 depigmented-small bristle 1384 671 48.48 depigmented-two joint 1494 744 49.80 peach-polychaete 2477 1139 45.98 (5) peach-rough 3572 960 26.88 peach-small bristle 1955 801 40.97 peach-two joint 1388 663 47.77 polychaete-rough 2954 1352 45.80 (2) polychaete-small bristle 467 7 1.50 (1) polychaete-two joint 5719 1014 17.73 rough-small bristle 1292 594 45.98 rough-two joint 3696 1756 47.51 small bristle-two joint 875 179 20.46 Total Linkage Determinations-63,927 eyes. Cardinal-sepiaoid was crossed to cardinal-rough and the ease with which · rough-sepiaoid cross-over chromosomes were picked up in the F3, as compared to the difficulty in picking up depigmented-sepiaoid cross-over chromosomes, leaves no doubt that sepiaoid lies to the right of depigmented and is the terminal gene at the right end of the chromosome as thus far mapped. Eventual data on de­pigmented sepiaoid will probably move sepiaoid a little further to the right. Cer­tainly if new mutants are picked up, lying between rough and cardinal, the total map length will be considerably ·greater. As for the X chromosome, the tempo­rary locus number of each second chromosome gene may be found by consulting the map, Figure 2. Description of the mutants follows. two-joint (tj) . From stock 160. Of the five tarsal joints all but the proximal and distal are removed from all legs. Flies are sluggish but show close to normal viability and fertility. However, two-joint flies from moldy cultures are never able to clean mold spores off their bodies and special precautions should be taken to prevent mold by frequent transfer of flies to fresh medium. Dr. J. D. Kriv­shenko (oral communication) states that he has the same mutant in D. busckii. It is otherwise unknown in any Drosophila species. polychaete (pc). From stock 468. There may be extra bristles in different bris­tle groups on head and thorax; but there are always two or more well defined presutural bristles in the acrostichal rows next to the outer ones as found in wild D. putrida and D. testacea. In well nourished flies there are also bits of un­attached venation near the distal end of the wing between L 2 and L 3. An al­most certain homologue is known in linkage group II in D. hydei, showing both the bristle and venation characters. small bristle (sb). From stock 339. Bristles about half normal length. Flies small and late emerging. This character masks polychaete in some flies, the pre­ suturals in polychaete-small bristle being reduced to size of acrostichal hairs. In running linkage tests on polychaete-small bristle only non small bristle flies were classified. This procedure, which eliminates one of the reciprocal cross-over and non cross-over classes is necessary where there is epistasis. taxi (tx). From stock 719. Wings generally held out almost at right angles to the long axis of the body; tend to be convex dorsally. Sometimes the wings are not so spread, but in these flies recognition is possible by the slightly smaller, narrower wings which are dusky. The locus is very close to curled. In several large scale experiments no cross-over chromosome bearing the two mutant loci has been recovered. An almost certain homologue of taxi in melanogaster and hydei. curled (cu). From stock 262. Wings strongly upcurved and slightly divergent. Post-scutellar bristles stand erect and cross at a sharp angle. Flies slightly darker than wild-type. An excellent homologue of curled in melanogaster and simulans. peach (p). In stocks 42 and 295. A pinkish peach eye color, easily classified. Darkens with age. Probably a homologue of similar mutants in melanogaster and hydei. rough ( ro). In stock 460. Eye texture very rough with facets disarranged. Eye size reduced depending on temperature. At 20° C. the eyes are very small with few facets. At 30° C. the eyes approach normal size but are rough. A good homologue of mutants in melanogaster and hydei. cardinal (cd). In stock 90 and four others. A bright scarlet color in young flies, and with a long, light shadow across the eye. Darkens rather rapidly with age so that it is difficult to classify in old flies. Homozygous cardinal-brownlll is a light lemon color in newly emerged flies, and cinnabar-brownlll is about the same color; whereas vermilion-brownlll is white, and scarlet-brownlll is almost white. Cardinal is a good homologue of cardinal in melanogaster and hydei. depigmented (dp). In stock 529. In wild-type mulleri the lower apical corners of the tergites of the abdomen show small dark gray triangular areas, very well defined. In depigmented most or all of this dark pigment is absent. The character · is, however, more extreme in males and somewhat variable in females. For this reason only males were used in the linkage counts. No homologue known, but mutant resembles wild-type pigmentation in some other species of the mulleri subgroup. sepiaoid (sed). In stock 144. A rich, dark purplish-sepia color. Probable homo­ logue of sepiaoid in melanogaster. brown-like. In stock 61. A dull brown eye color, best classified in young flies. The mutant was lost before its locus in the chromosome could be determined. It is therefore not given a symbol. The Map of Chromosome II The map of chromosome II presented in Figure 2 and based on the linkage data in Table 3 is 172.27 units in length. When and if mutants are found be­tween small bristle and curled, and between rough and cardinal many units will undoubtedly be added to the present map. Chino (1936) published the linkage maps of D. virilis. Chromosome II was 210.5 units long. Since then he has sent in several records to Drosophila Information Service with new mutant loci mapped. His last list (Chino, 1940) in D. I. S. includes loci at 256.6 and 257.6 in chromo­some II. This is at present the longest map reported in any species of Drosophila. Significantly, however, no new loci have been added to lengthen the map of the X in this species, 170.5 units as published by Chino (1936). As our map of the X in mulleri is almost this long, with long unmapped regions, it seems likely that the mulleri autosomes will also turn out to give linkage maps as long or longer than those in virilis. Our map of choromosome II is based on the location of 10 genes. Chino's 1936 map of virilis II, 210.5 units long, is based on the location of 21 genes. As the ratio of length of the map of the X to length of the maps of the autosomes seems to hold fairly well from species to species (with the obvious ex­ception of such species as pseudoobscura, where an extra limb has been added to the X) this seems to be a reasonable hypothesis. At least we would predict that the mulleri autosomes will be a bout as long or even longer than the virilis autosomes. DISCUSSION AND CONCLUSIONS As stated in the introduction it was our purpose to make a comparative study of the genetics of the two species, D. mulleri and D. hydei, as a supplement to the cytological work of Wasserman ( 1954) . An inspection of the linkage maps shown in Figure 1 reveals this interesting point. The mulleri X is definitely longer than the hydei X. This is no spurious conclusion based on sampling errors. There is a real difference, mulleri 167.2 units, hydei 116 units. And the pre­sumption is, based on long unmapped regions, that the discrepancy in length will be greater, not less, with accumulation of more data. Our data on the linkage maps of the hydei autosomes, which are not being published here, but which are based on the location of about as many genes per autosome as for chromosome II of mulleri, show hydei chromosome II, 103 units, chromosome III, 127 units, chromosome V, 100 units of map distance. These figures are certainly tentative, but they do indicate, on the basis of the mulleri II map, that the autosomes in hydei give considerably shorter linkage maps than in mulleri. We are forced to conclude that identity in banding pattern of the salivary chromosomes in two species does not lead to identity in recom­bination values. Of more interest is the matter of the order of the gene loci in two rather dis­tantly related species, hydei and mulleri, which have retained the same banding pattern in the X. It will be noted that the four homologous gene loci, vermilion, white, light, and bobbed, are in the same order in the two species and spaced about as would be expected on the basis of freer crossing-over in the mulleri X. But there is a mutant type, sable, certainly as good a parallel or suspected homo­logue as one could wish on morphological grounds, which does not fit into the Studies in the Genetics of Drosophila picture. Sable in hydei is between white and light, while in mulleri it lies be­tween light and bobbed and closer to bobbed. We shall therefore suspend judg­ment on the matter of the gene order in the two species until more homologous loci can be compared. Of course there are two mimic loci, miniature and dusky, which have remained close together in all Drosophila species in which both have been found. There are also two other mimic loci, forked and singed, which have shown very different relative positions in the few species where both have been found. It is interesting but perhaps idle to speculate at this point on whether there are two mimic sable loci. We conclude that the evidence is fair, though not incontrovertible, that the gene order has remained essentially the same in the X chromosomes of these two species, but that recombination values have changed in one or the other or both. ACKNOWLEDGMENTS The author wishes to thank particularly Mr. James Jolliff. who made many of the linkage determinations on the X chromosome loci. He also thanks Mr. Thomas Gregg for calling his attention to the mode of inheritance of the mutant termed carnation in D. hydei. Both to the College of Wooster, which made a year's research leave possible, and to the ,staff of the Zoology Department of The University of Texas, who provided ample facilities and warm hospitality, go his profound thanks. The latter part of the work was supported in part by a grant from the Wilson Fund, and he wishes to thank Dr. Robert Wilson for making this fund available. REFERENCES Chino, Mitsushige. 1936. The genetics of Drosophila virilis. Jap. Journ. Genet. 12: 189-210. ----.194-0. Mutants of Drosophila virilis. D. I. S. 13:62-63. Spencer, Warren P. 1949. Gene homologies and the mutants of Drosophila hydei. Genetics, Paleontology, and Evolution: 23-44. Princeton, N. J. Princeton Univ. Press. ----. 1957. Genetic studies on Drosophila mulleri. I. The genetic analysis of a population (this bulletin). · Stone, Wilson S. 1955. Genetic and chromosomal variability in Drosophila. Cold Spring Harb~r Symp. on Quant. Biol. 20:256-270. Sturtevant, A. H . 1929. The genetics of Drosophila simulans. Carneg. Inst. Wash. Puhl. 399: 1-62. Sturtevant, A. H ., and E. Novitski. 1941. The homologies of the chromosome elements in the genus Drosophila. Genetics 26: 517-541. Warters, Mary. 1944. Chromosomal aberrations in wild populations of Drosophila. Univ. Texas Puhl. 3335: 129-174. Wasserman, Marvin. 1954. Cytological studies of the repleta group. Univ. Texas Puhl. 5422: 130-152. Weinberg, Roger. 1954. The chromosomes of Drosophila macrospina and comparisons of the chromosome elements with other species. Univ. Texas Puhl. 5422: 153-162. XVI. Relationships between Species Groups of the Subgenus Pholadoris, Drosophila (Diptera: Drosophilidae) WHARTON B. MATHER1 INTRODUCTION The subgenus Pholadoris has been divided into five species groups on morpho­logical grounds (Mather 1955): victoria, coracina, maculosa, mirim and levis groups. Further, four species of the coracina group, Drosophila cancellata Math­er, D. lativittata Malloch, D. enigma Malloch and D. novopaca Mather have been shown to form an interbreeding network (Mather, 1956). The purpose of the present work was to test further the biological reality of these species groups by attempted hybridization, and if possible to throw light on the biological re­lationships of the groups. The species groups established by Burla (1954) have not been considered here, since stocks of the species concerned were not available. MATERIALS AND METHODS The stocks used in this study are listed in Table 1. The methods used are those previously described (Mather 1955, 1956). Ten pair matings both ways, changed weekly, were run in vials for one month and replicated 10 times. TABLE 1 Stocks of Pholadoris species used in this investigation. Species group Species Stock No. Origin victoria victoria 1865.3 Utah corac1na cancellata Z372.1 Moggill, Queensland coracina enigma 2372.3 Moggill, Queensland coracina lativittata 2372.4 Moggill, Queensland coracma novopaca 2372.7 Noosa, Queensland maculosa novamaculosa 2372.6 Moggill, Queensland m1nm latifasciaeformis 1975.4 Brazil levis bryani 2372.5 Maroochydore, Queensland RESULTS AND DISCUSSION The interspecific crosses attempted are set out in Table 2. Unfortunately D. coracina Kikkawa and Peng, the fifth member of the coracina species group, was not available for these tests but its biological relationships will be reported on in a subsequent publication. No tests have been carried out between the levis and mirim groups on the one hand and the victoria, maculosa and coracina groups on the other due to their considerable morphological divergence. D. novopaca is the one member of the coracina group which will cross with the other three available species of the group; therefore crossing was attempted be­tween this species and D. victoria Sturtevant in the related victoria group. Present address: Zoology Dept., University of Queensland, Australia. TABLE 2 Attempted crosses between species of the subgenus Pholadoris. Attempted Sexual Insemination cross Isolation• reaction victoria (; X novopaca 'i' victoria 'i' X novopaca (; victoria (; X novamaculosa 'i' victoria 'i' X novamaculosa (; cancellata (; X novamaculosa 'i' cancellata 'i' X novamaculosa o enigma (; X novamaculosa 'i' enigma 'i' X novamaculosa (; lativittata (; X novamaculosa 'i' lativittata 'i' X novamaculosa (; novopaca (; X novamaculosa 'i' novopaca 'i' X novamaculosa (; latifasciaeformis (; X bryani 'i' latifasciaeformis 'i' X bryani (; 15/15 23/23 40/ 42 31/31 13/13 13/13 19/ 19 15/ 16 15/ 15 15/ 15 13/ 13 15/15 618 24/24 present absent absent • Fraction of females dissected which did not show sperm in their reproductive tracts after exposure to males for one month. Crosses were also attempted between D. novamaculosa Mather and D. cancel­lata, D. lativittata and D. novopaca with which the former species is sympatric. The failure of hybridization between the species tested lends support to the erection of the groups on morphological grounds. However, there is a lack of complete sexual isolation between D. bryani Malloch and D. latifasciaeformis Duda, between D. enigma and D. novamaculosa, and between D. victoria and D. novamaculosa; as is shown in Table 2, there were a few cases of insemination in crosses involving these pairs of species. Thus a close biological relationship existing between the mirim and levis groups on the one hand, and the maculosa, ·coracina and victoria groups on the other is indicated. An insemination reaction in the two cases where victoria <; <; mated with novamaculosa 'i' 'i' is interesting since it has been shown that in intraspecific matings of victoria there is a strong homogamic insemination reaction (Wheeler, 1947). On the other hand, in the two cases of mating between latifasciaeformis <; <; and bryani 'i' 'i' , and the single case of mating between enigma 'i' 'i' and novamaculosa <; <; ,an insemination reaction was not observed. However, noth­ing is known of the intraspecific insemination reactions in these species. SUMMARY The failure of hybridization between species groups of the subgenus Phola­doris established on morphological grounds supports the biological reality of these groups. However, sexual isolation is incomplete between the levis and mirim groups, between the maculosa and coracina groups, and between the maculosa and victoria groups, indicating the close biological relationships of the cross mating groups. ACKNOWLEDGMENTS Grateful acknowledgment is made to Professor Wilson S. Stone of the Genetics Foundation University of Texas where this work was carried out while the ' ' author was on sabbatical leave from the University of Queensland, Australia. REFERENCES Burla, H. 1954. Zur Kenntnis der Drosophiliden der Elfenbeinkiiste. Rev. Suisse Zool. 61: 1-218. Mather, W. B. 1955. The Genus Drosophila (Diptera) in Eastern Queensland. I. Taxonomy. Austr. Jour. Zool. 3:545-582. -----. 1956. The Genus Drosophila (Diptera) in Eastern Queensland. IV. The Hybridi­zation Relationships of four species of the Pholadoris subgenus. Austr. Jour. Zool. 4:90-97. Wheeler, M. R. 1947. The insemination reaction in intraspecific matings of Drosophila. Univ. Texas Publ. 4720:78-115. XVII. Genetic Relationships of Four Drosophila Species from Australia (Diptera: Drosophilidae) WHARTON B. MATHER1 INTRODUCTION Drosophila serrata Malloch, D. pseudotakahashii, sp. nov., D. bryani Malloch and D. buzzatii Patterson and Wheeler from Australia are not closely related morphologically to any other species so far recorded from this area (Mather 1955) . By breeding with conspecific strains or with the most closely related ipecies, morphologically, from other parts of the world, information on the phylogeny of these species occurring in Australia has been investigated. MATERIAL AND METHODS The stocks used were those indicated in Table 1. The methods used were simi- TABLE 1 Stocks utilized in this study. Species Stock No. Origin serrata 2372.8 Greenslopes, Queensland kikkawai 2363.3 Katmandu, Nepal pseudotakahashii 2372.9 Samford, Queensland takahashii 2363.4 Katmandu, Nepal bryani 2372.5 Maroochydore, Queensland bryani 2370.10 M ajuro, M arshall Islands buzzatii 2372.10 Moggill, Queensland buzzatii 190 Carpentaria, Italy buzzatii 2093.11 Beirut, Lebanon lar to those described previously (Mather 1955, 1956b, 1956c). Ten pair matings, both ways, replicated ten times and run for a month with weekly food changes were attempted for D. serrata X D. kikkawai Burla, and for D. pseudotakahashii X D. takahashii Sturtevant. Duplicate matings both ways were extended to the F2 for D. bryani X D. bryani and for D. buzzatii X D. buzzatii crosses. The psewfutakahashii X takahashii hybrid F1 was back-crossed to the parents as indicated in Table 3. RESULTS AND DISCUSSION Drosophila serrata Malloch. D. serrata Malloch 1927:6. D. serrata, Mather 1955: 565. On morphological grounds this species is most closely related to D. kikkawai Burla (Mather 1955), the most obvious points of contrast being the sex-combs and certain features of the internal male genitalia (Table 2) . Tests indicated that these species never produce hybrids, nor were sperm detected in the female 1 Present address: Zoology Dept., University of Queensland, Australia. TABLE 2 Chief contrasting characters of D. serrata and D . kikkawai. Hypandrial bristle length Species Sex-comb teeth Penis Length of penis serrata 34+ 18 rounded 0.5 kikkawai 25+ 18 pointed 1.0 genital tracts of 54 kikkawai and 20 serrata dissected after exposure to males for four weeks. However, when virgin females were placed with the males, the latter immediately very vigorously attempted to mate. That there was sometimes suc­cessful mating was indicated by the fact that sperm were occasionally found in the female genital tract when dissected 30 minutes after placing them together. Thus sexual isolation is not as complete as would be concluded by the results of dissections made after four weeks of exposure. The explanation of this paradox may be that the sperm have a very short life in the alien female and that alien species lose interest in mating after the initial attempts. It is of interest that no sign of an "insemination reaction" was found in any of the flies dissected. Thus, D. serrata and D. kikkawai are quite distinct species and although closely related morphologically, very effective isolating mechanisms have developed between them. Drosophila pseudotakahashii, sp. nov. D. takahashii, Mather 1955:568. The stock designated Drosophila takahashii Sturtevant from Australia (Ma­ther 1955) when crossed with takahashii from Nepal produces hybrids only when the female parent is from Australia. These hybrids are produced in approxi­mately equal numbers of both sexes, unlike the situation in other interspecific crosses where females far outnumber the males (Mather 1956c). They are inter­sterile, due apparently to lack of motile sperm in the testes of the hybrid males. The F1 females, however, can be backcrossed to both of the male parents (Table 3) . Further, the salivary chromosomes of the F1 hybrids show very poor pairing. Thus although some "gene flow" would be possible if these species have a contact zone, the above data would indicate that these two stocks are distinct species. D. takahashii, D. lutea Kikkawa and Peng, and D. nepalensis Okada, all mem­bers of the takahashii subgroup of the melanogaster species group, can best be separated morphologically by differences in the internal male genitalia (Okada 1955). According! y the internal male genitalia of both of the above stocks were examined and it was found that the stock from Nepal was indeed takahashii Sturtevant, but the stock from Australia differed in that the basal branch of the posterior paramere was vestigial and the basal conical process of the novosternum was long and serrated instead of being short and conical. Unfortunately stocks of lutea and nepalensis were not available for hybridization tests but the morpho­logical differences of the Australian stock from the three established species of the subgroup are considered sufficient to raise it to specific rank (Table 4). In both the F1 and the backcrosses of the female hybrids to both the male par­ents , the basal branch of the posterior paramere was present and the basal conical process of the novosternum was elongated and serrated, although in the back­crosses the degree of expression of these characters was variable. This would indi­cate that these characters are controlled multifactorially. In hybrids the alleles controlling the basal branch of the posterior paramere from takahashii are domi­nant over the alleles from pseudotakahashii. On the contrary, the alleles control­ling the basal process of the novosternum from takahashii are recessive to the alleles from pseudotakahashii. In this case, unlike the case of body markings in the coracina species group (Mather 1956c), outstanding morphological differ­ences between species are controlled multifactorially, similar to the situation found in the machaon group of butterflies where the majority of outstanding differences are also multifactorially controlled (Clarke and Sheppard 1955) . Drosophila hryani Malloch. D. bryani Malloch 1934:310. D. levis Mather 1955:561. D. bryani, Mather 1956a:65. The synonymy of D. levis Mather with D. bryani Malloch was suggested on morphological evidence despite the difference in known geographical range TABLE 3 Results of crosses between D. takahashii and D. pseudotakahashii. Fertile Progeny J genitalia Attempted cross vials J 'i' dissected Remarks takahashii 3 X pseudotakahashii 'i' pseudotakahashii J X takahashii 'i' 5/10 0/ 10 96 0 107 0 17 giant chromosome pairing poor in hybrids; total progeny of 4 week test. ( takahashii J X pseudotakahashii 'i' ) J x ( takahashii J X pseudotakahashii 'i' ) 'i' 0/2 0 0 10 J 6 were dissected; showed motile sperm in none testes. (takahashii J X pseudotakahashii 'i' ) 'i' x takahashii J 4/4 15 21 10 total progeny is result of first week of attempted hybridiza­ti on. (takahashii 3 X pseudotakahashii 'i' ) 'i' x pseudotakahashii J 3/4 38 47 17 total week ti on. progeny is result of first of attempted hybridiza- TABLE 4 Chief contrasting features of the takahashii subgroup. basal conical common duct black Jlatch basal branch, process of of Malpig tubes; Species on wmg decasternum post. paramere novosternum post./ant. takahashii absent narrow, pointed short, serrate short, conical lutea absent narrow, truncate long, serrate short, serrate (at tip only) nepalensis present broad, truncate long, serrate short, serrate <1 pseudotakahashii absent narrow, pointed vestigial long, serrate 1 (Mather 1956a). A stock of bryani from Australia has been found to be com­pletely cross-fertile to the F2 with a stock of the same species from the Marshall Islands, thus adding support to the synonymy of these species. Drosophila buzzatii Patterson and Wheeler. D. buzzatii Patterson and Wheeler 1942: 97. D. versicolor Mather 1955:573; 1956a; 1956b. Despite certain morphological differences a stock of D. versicolor (Mather 1955, 1956a, 1956b) will freely interbreed with two stocks of buzzatii (Table 1) thus showing that versicolor is synonymous with buzzatii. Further, the Aus­tralian strain has the j inversion in the 2nd chromosome which has previ­ously been recorded only from Lebanon (Wasserman, 1954). It has been as­sumed that buzzatii was introduced into Lebanon and Italy from South America but Wasserman points out that there is insufficient data to show whether or not the j inversion arose in South America or in Lebanon. However, it seems more likely that buzzatii was introduced into Australia from South America (possibly on cactus) than from Lebanon, thus giving some support to the hypothesis that the j inversion did in fact arise in South America. SUMMARY By breeding tests it has been established that: 1. D. serrata Malloch is a distinct biological species from D. kikkawai Burla. 2. The fly previously recorded as D. takahashii Sturtevant from Australia is in fact a new species, D. pseudotakahashii, which is morphologically distinguish­able from takahashii by two features of the internal male genitalia which are controlled multifactorially. 3. The synonymy of D. levis Mather with D. bryani Malloch, previously es­tablished on morphological grounds, is confirmed biologically. 4. D. versicolor Mather from Australia is synonymous with D. buzzatii Patter­son and Wheeler, and contains an inversion previously recorded only from Lebanon. · ACKNOWLEDGMENTS Grateful acknowledgment is made to Dr. Wilson S. Stone, Professor of Zo­ology, Genetics Foundation, University of Texas, where this work was carried out while the author was on sabbatical leave from the University of Queensland, Australia. REFERENCES Clarke, C. A. and P. M. Sheppard. 1955. A Preliminary report on the genetics of the machaon group of swallowtail butterflies. Evol. 9: 182-201. Malloch, J. R. 1927. Notes on Australian Diptera. X. Proc. Linn. Soc. N. S. W. 52(2): 1-16. ----. 1934. Drosophilidae, Ephydridae, Milichiidae. Insects of Samoa, Pt. VI., Diptera. 8: 267-328. . Mather, W. B. 1955. The Genus Drosophila (Diptera) in Eastern Queensland. I. Taxonomy. Austr. Jour. Zool. 3:545-582. ----. 1956a. The Genus Drosophila (Diptera) in Eastern Queensland. II. Seasonal Changes in a natural population 1952-1953. Austr. Jour. Zool. 4:65-75. Studies in the Genetics of Drosophila 1956b. The Genus Drosophila (Diptera) in Eastern Queensland. III. Cytological Evolution. Austr. Jour. Zool. 4: 76-89. ----. 1956c. The Genus Drosophila (Diptera) in Eastern Queensland. IV. The Hybridiza­tion Relationships of four species of the Pholadoris subgenus. Austr. Jour. Zool. 4:90-97. Patterson, J. T. and M. R. Wheeler. 1942. Description of new species of the subgenera Hirto­drosophila and Drosophila. Univ. Texas Puhl. 4213:67-109. Okada, T. 1955. Fauna and Flora of Nepal, Himalaya (Kyoto) 1:387-390. Wasserman, M. 1954. Cytological Studies of the repleta group. Univ. Texas Puhl. 5422: 130-152. XVIII. A New Drosophilid from Australia (Diptera) MARSHALL R. WHEELER Through the courtesy of Dr. F. I. Van Emden of the Commonwealth Institute of Entomology, London, I have had the opportunity of studying four specimens of a beautiful new drosophilid from the C. I. E. collection. The flies were col­lected on Mt. Tambourine, Queensland, Australia, and were apparently reared from Endiandra sp., a member of the Lauraceae. The holotype and two para­types are being returned to Dr. Van Emden for deposit in the British Museum (Natural History); the third paratype is in the Drosophila Type and Reference Collection of the Genetics Foundation, University of Texas. All four specimens are females, but since striking sexual dimorphism is so rare in this family, it is probable that the males will be recognizable from the description and figures given here. Tamhourella Wheeler, new genus Type species: Tambourella endiandrae Wheeler, new species. Small species with highly marked wings and aberrant wing venation. Arista plumose; carina high and exceptionally narrow; postvertical bristles minute; acrostichal hairs absent; anterior dorsocentral placed at sutural level; no pre­sutural bristle; a single postalar; basal scutellars small; no mesopleurals; two­sternopleurals. Wings as in Figure 2; costa reaching 4th vein; 2nd vein bent abruptly to costa apically; 5th vein incomplete at apex but continued by fusion with the long, undulant posterior crossvein. The veins on all specimens show a strong+ and-character: costa +,2nd-, 3rd+, 4th-, 5th, and posterior crossvein +. The generic name, derived from the name of the type locality, is considered to be feminine. Tamhourella endiandrae Wheeler, new species 'i'. Front broader than long, yellowish tan with dense pollinosity, less so along a narrow area between orbit and large frontal triangle; orbits elongate, reaching anterior margin, the orbital bristles placed rather far forward (Fig. 1), the middle orbital minute or absent, the proclinate about 0.4 length reclinate, Il1G. 1. Tambourella endiandrae, head in profile. the latter about 0.9 length ocellars. Bases of ocellars clearly within the triangle formed by the ocelli. Both inner and outer verticals strong, the latter about 0.6 length of the former. Vertex elevated, mound-like, around bases of vertical bris­tles. Postverticals minute, cruciate, placed on rear of head. Antennae tan, 2nd joint with small hairs of which two are more prominent; 3rd joint a little darker, short pubescent. Arista with 5 dorsal and 3 ventral branches in addition to the terminal fork, the branches rather long and bent at their tips. Face glassy hyaline and yellowish on all four specimens; carina very narrow and sharp, ceasing abruptly before level of antennal apex, with the lower margin of face again elevated into two mound-like swellings, leaving a low median crease between them. Two thin orals, nearly of equal length. Cheeks narrow below the eyes, greatly widened behind, mostly tan in color becoming blackish along lower margin. Posterior cheeks strongly pubescent when viewed from certain angles. Clypeus hyaline, narrow; oral opening rather large, ephy­drid-like; palpi narrow, brown. Mesonotum opaque tan, strongly pollinose, the disc with two darker brown areas (variable in extent), one between posterior dorsocentrals and another anterior to it; scutellar disc with a similar brown mark when seen from above. No evident acrostichal hairs, but there are a few scattered hairs in the dorso­central rows and laterally. Two pairs of strong dorsocentrals, the anterior pair placed at the sutural level; two pairs of scutellars, the basal ones small and thin, about 0.25 length apical ones. No presutural bristle; one small humeral; two notopleurals; two supraalars, one postalar (the 2nd one represented by a small hair). Side of mesonotum with darker brown areas, especially around bases of supraalars; pleura brownish, rather shiny and hyaline, especially over meso­pleura and center of sternopleura. A short anterior sternopleural bristle and a long stout posterior one. Knobs of halteres dark. Abdomen of all specimens shriveled but evidently the basal 3 (or 4) tergites are dark with thick grayish to grayish golden pollinosity, the remaining (apical) tergites being dark and shining. Oviposi~or tan with small stout teeth along both upper and lower margins. Legs mostly collapsed, apparently pale tan with darker coxae and femora. An evident small apical bristle on 1st tibia, apical and preapical on 2nd tibia. First femur with a row of about 15 very small thornlike spines along inner side, each spine associated with a tiny hair. I mm FrG. 2. Tambourella endiandrae, wing, semi-diagrammatic. Wings generally brown with discrete white areas (Fig. 2). Costal index about 1.1; 4th vein index about 0.9. Apex of 1st costal section with a single small apical bristle; 3rd costal section with the small black spines on the basal 0.8. Costa reaching 4th vein; distal costal break shallow, the costa not prolonged as a lappet. Anal vein absent. Wing length about 2.2 mm.; overall length (tip of head to tip of wing) about 3.5 mm. Types.-Holotype 'i' and three paratype 'i' 'i', all bearing labels as follows: (1) "ex Endiandra sp; Mt. Tambourine; 30-12--52; May" ; (2) "731"; (3) "Com. Inst. Ent. Coll. No. 14691." One specimen bears the notation "killed 16­1-53"; if this means that the flies were kept alive for some two weeks, then the glassy hyaline character of the face and clypeus should not be interpreted as teneral peculiarities. XIX. Genetic Variation of lsoxanthopterin Content in Drosophila melanogaster1 2 FORBES W. ROBERTSONAND HUGH S. FORREST INTRODUCTION Hadorn and Mitchell ( 1951) showed that Drosophila melanogaster contained a number of fluorescent substances which could be readily separated into more or less distinct spots by paper chromatography of flies squashed directly on to the paper. Several of these compounds have since been identified as pteridines (For­rest and Mitchell, 1954, 1955; Viscontini, 1955), all containing the basic struc­tur~ I (Fi::;. 1), but s:10wing various differences in the side chains R1 and R,. Be· "Y"Y"~oe0N# Rz ~.) "l"Y")R, OH OH I lI FIGURE I. cause of their close chemical relationship, Forrest and Mitchell ( 1955) postulated that these compounds represent individual steps in a biochemical pathway lead­ing to the red eye pigments characteristic of Drosophila. However it seems likely that one of them, isoxanthopterin (II; Fig. 1), is not directly on this pathway but is produced from one of the intermediates on it, namely 2-amino-4-hydroxypteri­dine (III; Fig. 2). Schematically this hypothesis can be written as shown in Fig. 2. The evidence for this scheme is based on chromatographic examination of mu­tants of Drosophila (Hadorn and Mitchell, 1951; Forrest, unpublished), and also on the demonstration of the virtual absence of isoxanthopterin in the mutants rosy, maroon and maroon-like (Forrest, Glassman and Mitchell, 1956; Hadorn, 1956). These do not lack the red eye pigments, although they appear to have greatly reduced amounts, whereas the sepia mutant, for example, lacks the red eye pigments and "accumulates" a yellow pteridine which is therefore assumed to be their immediate precursor. Since the rosy, maroon and maroon-like mutants have reduced amounts of red pigments and of isoxanthopterin, it would be reasonable to assume that the latter 1 The work reported here was supported in part by a grant from the Welch Foundation, Houston, Texas. 2 Visiting Professor of Zoology, The University of Texas, 1956-57; Member of the Scientific Staff of the Agricultural Research Council of Great Britain. NHr~N I N) YELLOW RED +­ -x-v--COMPOUND PIGMENTS N sepia ~ N# OH m maroon moro-on-like rosy +')oH NrN~ N ~ # N OH FIGURE 2. had some effect on the production of the pigments. This has not been demon­strated on an enzymatic level, although some effects of isoxanthopterin on pig­ment production in other systems have been claimed. Thus Polonovski, Gonnard and Baril ( 1951) stated that it inhibited the initial oxidation of dihydroxyphen­ylalanine (DOPA), but accelerated the production of melanin at the expense of the red intermediates, and more recently, Oshira, Seki and Ishazaki (1956) pro­pounded a scheme for its activity in the control of the colour of the elytron in the lady-beetle. It would be possible, of course, to attribute the apparent relationship between red eye pigment and isoxanthopterin in Drosophila to the functioning of the enzyme, xanthine oxidase, which is responsible for the production of isoxan­thopterin (Forrest, Glassman and Mitchell, 1956) but which could also act as an oxidase or dehydrase in the process of red eye pigment formation. As is obvious, however, information on the biological function of isoxanthopt­erin is inadequate, although there are some indications that it may play an im­portant role. Thus it appears to be especially abundant in certain tissues of Drosophila, especially the testes, thereby producing a characteristic sex difference (Hadorn and Mitchell, 1951), although females also carry the compound in easily detectible concentration. Also during the life-cycle of Drosophila melano­gaster, there is a rapid and spectacular rise in concentration during the pupal period. It has also been suggested (Haddow, 1955) , that its isomer, xanthopterin, plays an important part in cell division in renal tubular epithelium and, on this basis, that other specific pteridines might also be involved in the growth of other types of tissue. So far attention has been confined to the isoxanthopterin content in mutant stocks and at least three genes have been shown to influence the content. It is a reasonable inference that many other genes will also intervene in the production of this compound and it would be interesting to know how far non-mutant, wild stocks differ in isoxanthopterin level, since this would provide a basis for assessing the relative effects of different mutant genes and would indicate how far differ­ences in genetic background are likely to influence the individual content. Indeed, an identifiable chemical compound, subject to the influence of many gene differ­ences, constitutes rather novel material for the study of quantitative inheritance, since there is the ultimate prospect of relating the genetic to the underlying bio­chemical behaviour. If isoxanthopterin, or some precursor which is related quantitatively to the final isoxanthopterin level, turns out to be important in metabolism or differentiation, evidence of genetic variation and of genetic be­haviour will acquire additional interest. Isoxanthopterin is eminently suited for such comparisons since it can be readily isolated from individual flies and then estimated accurately. The present paper deals with the isoxanthopterin content in a number of non­mutant inbred lines and wild stocks of diverse origin. Also some of the inbred lines have been crossed to determine the relation between the F1 and the parent lines. The effects of variation in body size have been estimated by comparing the isoxanthopterin content in selected strains which differ in size. MATERIAL AND METHODS The technique for chromatography was essentially that of Hadorn and Mit­chell (1951) . Individual flies were squashed on Whatman No. 1 filter paper on a line drawn 1.5 cm. from one edge and each at a distance of 1.5 cm. The paper was developed, using n-propanol, 1 % ammonia (2: 1) as the solvent, for about 16 hours, after which the chromatogram was dried and the areas corresponding to isoxanthopterin were located by their fluorescence in U.V. light. These were cut out and the fluorescent material was eluted from them with 0.5 N ammonia. Fluorescent measurements on the eluting fluid were made with a Farrand Model A Fluorometer using the following filters: Primary filter, Corning No. 5860; Secondary filters, Corning Nos. 3389 and 4308. A weighed sample of isoxan­thopterin was used as the standard. Two dimensional chromatograms were occasionally run (see text). In these cases, individual flies were squashed at one corner of a rectangle of filter paper (15x20 cm.). The paper was developed in the shorter direction with n-propanol, 1 % ammonia (2:1), and in the longer direction with 5% acetic acid. The isoxan­thopterin was located and eluted as described above. The isoxanthopterin content of individual flies obtained from such chromatograms was lower than from one dimensional ones, as would be expected; but the results were consistent within a group. Since the two dimensional technique is much more laborious, especially when handling large quantities of flies, it was therefore abandoned as a general procedure. The genetic stocks included 6 wild stocks descended from wild flies caught in widely separated localities and kept in the laboratory in mass culture. Fifteen inbred lines were also compared; these were derived by long-continued brother.­sister mating from various wild stocks or from strains selected for large or small body size (Robertson and Reeve, 1952; Robertson, 1956). Five of the inbred lines were intercrossed to provide 10 F1 groups. All flies were reared under optimal conditions on the usual cornmeal-molasses medium, well supplied with yeast; 70 eggs per vial were set up to eliminate crowding. The cultures were kept at 22-23° C. The isoxanthopterin content for each genotype is based generally on data from 10 flies. Within about 18 hours after emergence from the pupa, a sample of 20 flies was weighed. The flies were aged about 2Yz days before being chromato­graphed. The isoxanthopterin content is a good deal higher in males than females, some­times as much as 15 times greater. Since the coefficient of variation works out at about the same in the two sexes (17-18% in the present determinations) a logarithmic scale is indicated. However, since the analysis is confined to com­parisons within one sex or the other, the range is not great enough for such a transformation to make much difference and so the data are expressed in micro­grams (µg.) per fly or per milligram weight. RESULTS (a) Isoxanthopterin content and body size. The first point to consider is whether the total content per fly or the amount per unit weight should be the basis for comparison. To answer this question, isoxanthopterin was determined on selected large and small and also unselected, normal-sized flies from the Pobla de Lillet stock; only males were studied in this test. Mass selection had been car­ried out for some 10 generations and there was a substantial difference in size between the large and small strains, 0. 78 mg. in the former, 1.12 mg. in the latter. Fig. 3 shows the isoxanthopterin content per fly plotted against body weight. The graph falls close to the line of proportional increase, so that isoxan~hopterin con­ ·50 FIGURE 3. Average lsoxonthopterin Content per Mole in Lorc;ie, Small and Unselected Strains. ~ u. 0:: ILi CL z it: ILi li:: :40 0 :c 1-­ z ~ x 0 ~ 0 .35 0.7 0.8 0.9 1.0 I.I BODY WEIGHT OF MALES (m9.) tent per milligram body weight is virtually the same in the large and small flies. Since appreciable differences in size occur among the inbred lines and wild stocks described below, it would be misleading to deal in terms of total content per fly and so the data are expressed in terms of isoxanthopterin per milligram weight. (b) Differences between inbred lines. Six of the lines used were inbred from various wild stocks; the lines are referred to as Nb2, R", OrR, C6 and C,,, while one line carries vermilion in what was originally an OrR background (OrR v) . The other lines, inbred from selected strains, include CL1, RL4, IL4 and LT0 from different large strains, and CS1' ES1 2 and IS2 from small strains. In addition, two lines, EL1 and ES1 were homozygous for white-eyed alleles which had arisen by mutation during inbreeding. The average isoxanthopterin content per milligram for the 15 lines is listed in Table 1. Bearing in mind that the standard error of the difference between means, based on the pooled variance, is 0.033, it is obvious that significant and substantial differences exist. Excluding the white-eyed lines the values range from 0.690 µg. in IL4 to 0.312 µg. in RL4, with an average of 0.408 1~g. for the 13 lines. There is no evidence of characteristic differences between inbred lines derived from large as opposed to small strains. T ABLE 1 Isoxanthopterin Content in Males of Inbred Lines Line 11-g. per mg. body weight Line 11-g. per mg. body weight c, c, R, Nb, 0.434 ± .039 0.41 3 ± .014 0.374 ± .027 0.327 ± .006 CL,RL., IL, LT, 0.366 ± .024 0.312. ± .001 0.690 ± .009 0.412. ± .012. OrR 0.412. ± .023 OrRv 0.404 ± .018 cs. 0.358 ± .028 EL. (whi te-eyed) ES, (white-eyed) 0.035 ± .006 0.184 ± .014 ES,, IS, 0.407 ± .021 0.392 ± .018 The variance of the determinations on individuals of the same genotype is rather high; the average coefficient of variation is approximately 17%. Since the isoxanthopterin content of each fly is divided by the average of the weighed sample, rather than its own weight, the variance will be compounded of individ­ual variation in size, true variation in relative isoxanthopterin content, and also errors of determination. General experience suggests that the last two sources of variation are likely to be the most important. The lower values recorded for the pair of white-eyed lines are not unexpected (Hadorn and Mitchell, 1951; Forrest, unpublished), but although the value found in ES1 is well below the wild-type range, it is nevertheless substantially higher than has been found in other tests on stocks carrying a white-eye allele. It has been shown by Judd (unpublished) that this effect is comparatively independent of genetic background, so we appear to have here a new allele at the white locus, characterized, so far, only by its association with a relatively higher isoxanthopt­erin content. (c) Wild stocks and crosses. In this test determinations were carried out on both males and females of 6 unrelated wild stocks, 5 of the inbred lines from un­ The University of Texas Publication selected stocks, together with the 10 possible crosses between them. All the cul­tures were set up together and the chromatograms were run at the same time. The average values for the different genotypes are set out in Table 2. Comparison of the values for males of the 5 lines also stud.led in the earlier test shows fair agreement, although the values in the second test tend to be a little higher. The biggest discrepancy occurs in the R2 line, with estimated values of 0.374 ± .027 and 0.519 ± .030 in the first and second tests respectively. The reason for this difference is unknown, except that there are indications that this line may be atypical in that the isoxanthopterin content appears to increase to its maximum level during the first day or so after emergence. T ABLE 2 Isoxanthopterin Content in Wild Stocks, Inbred Lines and Crosses 11-g. per mg. body weight Males Females Wild Stocks Pobla De Lillet 0.507 ± .027 0.032 ± .004 Stephenville New Mexico 0.297 ± .015 o.549 ±.om 0.059 ± .002 0.058 ± .005 Pennsylvania Hawaii 0.480 ± .008 0.485 ± .011 0.037 ± .002 0.057 ± .005 Texas 0.449 ± .010 0.049 ± .003 Average 0.461 0.048 Inbred Lines c, R, 0.485 ± .011 0.519 ± .030 0.068 ± .004 0.074 ± .004 Nb, OrR 0.369 ± .023 0.447 ± .020 0.061 ± .005 0.062 ± .002 OrRv 0.404 ± .015 0.081 ± .003 Average 0.445 0.069 Crosses C,x R, 0.372 ± .021 0.050 ± .002 C, X Nb, 0.410 ± .037 0.057 ± .003 C, X OrR 0.462 ± .031 0.057 ± .004 C, X OrRv 0.392 ± .015 0.056 ± .003 R,x Nb, 0.452 ± .020 0.053 ± .003 R, X OrR o.464 ± .om 0.056 ± .003 R, X OrRv 0.416 ± .013 0.050 ± .003 Nb, X OrR 0.374 ± .016 0.065 ± .006 Nb, X OrRv 0.306 ± .038 0.073 ± .002 OrR X OrRv 0.433 ± .019 0.083 ± .003 Average 0.408 0.060 The standard error of the difference between means, from pooled variances is 0.032 for males and 0.005 for females. It is evident, therefore, that appreciable differences occur between wild stocks. In males the range is from 0.297 in Stephenville to 0.549 in New Mexico, while in females the lowest and highest values are found in Pobla de Lillet and Stephenville: 0.032 for the former, 0.059 for the latter. The average content per mg., is about 9-10 times as great in males as females. The value quoted for males of Pobla de Lillet is appreciably higher than the value found in the first test relating to the large, small and unselected strains: 0.507 versus 0.441. However, these values are not strictly comparable, since the latter was based on two-dimensional chromatograms which may be expected to afford lower estimates of isoxanthopterin and this probably accounts for the apparent discrepancy. Studies in the Genetics of Drosophila An interesting feature appears in the lack of correlation between the content in males and females. Thus for the entire set of comparisons-stocks, lines and crosses-the correlation works out at -.237, an insignificant value. It might be argued that it is unfair to include the inbred lines which may be atypical; how­ever, the picture is not altered if they are excluded (r -.276) . This lack of agree­ment between the sexes will be considered later. Turning now to the crosses between lines, we find a distinct tendency for the content per mg. to be higher in progeny than parents. The differences between F1 and mid-parent values are listed in Table 3; the average difference in males of 0.037 is clearly significant at the 0.05 level, while in females, the average value of 0.009 falls just below significance. T ABLE 3 Difference in Isoxanthopterin Content between Inbred Lines and Crosses Between the Lines Cross Mid-P arent Content- F, Content Males Females C. X R, + o.13o + 0.021 C. X Nb, + 0.011 + 0.007 C, X OrR + 0.004 + 0.008 C. X OrRv + o.os2 + o.oi8 R,X Nb, -0.008 + 0.014 R, X OrR + 0.020 + 0.012 R, X OrRv + o.o46 + 0.028 Nb, X OrR + 0.034 -0.004 Nb, X OrRv + 0031 -0002 OrR X OrRv -0.008 -0.01 2 Average 0.037 0.009 Finally, the regression of the content in F, on the average value for parent lines may be expected to provide some indication of genetic behaviour. With a fully additive situation, the regression will approximate to unity, while non­additive effects will lower it to varying degree, depending on their nature and relative magnitude. In the present instance the regression for males and females works out at 0.73 ± .42 and -0.25 ± .75 respectively. Thus in females there is no evidence of correlation between the concentration in progeny and average concentration in parents. It is improbable that any particular significance can be attached to the negative sign. In males, however, there is evidence of positive association between parent and progeny, although the regression is not statisti­cally significant, probably because of non-additive interactions which occur in the various crosses. It seems reasonable to conclude that non-additive genetic effects are important in the control of isoxanthopterin content. DISCUSSION The substantial differences in isoxanthopterin content between wild stocks and various inbred lines indicate that many gene differences are involved and so the level of concentration of this compound can be added to the the list of "quantitative characters" available for experimental study. It follows that stand­ardization of the genetic background is essential for comparison of the effects of different mutant genes. The University of Texas Publication The number of comparisons between parent lines and their crosses are too few for very definite assertions, but there does appear to be a considerable de­parture from an additive system. The indication of a relatively more additive situation in males as opposed to females is probably related to their higher con­centration of isoxanthopterin. The most interesting aspect of the crosses refers to the tendency for relative content to be lower in the F1 than in the parents. At first sight this might suggest that the isoxanthopterin content was influenced by inbreeding, being higher in the homozygous lines, relatively lower in the crosses, which, of course, will be more nearly like wild-flies in growth and reproduction (Robertson and Reeve, 1955) . It is a little difficult to see why the changes in growth which accompany inbreeding should be reflected in a higher isoxanthopterin content, although it may occur. However, there is an alternative and, at present, more attractive explanation. Recent work by Robertson ( unpub­lished) has indicated that the genetic variation of body size in Drosophila may be due to changes in either cell number or size -0r both and that variation of body size originating in different kinds of genetic change are associated with character­istic changes in one or the other of the primary components of body size. Thus changes in size due to selection are due to changes in cell number, whereas the 'reduction in size due to inbreeding and its restoration to the average level of wild stocks by outcrossing are associated with corresponding changes in cell size. If isoxanthopterin content is primarily correlated with cell number, and the more or less proportional differences in content in comparisons between large, small and unselected strains support this view, and if the F1 flies have relatively larger cells than the parents, then we could expect isoxanthopterin per unit body , weight to be lower in the crosses. Hence a fuller understanding of the inherit­ance of this compound in crosses involves an understanding of the behaviour of cell size and number as well; experiments are in progress to throw further light on this problem. In view of the somewhat variable degree of heterosis in crosses between lines, it might be thought that the total content per fly rather than per mg. would provide a better basis for comparing parent and progeny. However, since the regression of F1 on mid-parent value works out at 0.73 ± .41 in males and -0.68 ± .79 in females, the situation remains the same. From the variation in isoxanthopterin revealed in these tests, it appears that the biosynthetic pathways which lead to the formation of this compound are as­sociated with a varietv of equilibria which are reflected in the differences be­tween strains. It would be particularly instructive to know how the other related compounds, which are probably involved in the same system, also vary, since this would help us to understand the significance of variation in isoxanthopterin and probably afford further clues to the characteristic sex difference. It is likely that isoxanthopterin is not confined to the testes in males, but may exist in other tissues at about the same concentration as that found in females, but the be­haviour of genetic variation in this respect would be obscured. The genetic vari­ation of the content in the testes may differ in behaviour from variation in other tissues and this may be related to the apparent discrepancy between the sexes in the regression of F1 on mid-parent level in the crosses between lines, and also to the lack of correlation between males and females. Studies in the Genetics of Drosophila SUMMARY 1. Isoxanthopterin content has been determined by paper chromatography on a number of wild strains, inbred lines and crosses between lines. 2. Increase or decrease of body size by selection is accompanied by more or less proportional changes in isoxanthopterin and so the comparisons are based on the content per mg. body weight. 3. Substantial differences in isoxanthopterin content exist between wild stocks and inbred lines and many gene differences are probably involved. 4. No correlation has been found between the content in males and females. 5. Crosses between lines suggest that non-additive genetic interactions are important in the control of isoxanthopterin level. This is particularly evident in females, less so in males; the apparent sex difference in this respect is prob­ably related to the much higher concentration in males. 6. Content per unit weight tends to be lower in the F1 of crosses than in the parent lines and it is suggested that this may be due to the relatively larger cells in such crosses, compared with the homozygous lines. 7. An allele of white-eye has been identified which is responsible for a higher isoxanthopterin content than has been previously found for other alleles which at first sight appear to be phenotypically identical. REFERENCES Forrest, H. S., and H. K. Mitchell. 1954. Pteridines from Drosophila. I. Isolation of a yellow pigment; II. Structure of the yellow pigment. J. Amer. Chem. Soc. 76: 5654, 5658. ----. 1955. Pteridines from Drosophila. III. Isolation and identification of three more pteridines. J. Amer. Chem. Soc. 77:4865. Forrest, H. S., E. Glassman, and H. K. Mitchell. 1956. Conversion of 2-amino-4-hydroxypteridine to isoxanthopterin in D. melanogaster. Science 124: 725. Haddow, A. 1955. The biochemistry of cancer. Ann. Rev. Biochem. 24: 729. Hadorn, E. 1956. Patterns of biochemical and developmental pleiotropy. Cold Spring Harbor Symp. on Quant. Biol. 21:363. Hadorn, E., and H . K. Mitchell. 1951. Properties of mutants of Drosophila melanogaster and changes during development as revealed by paper chromatography. Proc. Nat. Acad. Sci_ 37:650. Oshima, C., T. Seki, and H. Ishizaki. 1956. Studies on the mechanism of pattern formation in the elytra of lady beetles. Genetics 41 :4. Polonovski, M., P. Gonnard, and A. Baril. 1951. The action of pterin derivatives an::! of · ribo­flavin on melanogenesis in vitro. Enzymologia 14:311. Robertson, F. W. 1955. Selection response and the properties of genetic variation. Cold Spring Harbor Symp. on Quant. Biol. 20: 166. Robertson, F. W., and E. C. R. Reeve. 1950. Studies in Quantitative Inheritance. I. The effect of selection of wing and thorax length in Drosophila melanogaster. J. Genetics 50·414. ----. 1955. Studies in Quantitative Inheritance. VIII. Further analysis of heterosis in crosses between inbred lines of Drosophila melanogaster. Z. indukt Abstamm. u. Vererb. 86:439. Viscontini, M., M. Schoeller, E. Loeser, P. Karrer, and E. Hadorn. 1955. Isolierung fluoreszie­render Stoffe aus Drosophila melanogaster. Helv. Chim. Acta 38:397. Viscontini, M., E. Loeser, P. Karrer, and E. Hadorn. 1955. Fluoreszierende Stoffe aus Drosophila melanogaster. 2. Mitteilung Helv. Chim. Acta 38: 1222. XX. The Production of Attached-X Chromosomes in Drosophila hydei THOMAS G. GREGG INTRODUCTION The first known case of attached-X chromosomes in Drosophila was discovered in Drosophila melanogaster by L. V. Morgan (1922). Later, E. G. Anderson (1925) succeeded in producing an attached-X female which was heterozygous for a number of recessive mutants. This enabled him to study crossing-over in attached-X chromosomes. From this study he determined that crossing-over in attached-X chromosomes occurred with the same frequency as crossing-over in free X chromosomes, and furthermore, that crossing-over takes place in the four strand stage with only two chromatids involved at any level. He also showed that any single cross-over involves two homologous chromatids at random, regardless of which chromatids were involved at a different level. These results of Anderson were substantiated by Sturtevant ( 1931 ), Emerson and Beadle ( 1933), and Beadle and Emerson ( 1935) . In addition, Beadle and Emerson were able to show that two and four strand double exchanges occurred with approximately equal frequencies, as did two and three strand double . exchanges. Anderson also noted that the frequency with which the heterozygous recessives became homozygous, as the result of non-reciprocal cross-overs, was a function of the distance of each mutation from the spindle attachment, so that the mutations more distant from the attachment appeared homozygous more frequently. Mark­ ers approximately 40 units from the centromere became homozygous with a fre­ quency approaching 16. 7 per cent. This is the frequency of homozygosis that would be expected if the chromatids at a given locus were assorted at random .during meiosis. · L. V. Morgan (1925), M. M. Rhodes(1930) and Beadle and Emerson (1935), however, have data to show that the frequency of homozygosis for mutations near the distal end of the X chromosome is definitely greater than the 16.7 per cent expected on a basis of random assortment of genes. Some of their values are as high as 20.3 per cent. Thus it seems that homozygosis does not increase in a regu­ lar fashion with 16.7 per cent as a limit, as Anderson had assumed. Since homo­ zygosis values greater than 16.7 per cent were observed it was concluded that sister strand crossing-over does not occur, or is not equivalent in interference to regular crossing-over, as stated by Beadle and Emerson (1935). The latter work­ ers also showed that there is a discrepancy between actual homozygosis fre­. quencies and expected frequencies, calculated on the assumption that sister strand crossing-over does not occur. In the present experiment it seemed theoretically possible to produce attached­ ::X: chromosomes in a second species of Drosophila, Drosophila hydei, since one ..arm of the large metacentric X chromosome is entirely heterochromatic. At­ taohed-X chromosomes in hydei should prove very interesting for two reasons. Studies in the Genetics of Drosophila First, an attached-X has never been found in any species of Drosophila other than melanogaster. Secondly, crossing-over in hydei is over twice as frequent as it is in melanogaster. The linkage map of the hydei X chromosome is 116 units long, while that of melanogaster is only 66 units long. Thus the comparison be­tween the actual and expected frequency of homozygosis could be worked on more profitably in hydei attached-X chromosomes. Welshans (1955) showed that when double crossing-over occurs in a short region of attached-X chromosomes, the types of double cross-overs recovered do not occur in the ratio expected. He finds that this is not inconsistent with the sister strand cross-over theory of Schwartz. More information on this point might be gained in properly marked hydei attached-X chromosomes, since double cross­ing-over within a short region would be more frequent in hydei than in melano­gaster. Attached-X chromosomes in hydei would, therefore, be interesting from the standpoint of comparing crossing-over in the attached-X chromosomes of the two species, and especially interesting from the standpoint of gaining more informa­tion about sister strand crossing-over and the frequency of homozygosis of re­cessive mutations. In addition, the attached-X chromosomes are useful tools in genetic research on other problems. PROCEDURE AND RESULTS To begin the experiment, sixty virgin females, homozygous white, less than 72 hours old, were subjected to X-ray treatment. The total dose given was 3000 r at the rate of 112 r per minute. The flies were exposed for a total of 26.8 minutes. A beaker suspended in ice water maintained their temperature between 13 and 17 degrees centigrade. During treatment, the flies were contained in celluloid capsules on the bottom of the suspended beaker. Twenty-four hours after treatment, the females were mass mated to wild type males. The age of the males varied from one to seven days. The flies were changed to fresh food each day so that any X-ray sensitive period in meiosis, as measured by non-disjunction, might be detected, if a large number of non-dis­ junction offspring were produced from the eggs laid on any particular day or group of days. Larval competition was held at a minimum by spreading eggs and larvae from overcrowded vials on to fresh food. The offspring from the 60 females are summarized in Table 1. Each non-disjunction male was individually mated to females homozygous for vermilion, scute, yellow, miniature, cherry and bobbed. All such males proved to be sterile as expected, since primary non-disjunctional males are usually XO. The super females were short-lived and inviable. Subsequent to the treatment of the 60 females just mentioned, 300 white fe­ males were given the same treatment except that the temperature was held lower, varying from seven degrees centigrade at the start of irradiation, to 13 degrees centigrade at the end of the irradiation period. The same mating pro­ cedure was followed for these females as for the first group. The offspring from the 300 females are summarized in Table 2. Non-disjunction males were again mated to mutant females and changed to fresh food several times. This time 49 of the males bred as normal wild type TABLE 1 * OFFSPRING FROM SIXTY WHITE-EYED IRRADIATED FEMALES Type of Offspring Days 1 2 3 4 and 5 6 7 8 9 10 11 1Z 13 14 15 16 17 18 Totals Regular'? Regular () Non-disjunction'? Non-disjunction () 16 30 0 1 63 67 1 super'? 3 15 21 0 2 584 571 1 super'? 2 283 348 0 0 231 339 0 6 367 405 0 3 403 491 0 3 344 448 1 super '? 0 300 256 0 0 374 492 0 1 475 275 0 3 434 408 0 0 366 174 0 0 95 18 0 0 58 95 0 0 67 64 0 0 4375 4502 3 super'? '? 22 ""-3 ~ c:::! ;:i§' ~­ TABLE 2 -Q.. ~ OFFSPRING FROM 300 WHITE-EYED IRRADIATED FEMALES ~ ~ Type of Offspring Regular'? Regular () Days 1 2 3 0 0 0 0 0 0 4 0 0 5 33 40 6 438 499 7 851 891 8 1926 1546 9 1948 1264 10 1330 1472 II 1255 917 12 2731 2531 13 1594 1809 14 2297 2371 15 2283 2397 Totals 16685 15737 ~ .._ §' ..... s· ;:i Non-disjunction'? (white­eyed) 0 0 0 0 0 1 1 2 3 0 1 0 0 0 0 8 Non-disjunction () (wild type) 0 0 0 0 0 11 13 37 17 19 8 13 3 6 11 138 Total Offspring 32569 males, thus giving mutant males and wild type females in the F1 generation. The remaining 89 males were sterile. This strongly indicates that the 49 normal males resulted from a contamination, while the remaining 89 were actually non­disjunction males. Even with this slight distortion in Table 2, it appears possible that a peak of non-disjunction occurs in eggs laid seven to nine days after X-ray treatment. This possibility is supported by the data in Table 1, although the number of flies in Table 1 is not large. Non-disjunction females were individually mated to wild type males. Six of the females produced white-eyed sons and wild type daughters, and so were dis­carded as not being attached-X. The other two, however, continued to produce non-disjunctional offspring; i.e., white-eyed females and wild type males, and proved to be of attached-X constitution. An attached-X stock was started from each of these females. To check for complications, the salivary gland chromosomes of six F 2 larvae from.each original attached-X female were analyzed for inversions, deletions or other chromosomal aberrations that might have been produced concurrently with the attched-X. No such complications were evident, and the normal hydei sali-· vary gland chromosome configuration of five arms extending from the chromo­center was present in all cases. The metaphase configuration of the chromosomes in the males from the at­tached-X stocks was four pairs of rods, a pair of dots, and a large V-shaped X chromosome, paired with a J-shaped Y chromosome. This is the normal hydei male configuration. In the attached-X females, the chromosome configuration is exactly the same as it is in normal hydei males. The normal hydei female con­figuration is four pairs of rods, a pair of dots and a large pair of V's. Thus it ap­pears that the heterochromatic arms were lost from the egg which produced each attached-X female. As a further investigation into the normality of the attached-X stocks, the sex ratios were checked and egg counts were made, in order to determine the per cent emergence. It was also hoped that some idea of the rate of detachment of the attached-X could be gained. This was done in the following way. (The attached-X stocks have been arbitrarily numbered as 1 and 2 so that they can be referred to more easily.) Ten four-to six-day-old attached-X females from stock 1 were mated individu­ally to wild type males. The flies were changed to fresh food vials daily, the number of eggs laid in each vial was counted, the pupae were counted, and the emerging flies were counted and classified. These data are shown in Table 3. Four of the females produced no offspring and are omitted from the table. In none of the females which produced offspring was there a significant devia­tion from the 1: 1 ratio of males to females. (Whether or not a given deviation was significant was determined by applying the chi-square test with one degree of freedom.) Likewise, there is no significant deviation from the 1: 1 ratio when all of the offspring from the females in Table 1 are totaled. Of the eggs laid, 31. 7 per cent developed into adults. The most eggs that could be expected to develop into adults would be 50 per cent, since YY zygotes are lethal and XXX super females rarely develop beyond the pupal stage. That only TABLE 3 Fertility and sex ratio checks in attached-X females from stock 1 Days 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Totals to -!>­to Female A Eggs Pupae White eyed 'i' Wild type () Other 44 16 9 5 0 93 62 24 29 2 super 'i' 95 40 18 17 0 63 36 20 10 0 102 60 27 25 0 92 56 19 15 0 95 50 19 8 0 0 0 0 0 0 54 7 1 1 0 0 0 0 0 0 28 0 0 0 0 0 0 0 0 0 666 327 137 110 2 Female B Eggs Pupae White eyed 'i' Wild type () Other Female C Eggs Pupae White eyed 'i' Wild type() Other 11 3 50 27 17 0 92 38 17 18 0 109 35 14 13 0 143 56 27 18 0 57 14 5 4 0 62 36 15 18 0 84 32 16 12 0 79 45 21 16 0 78 50 14 27 0 62 40 17 20 0 0 0 0 0 0 74 30 13 9 0 200 60 16 24 1+!+ 'i' 82 45 13 12 0 122 53 14 23 0 95 60 26 20 0 48 20 12 5 0 26 22 9 5 0 51 28 8 7 0 40 28 7 10 0 56 40 12 '14 0 21 17 5 8 0 132 56 21 19 0 80 48 18 17 0 1050 438 159 164 I 856 465 183 171 0 """'3 ~ (1) C::! ;:i..... ?€ ~ ..... ..... ~ ~ Female D Eggs Pupae White eyed 'i' Wild type () Other Female E Eggs Pupae White eyed 'i' Wild type () Other 16 15 29 25 12 8 24 19 111 16 2 10 0 99 30 10 10 0 121 42 15 12 0 0 0 0 0 0 69 26 7 13 0 108 46 16 11 0 54 17 8 8 0 11 4 48 29 10 0 80 40 8 14 0 101 50 25 24 0 83 25 9 5 0 99 40 13 13 0 72 24 9 9 0 62 28 15 8 0 128 40 11 15 0 106 52 21 15 0 16 0 1 0 0 48 33 8 9 0 0 0 0 0 0 63 38 14 12 0 96 0 0 0 0 82 48 12 10 0 60 0 0 0 0 61 37 13 14 0 810 230 69 86 0 943 450 257 203 0 """'3 ~ ~ "' ~ !:::: c::r­..._..... ~ ~ .......... c ;:i Female F Eggs Pupae White eyed 'i' Wild type() Other 16 16 19 18 11 J4. 108 43 15 20 3 super 'i' 26 5 1 2 0 95 29 9 14 1 super 'i' 87 33 10 19 0 100 60 30 22 0 54 28 12 4 0 84 50 15 15 0 110 56 11 19 0 41 26 7 9 0 34 23 9 5 0 47 30 9 11 0 144 55 16 18 0 930 438 190 206 4 super 'i' 'i' Grand Totals: Eggs Pupae 5335 2348 White eyed 'i' Wild type () Super 'i' Wilcltype'i' 995 940 6 l 31.7 per cent of the eggs developed probably indicates the presence of some lethal genes in the stock. Among the 1,935 total flies in Table 3, one apparently normal wild type fe­ male was produced by female B. This female may have resulted from a detach­ ment of the attached-X chromosomes, but since she died within a few days without offspring, it is impossible to be certain of her origin. Stock number 2 was tested in the same way as stock 1. The data are shown in Table 4. Only eight females were mated this time. One produced no offspring and is omitted from the table. Female G showed a significant deviation from the 1: 1 ratio of males to females . . Unfortunately, the presence of this deviation was not realized until after the offspring had been discarded. T ABLE 4 Fertility and sex ratio checks in attached-X females from Stock 2 Days 7 8 9 10 11 12 13 14 15 16 17 18 Totals ~Eggs 0 0 0 70 170 84 44 0 0 0 0 0 368 "'Pupae 0 0 0 34 46 45 28 0 0 0 0 0 150 ~ White eyed 'i' 0 0 0 14 20 15 11 0 0 0 0 0 60 ~ Wild type(I; 0 0 0 13 15 24 12 0 0 0 0 0 64 "'-Other 0 0 0 1 super 'i' 0 0 0 0 0 0 0 0 1 super 'i' i::ci Eggs 0 47 0 74 0 112 36 68 0 0 46 40 423 "'Pupae 0 0 0 40 0 25 0 0 0 0 0 0 65 ~ White eyed 'i' 0 0 0 18 0 10 0 0 0 0 0 0 28 ~ Wild type i1J 0 0 0 17 0 8 0 0 0 0 0 0 25 "'-Other 0 0 0 0 0 0 0 0 0 0 0 0 0 Eggs 24 88 113 85 114 71 72 150 50 36 56 128 987 0 "'Pupae 20 30 36 53 65 26 30 55 20 30 34 58 451 ~ White eyed 'i' 7 16 14 13 23 6 11 22 8 15 18 19 172 ~ Wild type(I; 10 11 13 21 24 14 8 18 7 13 13 15 169 "'-Other 0 0 0 0 0 0 0 0 0 0 0 0 0 49 83 118 140 82 87 92 108 33 60 65 80 997 48 50 50 46 18 52 20 34 44 42 443 Q Eggs "' Pupae 0 39 ~ White eyed 'i' 0 22 10 21 18 17 8 17 9 15 17 18 171 E: Wild type;t; 0 8 20 19 15 15 5 3 7 7 17 14 160 ~Other 0 0 0 0 0 0 0 0 0 0 0 0 0 42 0 69 68 90 0 79 147 33 52 73 38 691 ~Eggs ·"'Pupae 0 0 34 30 60 0 45 60 30 30 32 30 351 ~ White eyed 'i' 0 0 16 10 24 0 13 21 14 11 23 9 141 11 11 14 6 134~Wild type;t; 0 0 14 11 25 0 19 23 "'-Other 0 0 0 0 0 0 0 0 0 0 0 0 0 106 46 97 102 93 0 0 0 0 0 649"'-Eggs 109 96 58 45 45 0 0 0 0 0 304 "' Pupae 38 36 37 45 ~ White eyed 'i' 11 20 16 18 17 12 11 0 0 0 0 0 105 0 0 0 0 110 ~Wild type i1J 12 8 13 21 27 16 13 0 "'-Other 0 0 0 0 0 0 0 0 0 0 0 0 0 72 44 60 88 84 120 21 52 44 96 890C) Eggs 102 107 45 43 44 46 14 35 29 50 411 "'Pupae 0 34 35 36 28 18 16 17 7 15 12 18 176~White eyed 'i' 0 14 15 16 6 15 4 11 14 15 108 E: Wild type (I; 0 7 15 6 10 5 ~Other 0 1 w (I; 0 0 0 0 0 0 0 0 0 0 1 Grand totals Eggs 5005 Pupae 21 75 White'i' 853 Wild ;t; 770 Super'i' 1 White (I; 1 The University of Texas Publication Deviations shown by the other six females individually, and by all seven col­lectively, were not significant. Of the 5,005 eggs laid, 32.4 per cent developed into adults, again indicating the probability of some lethal genes in the stock. One white-eyed male was produced by female G. Although the male died within a few days, he probably originated from a detachment of the attached-X chromosomes. The fact that four stock 1 females and one stock 2 female gave no offspring is not considered at all· significant since individual matings in hydei are oft.en incompatible. Detachment of the attached-X in either stock is at best a rare occurrence. To check this further, 1,079 flies from stock 2 were examined for detachments. One. wild type female was found. This female was slightly rough-eyed and sterile, which indicates that she was probably a super female. Of 1,137 flies from stock 1, no::ie showed any possibility of having originated from a detachment. Since two stocks of attached-X were obtained by irradiation of white-eyed females, an attempt was made to produce an attached-X female heterozygous for a number of markers, suitable for cross-over studies. To do this the necessary mu­tant stocks were established and females, homozygous vermilion but heterozy­gous for several other mutations, less than 60 hours old were given 3,250 r at the rate of 740 r per minute. This treatment was given to several groups of females at both room temperature and lower temperatures but no cases of attached-X were detected. It is interesting to compare the X chromosome non-disjunction rate in this experiment with the spontaneous non-disjunction rate in the X chromosome of hydei, studied by Spencer ( 1930). The rate of non-disjunction in irradiated females is considerably higher than in the untreated females studied by Spencer. However this higher rate results in fewer nondisjunction female offspring from the irradiated parent, presumbably due to the tendency of tangled chromosomes to be lost in the polar bodies in meiosis. ACKNOWLEDGMENTS The writer wishes to express thanks to Prof W . S. Stone for advice and con­sultation during the course of the experiment. to Dr. L. E. Mettler for analvzing the salivary gland chromosomes, and to Prof. W. P. Spencer for providing all the Drosophila hydei stocks. SUMMARY White-eyed hydei females were subjected to 3,000 r of X-radiation. Among the offspring of the irradiated females, two attached X females were recovered. Analysis of the salivary chromosomes of F2 larvae from each attached-X fe­ male showed that no chromosome abnormalities were present other than the attached-X. The metaphase chromosome configuration of females from the at­ tached-X stocks is the same as the metaphase configuration of a normal male hydei. Detachment of the attached-X chromsomes is a rare occurrence. Studies in the Genetics of Drosophila REFERENCES Anderson, E. G. 1924. X-rays and the frequency of non-disjunction in Drosophila. Pap. Michigan Acad. Sci. 4: 523-525. -----, 1925. Crossing over in a case of attached-X chromosomes in Drosophila melano­gaster. Genetics 10:403-417. Beadle, G. W . and S. Emerson. 1935. Further studies in crossing over in attache:l-X chromosomes of Drosophila melanogaster. Genetics 20: 192-206. Bridges, C. B. and K. S. Brehme. 1944. The Mutants of Drosophila melanogaster. Washington. Carnegie Inst. Wash. 7. 257 pp. Emerson, S. and G. W. Beadle. 1933. Crossing over near the spindle fibre in attached-X chromo­somes of Drosophila melanogaster. Z. I. A. V. 65: 129-140. Morgan, L. V. 1922. Non-criss-cross inheritance in Drosophila melanogaster. Biol. Bull. 42:434­ 446. -----, 1925. Polyploidy in Drosophila melanogaster with two attached-X chromosomes. Genetics 10:148-178. Rhodes, M. M. 1931. The frequencies of homozygosis of factors in attached X-females of Dro­ sophila melanogaster. Genetics 16:375-385. Spencer, W . P. 1930. Primary non-disjunction in Drosophila hydei. Ohio Jour. Sci. 30:221-229. Sturtevant, A. H. 1931. Two new attached-X lines of Drosophila m elanogaster, and further data on the behavior of heterozygous attached-X's. Publ. Carnegie Inst. 421: 61-81. Welshons, W. J. 1955. A comparative study of crossing over in attached-X chromosomes of Drosophila melanogaster. Genetics 40: 918-937. XXL Effects of Irradiation on the Screw-Worm, Callitroga hominivorax (Coq.) GAIL KAUFMAN AND MARVIN WASSERMAN Since the eradication of the screw-worm, Callitroga hominivorax, from the island of Curac;ao by Baumhover and co-workers in 1954 (Bushland, 1955), workers i~ the fields of entomology and genetics have been studying the effects of irradiation on these flies with the intention of carrying out a project which would eradicate the screw-worm population from Florida. The work we have done in these laboratories, in conjunction with the U. S. Department of Agricul­ture's Bureau of Entomology and Plant Quarantine Laboratory at Kerrville, Texas, was performed in an effort to determine the following: 1) whether ir­radiated males would inseminate as many females as nonirradiated males when both were presented with a maximum number of virgin females; 2) whether there was any recovery of fertility in the irradiated males after they had used up the irradiated sperm; 3) whether there were any viable offspring from matings between irradiated males and non-irradiated females; 4) if spermato­genesis in irradiated males differed from spermatogenesis in nonirradiated, or normal, males; and, 5) what cytological effects the irradiation had on the cells in the testes. We wish to thank Dr. R. C. Bushland and his colleagues for supply­ing us with the. flies and pupae which were necessary for these experiments and for observing the egg masses laid by the females and the subsequent egg hatches. We also wish to thank Dr. Forbes Robertson for advising us on the best statistical analysis to use on the data compiled, and Dr. Wilson S. Stone for his help and advice throughout the experiments and for reading and criticizing the manu­script. METIIODS Half a population of five-day old C. hominivorax pupae were irradiated in air with 7500r at 250 KV, with a 1hmmCu-1mmA1 filter, from a Westinghouse X-ray machine. The irradiation rate was approximately 750r/ minute. These pupae were taken to Kerrville and reared at the USDA laboratory there. Upon emergence from the pupae cases, 100 of the irradiated males were numbered and each was put into a carton with 15 non-irradiated, or normal, virgin females from the same population. At the same time, 100 normal males from that popu­lation were also numbered and each put into a carton with 15 normal virgin females. At the end of one week, 10 cartons containing irradiated males and their females and 10 cartons containing normal males and females were brought to The University of Texas laboratory and the spermathecae of the females were checked for sperm content. At Kerrville, the remaining males of both groups were put into new cartons, each again with 15 normal virgin females, and the females which had been exposed to these males previously were allowed to lay their eggs. The egg masses were observed and a count was made of the subse­quent egg hatches. At the end of the second and third weeks, the same process Studies in the Genetics of Drosophila was repeated. Three experiments were run in this manner, each one lasting for three weeks only, since most adults live for only three weeks after emergence from the pupae cases. For the cytological studies of the irradiated male testes, 5-day old pupae were irradiated as described above. Starting with one-half hour after irradiation, the testes were dissected out in saline solution, fixed and stained with aceto-orcien, and squashed. Slides were made every half hour for three hours and then every hour up to 15 hours after irradiation. At 28 hours after irradiation, slides were again made. Some pupae were also given 1160r at 210KV, at a rate of 340r/ ffiinute, with the same filter, in order to see what effects a lower dosage had on the testes. Slides of the normal testes were prepared in the same manner as slides of the irradiated testes. For the comparative studies of the testes themselves, we attempted to keep the organs as nearly intact as possible and did not squash them, although they were fixed and stained as were those used in the cytological studies. EFFECTS OF IRRADIATION ON INSEMINATION RATE Results and Discussion The data for the three insemination studies are presented in Tables 1, 2, and 3. The males are identified by their numbers, and the number of virgin and number and per cent of inseminated females is given for each male. We generally re­ceived a random sample of the population here, with little effort being made to get ten consecutively numbered males. For each week the per cent of females inseminated was calculc,ited for each individual male and the over-all percentage of inseminated females for that particular group was also calculated. In some cases there were one or two dead females in the cartons and these were not checked for sperm content, since we had no way of knowing how long they had been dead. When there were either two males or a dead male in the carton, none of the females were checked. It is obvious from a cursory glance at the data that the three populations studied differed from one another, and at times quite significantly so. Also, within each population and within each week's lot of males, there was a great deal of variation among the individual males of both types in insemination activity. The flies in the first population, experiment 1, did not survive as well as flies of the second and third groups. The irradiated males began dying in large num­bers in the second week, and by the third week there were large numbers of males dead in both the irradiated and normal groups. In the first week of experiment 2, the flies were brought to our laboratory several days in advance of the end of the week and many of the males and fe­males died here in the lab, the greatest percentage of deaths being in the irradi­ated males. Whether it was the change in environment or a shortage of water, we do not know with certainty, but the high mortality and poor condition of the re­maining flies unfortunately skewed our results in this particular phase of the experiments. In experiment 3, all irradiated males were dead by the end of the third week. There are several reasons for high numbers of early deaths: a change in the environment, scarcity of food and water, and parasites. Throughout the experi­ TABLE 1 Data from insemination studies, Experiment 1. Normal Inseminated Virgin %Insem. Irradiated Inseminated Virgin %Insem. (; (; 'i' 'i' 'i' 'i' 'i' 'i' (; (; 'i' 'i' 'i' 'i' 'i' 'i' No. 3 5 9 35.7 No. 3 5 10 33.3 6 5 9 35.7 6 7 8 46.6 8 8 6 57.1 10 4 9 30.7 1st 10 (2,t; )* . . 11 6 8 42.8 week 11 5 10 33.3 16 4 11 26.6 15 6 9 40.0 21 7 7 50.0 17 3 12 20.0 22 4 11 26.6 20 1 14 6.6 24 4 11 26.6 22 7 8 46.6 26 4 11 26.6 24 8 6 57.1 27 6 9 40.0 48 83 Av. 36.6 51 95 Av. 34.9 4 5 10 33.3 8 5 9 35.6 13 6 9 40.0 9 2 13 13.3 23 4 11 26.6 12 0 14 0.0 23 4 11 26.6 12 0 14 0.0 2nd 33 7 7 50.0 13 D week 34 4 10 28.5 18 (3,t; ). 37 1 12 7.6 36 D 38 2 13 13.3 37 D 39 2 13 13.3 38 D 40 5 9 35.6 44 D 48 5 10 33.3 53 D 50 6 8 42.8 60 D 47 112 Av.29.5 7 36 Av.16.2 3rd wee];; 74 71 35 51 57 65 87 79 97 D D D 0 0 D D D 0 15 13 15 0.0 0.0 0.0 48 74 76 77 82 91 94 96 78 2 D 0 0 D D D D D 13 15 15 13.3 0.0 0.0 88 84 92 93 2 D D D 12 14.3 2 43 Av. 4.4 2 55 Av. 3.5 D =no test; dead male. •=no test; males in carton. ments, from time to time there would be an outbreak of parasites at the hatchery in Kerrville, and it is quite possible that these parasites were responsible for some of the mortalities and the general poor condition of many of the adults. It is perhaps significant, however, that those flies which were more quickly affected by adverse conditions and which died more quickly and in greater numbers were the irradiated males. For a statistical comparison of the effectiveness of the irradiated males against the normal males in the number of females inseminated, we ran chi square tests on our data. The results are presented in Table 4. There were three tests which showed significant differences between the insemination rates of the two sets of males-the first and third weeks of experiment 2 and the second week of ex­periment 3. The first week of experiment 2 may, due to the high mortality in both groups, be ignored. Also, the data for the third week of the same experiment are based on a small sample of irradiated flies, as compared to the sample of the normal males, which, because of the great degree of individual variation in these animals, may account for some of the difference. For the significance in the chi square value in the second week of experiment 3 we have no explanation other than the fact that the irradiated males died much more rapidly in this ex­periment. It is significant, we believe, that the chi square values are the smallest, and the probability range the highest, in the first week of experiments 1 and 3. This would probably be the critical week in competition between released irradiated flies and the normal flies in nature. It is also the week when the insemination activity of the normal flies is the highest. Although we found that during this week the irradiated males could inseminate as many females as the normal males TABLE 2 Data from insemination stuclies, Experiment 2. Normal Inseminated Virgin %Insem. Irradiated Inseminated Virgin %Insem. $$ P > 0.75 12.39 0.005 > P 0.177 0.75 > P > 0.50 2 3.03 0.10 > P > 0.05 0.69 0.50 > P > 0.25 25.36 0.005 > P 3 0.63 0.50> P > 0.25 4.38 0.05 > P > 0.02 .... .. .. .. .... when they were isolated with a maximum number of females in individual car­tons, our data do not show what the insemination rate of the two groups would be in a situation where both types of males were exposed to the same females at the same time. (For further information on this aspect, see Bushland, 1951.) The decline in activity of both types of males during the second and third weeks, as they got progressively older, can best be appreciated by the graph in Figure 1. Here we have taken, for experiments 1 and 3, the insemination rate of the normal males in the first week of each experiment and let this represent 100 per cent efficiency for that experiment. It is obvious from this graph that during the first week the efficiency of the irradiated males in insemination is favorably comparable to that of the normal males. Both groups in both populations decline in per cent of females inseminated in the second and third weeks, with the irra­diated males showing the greatest decline. In the third week of experiment 1, the per cent of females inseminated by the normal males shows an unexpectedly steep drop. This could well be a reflection of the extremely high mortality of the three-week old flies in this particular population, indicating that possibly those males which were still alive, when we checked for sperm in the females, were in 100 90 80 ~ 0 z 70 l&J 0 IL IL 60 la.I 3 0 z 50 ti z 40 ~ l&J U) z 30 ~ l&J 20 0 a: l&J ~ 10 0 WEEK WEEK WEEK I 2 3 Fm. 1. A comparison of efficiency of insemination between normal and irradiated males. poor condition. We have no data for the inseinination rate of the irradiated males for the third week of experiment 3, since those males were all dead when we received the cartons in the laboratory. At Kerrville, the egg masses laid each week by the females, and the subsequent egg hatches, were counted. Because several of the females from a particular male laid their eggs in the same cage and quite often one on top of another, the counts were often approximations. Since even virgin females lay, theoretically, a full complement of eggs, we would have expected the percentage of egg masses hatch­ing to follow fairly closely the percentages of females inseminated, for the per­centage of egg hatches was figured on the number of egg masses which hatched divided by the total number of females in the cage. There was a discrepancy, however, between our figures for the per cent of females inseminated by normal males and the Kerrville figures for the per cent egg hatch for the normal popu­lations studied. Part of this discrepancy may have been due to the gregariousness of the egg-laying females and the consequent necessitated approximations in the egg mass and egg hatch counts. The most significant observation on the egg hatches was with females which had been mated to irradiated males. Although these females laid as many egg masses as did the females which had been mated to normal males, no eggs hatched from their egg masses. The irradiated males were completely sterile and showed no recovery of fertility after irradiation. This, coupled with the fact that the insemination rate of these males in the first week after emergence from their pupae cases compares favorably with that of the normal males, adds to the like­lihood of possible future eradication of the screw-worm from Florida and South­eastern United States. EFFECTS OF IRRADIATION ON THE TESTES Results and Discussion We have compared the normal testes with the testes of the irradiated flies. There is variation from fly to fly The following account is a general description of the sequence of events in the aging of the flies. An examination of 5-day old pupae revealed two ovoid testes less than one millimeter in length. Approximately one-tenth of the testis was composed of spermatogonial cells, four-tenths spermatocytes, and one-half sperm and sperma­tids. During the next two days there was very little growth. A layer of tissue composed of binucleate cells formed and surrounded the testis. This layer of cells persisted throughout the life span of the flies. The testes had begun to turn golden yellow. Examination of normal adults, ten days after emergence, revealed some spe~­matogonial cysts. Approximately one-fourth of each testis contained spermato­cytes. Sperm and spermatids filled up the remainder. The irradiated testes at this age had much more pigment, were dark orange in appearance, and were elon­gated rather than ovoid. Only sperm were present in the irradiated testes. The testes of 17 -day old normal males were elongated and orangish, looking somewhat like those of the 10-day old irradiated flies. There were still some meiotic cells, although most of the testis was filled with sperm. The irradiated males had very elongated, thin, almost collapsed testes, which were highly pigmented. Very few sperm were present. Normal males at 24 days were found to have some meiotic cells present in the testes. There was essentially no change from the 17-day old males, except that the testes were beginning to empty. Some sperm were still observed in the testes of the irradiated flies at this time. EFFECTS OF IRRADIATION ON SPERMATOGENESIS Results and Discussion The normal karyotype of the male screw-worm is five pairs of autosomes and the X and Y chromosomes. The Xis an acrocentric with a large terminal satel­lite. The Y is a short rod. One of the pairs of autosomes is a metacentric with arms of equal length. Three other pairs are metacentrics with unequal length arms. The fifth pair of autosomes is J-shaped, having one of the arms very short. Figure 2 shows an idiogram of the haploid set. Both the X and the Y are included. Figure 3 shows a metaphase of a spermatogonial cell. A metaphase plate of the female, taken from a brain cell, is shown in Figure 4. The X and Y are indicated in each camera lucida drawing. In the meiotic divisions a definite equatorial plate was observed in metaphase. In the first division the sex bivalent was seen in the center of the equatorial plate, surrounded by the autosomes. The X and Y were oriented toward the poles, attached by their terminal ends. No chiasmata were seen. The first division was reductional for the sex chromosomes. Figure 5 is a camera lucida drawing of a first division metaphase. An early anaphase is shown in Figure 6. The X and the Y appeared negatively heteropycnotic. The chromosomes were somewhat smaller in the second meiotic division. One could usually differentiate between those cells with the X chromosome (Figure 7) and those with the Y chromosome (Figure 8) in the second metaphase. From the pupae irradiated with 7500r, a series of slides was made beginning with one-half hour after irradiation and continuing until 15 hours after. Several slides were made 13 hours after this to see if there were any marked changes in cytological effects with a longer period of time postirradiation. We checked over 1350 cells in anaphase, telophase and daughter cell stage and we also checked about the same number of metaphase plates. After a dose of 7500r, there were no normal divisions observed in either meiosis I or II. All of the cells seen from the anaphase stage on had at least one chromosome bridge, and many had from 2-4 distinct bridges. The higher number of bridges was generally more evident in middle and late anaphase. In early telophase, quite often bridges were seen which appeared to have broken im­mediately before, or perhaps, upon fixation (see Figure 9). Camera lucida draw­ings of several types of bridges are shown in Figures 9-13. In Figure 10, a typical anaphase cell, it will be noted that there is no clear line of demarcation between the chromosomes towards the poles and throughout part of the bridge area. The vast majority of anaphase cells, in both meiosis I and II, exhibited this charac­teristic, which we attribute to the phenomenon termed "stickiness" by various authors (Sax and Swanson, 1941; Carlson, 1954). At no time did we see an acentric fragment in the 1350 cells that we studied. This does not, however, , I y x 2 3 y.!x ~~ 1(( FIG. 3 y ~~1c~ x FIG.5 _. t'J.-x:.,.$ FIG. 7 4 5 6 FIG. 2 ;If,r;. tt xx .Olmm. FIG.4 tffJi ~ -v FIG.6 .,~ '-"~< FIG.8 FIG.9 FIG. 10 FIG.II PLATE 2 The post-irradiation chromosomes of Callitroga hominiuorax. Fie. 9. Cell in telophase, showing bridge formation. 9 hrs. after irradiation with 7500r of X-rays. FIG. 10. Cell in late anaphase, showing stickiness effect. 13 hrs. post-irradiation, 7500r. Fie. 11. Unequal division, late telophase cell. 28 hrs. post-irradiation, 7500r. invalidate the proposal by Sax and Swanson ( 1941) that the acentric fragments which they observed in Tradescantia were due to stickiness and subsequent break­age of sister chromatids. It is quite likely that our material was given such a high dosage that not only the chromatids and chromosomes exhibited stickiness (Carlso.n, 1954), but that any fragments which were broken off also tended to adhere to the centric chromatids or chromosomes and were taken along to the poles in the division process. Muller ( 1954) suggested that in spermatocytes there are spatial difficulties involved in both recombination and restitution of broken chromosomes or chromosome fragments. While it is true that the spermatocyte chromosomes appear to be more widely separated than those in the spermato­gonial cells, we feel that acentric fragments were able to overcome the spatial difficulties of such recombinations, as were centric fragments, possibly because of the fact that a dosage as high as 7500r would speed up the molecular motion to a very great extent. There would thus be a greater chance for recombination between centric fragments, along with a greater chance of the acentric fragments coming into contact with centric portions of chromatin and being taken along to the poles in this way. Whether any given fragment would thus go to the right pole would probably be a matter of chance. Obviously unequal divisions were evi­dent in many of the cells (Figure 11). These, coupled with the fact that we ob- PLATE 1. The chromosomes of Callitroga hominivorax. FIG. 2. Idiogram of the chromosomal complement. FIG. 3. A metaphase of a spermatogoni'll cell. The X and the Y are shown. FIG. 4. A metaphase plate of a female ganglion cell showing two X chromosomes. FIG. 5. Metaphase I of the male. The X and Y show terminal association. FIG. 6. Early anaphase I of the male. FIG. 7. Polar view of metaphase II. This cell has an X chromosome. Frn. 8. Polar view of metaphase II. This cell has a Y chromosome. served, on several occasions, three nuclei joined by bridges, as in Figure 12, have led us to believe that perhaps a primary spermatocyte had undergone a division which resulted in two aneuploid nuclei joined by a bridge, and that the bridge remained while one of these nuclei divided again, with a bridge formed in the second division also. Figure 13 illustrates an intermediate stage in this process. Here are shown two meiotic cells, the nuclei of which are dividing, but which are also joined by a definite bridge from the previous meiotic division. Although the final division of these two cells, with bridge formation in each, would give four nuclei·connected by bridges, we unfortunately never observed cells which por­trayed the final stage of this particular phenomenon. Another explanation of the phenomenon of 3 or 4 nuclei joined by bridges would be the formation of a multi­polar spindle. As far as we know, however, only extremely high dosages of X­irradiation have been known to produce this effect on the spindle (Henshaw, 1940 and 1941 ). FIG. 12 FIG. 12. Three nuclei joined by chromosome bridges. 11 hrs. after irradiation, 7500r. The cause of the bridges is in some cases undoubtedly chromosome stickiness. We do feel, however, that even though we never saw an acentric fragment in any of these cells, there was probably a certain amount of recombination, especially in those cells which were in the early stages of division when irradiated. At a lower dosage of irradiation, which will be discussed later 'in this paper, we did find acentric fragments with bridge formation. In his 1954 paper, Carlson mentioned the fusion of Chortophaga chromosomes, in prometaphase, metaphase or anaphase, if they were irradiated at those stages. Evans (1956) produced this same clumping, in Chortophaga, by cooling the ma­terial he studied. Carlson also stated that the chromosomal mass thus formed "elongates in the direction of the poles and appears to be divided by a pressing inward of the cleavage furrow". Although our experimental design eliminated observation of material immediately after irradiation, we did find clumping, or fusion, of the chromosomes in most of the metaphase plates seen, even up to 28 hours postirradiation (Figure 14). We also noticed that there were many divi­sions, as illustrated in Figure 15, in which the chromosomal mass seemed to be elongating toward the poles with a general thinning out between the two poles. We did not see a cell in which a noticeable cleavage furrow was apparently re­sponsible for the ultimate constriction and division of such a mass. The chromosomes in metaphase and anaphase, even at 28 hours postirradia­tion, looked coagulated. That is, instead of the arms being the same diameter Studies in the Genetics of Drosophila ~hroughout, in places they seemed pinched in and in other areas,. swollen. This is, perhaps, a similar phenomenon to that noted by Carlson (1940) in.his studies ?.n mitosis in the Chortophaga neuroblast, in which he saw chromosomes with a .beaded" appearance after an X-ray dosage of 250r. Although he states that, smce these chromosomes went through mitosis in an apparently normal fashion afterwards, this was a reversible change as opposed to a degenerative one, we feel that the coagulated chromosomes we saw in Callitroga pupal testes represent a ?egenerative change and that the constricted areas were possibly breakage pomts. This latter has previously been suggested by White (1937). We base our FIG.14 Fie. 15 f 8 f G~ I FIG. 18FIG. 16 FIG. 17 PLATE 3 ' The post-irradiation chromosomes of Callitroga hominivoraz. FIG. 13. Meiosis II division, showing bridge remaining from meiosis I and further bridges in the current division. 13 hrs. post-irradiation, 7500r. FIG. 14. Metaphase plate, showing clumping of chromosomes. 28 hrs. post-irradiation, 7500r. FIG. 15. Elongation in late anaphase of pre­viously clumped chromosome mass, Yz hour after irradiation, 7500r. FIG. 16. Degenerating nuclei, 28 hrs. post-irradiation, 7500r. FIG. 17. Chromosome fragment in late anaphase cell, 2Yz hrs. after irradiation with 1160r. FIG. 18. Chromosome fragments and bridges in anaphase cell, 2% hrs. after irradiation with 1160r. belief that the change was a degenerative one on the fact that we never found a normal meiotic division in process in all of the cells examined. There were also many degenerating nuclei evident at all postirradiation stages studied. The number seen seemed to increase at about 41/2 hours after irradiation, and at 5Yz hours there were large numbers of such cells. After that there was neither an apparent increase nor decrease in their number. A camera lucida drawing of several of these nuclei is shown in Figure 16. It is also evident in Figure 12 that the 3 nuclei joined by bridges are becoming vacuolated and are disintegrating. In addition, there were no more sperm in the slides made 28 hours after irradiation than there were in the half-hour slides, as far as noticeable increase in numbers is concerned. The individual sperm were not counted. It seems quite likely, from both these slides and the studies of the testes themselves, that the dividing cells and spermatogonial cells disintegrated before the formation of mature sperm. Because of the absence of fragments and great stickiness of the chromosomes after irradiation with 7500r of X-rays, we irradiated several dozen pupae with 1160r in order to see if a lower dosage had similar effects. As in those irradiated with 7500r, we noticed metaphase clumping, distortion of the chromosomes, stickiness, and many bridges. We examined about 100 cells in anaphase, ·telo­phase and daughter cell stage. We found three cells in late anaphase which may have been normal divisions. They had faint indications, however, of a "halo" effect which was found surrounding the bridges seen in other cells. This "halo", appearing as a light area running linearly between the poles, may have been the spindle, or may have been caused by the spindle. There were no chromosome bridges or indications of bridges present. We also found several cells in late ana­phase which showed possible deletions (Figure 17), and several others which contained acentric fragments, probably due to either translocations, isochromo­some formation, or deletions. An example of this type of cell is shown in the camera lucida drawing in figure 18. There were quite a few degenerating nuclei. On the whole, the visible chromosome distortion and damage seemed less marked. The clumping was not as extreme nor was the stickiness as widespread. In conclusion, the 7500r X-ray dosage apparently caused permanent damage to both meiotic cells and spermatogonia. The large number of degenerating nuclei accounts for the fact that there was no apparent increase in the number of sperm in 28 hours postirradiation over the number seen half an hour after irradiation. It also explains the decrease in the amount of sperm in the testes of irradiated males much sooner than in the normal males during the insemination tests. Furthermore, it is obvious from the fact that no meiotic cells were seen in the testes 12 days after irradiation, while normal males still had quite a few cells undergoing meiosis at this time, that the spermatogonia were adversely affected by the X-rays. The chromosomal aberrations and stickiness as late as 28 hours postirradiation furnish further evidence for this fact. That the irradiated sperm themselves carried a dominant lethal effect is indi­cated by the complete sterility of all matings in which the irradiated males were involved. This dominant lethal effect was probably due to broken or fused chromosomes which, upon union with the normal chromosomes of the egg, were unable to organize normal development. Studies in the Genetics of Drosophila SUMMARY 1. Screw-worm flies, Callitroga hominivorax, were irradiated with a dosage of 7500r of X-rays. 2. From the data compiled in our insemination studies, it was seen that in the first week after emergence from the pupae cases the irradiated males could in­seminate as many females as could the normal males when both types were iso­lated with a maximum number of normal virgin females. 3. No larvae developed from matings between irradiated males and normal females, even after three weeks of matings. This dosage produced complete sterility, with no recovery of fertility, in the male screw-worm. 4. The dominant lethal effect in the sperm was probably due to induced chromosome breakage. 5. The irradiation caused permanent genetic damage to both spermatogonial cells and spermatocytes, as well as to spermatid and sperm, as evidenced by the presence of stickiness and bridge formation in all of the dividing cells seen, the great number of degenerating nuclei observed, and the fact that there was no apparent increase in the amount of sperm in the testes after irradiation. 6. The bridges seen in the dividing cells could have been due to recombina­tions, or chromosome stickiness, or both. REFERENCES Bushland, R. C. and D. E. Hopkins. 1951. Experiments with screw-worm flies sterilized by X-rays. Jour. Econ. Entom. 44(5).:725. Bushland, R. C., A. W. Lindquist, and E. F. Knipling. Eradication of screw-worms through re­lease of sterilized males. Science 122:287-288. Carlson, J. G. 1940. Immediate effects of 250r of X-rays on the different stages of mitosis in neuroblasts of the grasshopper, Chortophaga viridifasciata. J. Morphol. 66: 11-23. ----. 1954. Immediate effects on division, morphology, and viability of the cell. Radiation Biology 11:763-817, McGraw-Hill Book Co., Inc., New York. Evans, W. L. 1956. The effect of cold treatment on the desoxyribonucleic acid (DNA) content in cells of selected plants and animals. Cytologia 21 (4) :417--432. Henshaw, P. S. 1940. Further studies on the action of roentgen rays on the gametes of Arbacia punctulata. VI. Production of multipolar cleavage in the eggs by exposure of the gametes to roontgen rays. Am. J. Roentgenol. Radium Therapy, 43:923-933. ----. 1941. The induction of multipolar cell division with X-rays and its possible sig­nificance. Radiology 36:717-724. Muller, H . J. 1954. The nature of the genetic effects produced by radiation. Radiation Biology 1(1) :351--473, McGraw-Hill Book Co., Inc., New York. Sax, K. and C. P. Swanson. 1941. Differential sensitivity of cells to X-rays. Am. J. Botany 28:52--59. White, M. J. D. 1937. The effect of X-rays on the first meiotic division in three species of Orthoptera. Proc. Roy. Soc. London, Series B, 124: 183-195. XXII. Genetic Studies of Irradiated Natural Populations of Drosophila1 ' 2 WILSON S. STONE, MARSHALL R. WHEELER, WARREN P. SPENCER, FLORENCE D. WILSON, JUNE T. NEUENSCHWA NDER, THOMAS G. GREGG, ROBERT L. SEECOF, CALVIN L. WARD3 Genetics Foundation, Department of Zoology, University of Texas The genetic structures of populations and their changes through time are important in the evolution of living forms. Few populations are sufficiently iso­lated from neighboring populations that they can be regarded as isolated units. Some islands or similarly isolated areas are of this kind. Very few of these iso­lated populations are satisfactory for the study of changes in the genetic structure of the populations. The Drosophila ananassae populations of the Marshall Is­lands and of the eastern Carolines provide this opportunity. In order to determine the changes in genetic structure of the populations through space and time, col­lections were made in 1955 and 1956 and these were analyzed in a number of ways. These analyses are given in the several sections of the paper. ACKNOWLEDGMENTS The authors wish to express their gratitude to Mr. Thomas Hardison, A.E.C. representative at the Pacific Proving Ground, for his continuous help and co­operation, to many civilian and military personnel at the Pacific Proving Ground who assisted us, and to the members of the administrative staffs of the Trust Territory of the Pacific Islands, especially Mr. Maynard Neass, District Adminis­trator, and Mr. Tony Cruz, Agriculturist, on Majuro, and to Mr. Henry Hedges, DistrictAdministrator on Ponape and his administrative assistant, Mr. Henry Takeshita; we wish also to acknowledge the hospitality and friendliness of the many other people on the various islands who made our visits both interesting and pleasant. We also desire to express our appreciation to Professor Robert W. Hiatt, Dean of the Graduate School, University of Hawaii and Director of the Eniwetok Marine Biological Laboratory, also to the members of the Division of Biology and Medicine of the Atomic Energy Commission in Washington who made the work possible through the support of the Atomic Energy Commission. The Welch Foundation provided funds for the X-ray machine used by Seecof. The Rockefeller Foundation provided funds for general support to the genetics laboratory and for support of some of the graduate students. We wish to express our indebtedness to Professor Forbes W . Robertson who was kind enough to as­sist us with the statistical analyses. 1 This work was supported by a contract with the Atomic Energy Commission [AT-(4-0-1)­1323]. 2 Department of Biology, College of Wooster, Wooster, Ohio. a Department of Zoology, Duke University, Durham, North Carolina. Studies in the Genetics of Drosophila SECTION I GEOGRAPHY, ECOLOGY AND RADIATION MarshallR. Wheeler, WarrenP. Spencer, Calvin L. Ward, and Wilson S. Stone Drosophila ananassae has a broad circum-tropical distribution and has been reported occasionally from more temperate climates. In the Micronesian islands it is clearly the dominant species of Drosophila. We have collected this species in the Marshall Islands and on Ponape, a "high island" in the eastern Carolines. Chart 1 gives the general location of the collecting areas in relation to Eniwetok ENIWETOK ATOLL 10° MAJURO .-, !Ao" : ATOLL ~' >~.~ ~ ' PONAPE ~'·~ KUSAIE ~ 160° 165° 170° The Marshall Islands area. and Kwajalein atolls. Our collections were made working out of the Atomic Energy Commission Pacific Proving Ground base on Parry Island, Eniwetok Atoll. The flies were shipped airmail or were carried directly to Austin for labora­tory analysis. Collections were made in August and early September, 1955, on Bikini, Rongelap, Rongerik and Majuro atolls; and in August, 1956, collections were made on Bikini, Rongelap, Rongerik and Majuro atolls, and on Ponape Island. Only Drosophila ananassae was found in the northern Marshall Islands, while in the south (Majuro) and on Ponape, several other .species occur. On Majuro, a few D. melanogaster and D. bryani were taken, but D. ananassae was extremely common. On Ponape D. ananassae, D. hypocausta, and D. spinofemora were all common, and D. melanogaster, D. bryani, and D. kikkawai occurred in much smaller numbers. The University of Texas Publication The natural food sources varied from island to island. On Bikini the ananassae population probably maintains itself primarily on the fallen fruits of Morinda citrifolia, with less extensive breeding occurring in fruits of papaya and possibly pandanus. On Rongelap, where large populations of ananassae were found, papaya and Morinda were common, and there were also several fruiting bread­fruit trees ( Artocarpus sp.). As has been pointed out by Gressitt ( 1954) the pro­ductivity of one rotten breadfruit in terms of individual Drosophila flies is enor­mous. In 1955 on Eniwetok Island, Rongerik Atoll, the only available food ap­peared to be the small fruit of Guettarda speciosa, and the population of ananas­sa.e was very small. In 1956 we were unable to collect the species on this island, but on the nearby island, Rongerik, a few hours collecting over Morinda fruits yielded a moderate number of flies. Majuro Atoll, in the southern Marshalls, had Morinda, papaya and breadfruit of :which Morinda fruits which had fallen into a pigpen proved to be the best for collecting purposes. On Ponape the opportunities for Drosophila breeding were more extensive, with considerable amounts of breadfruit and banana. There was some variation in the two years in the amount and type of fruit available. On Bikini, for example, papaya fruits were fairly common in 1955, while in 1956 there were very few and collections could be made only from Mo­rinda and Pandanus fruits although some flies were taken at baits of bananas and yeast mixtures. In the Marshall Islands the morning and evening temperatures have a mean around 78° F. The usual range is 77° to 87° (at midday) but more extreme tem­peratures do occur (data provided by the A.E.C.). This range corresponds to that reported by Pipkin (1953) for Moen Island, Truk Atoll. The humidity is gen­erally high, and although rains are frequent and heavy throughout the area, the northern Marshalls are clearly drier than the southern portions, while Ponape has the highest rainfall of all. Availability of food for natural breeding is seriously reduced on several islands by competitors, consisting primarily of rats and land crabs. On Eniwetok, for example, there was a small population of Drosophila in 1955 but by August of the following year the increase in numbers of rats was so extreme that little fruit remained available for flies. Three kinds of predators were fairly common: skinks, toads and spiders. On Ponape, toads (Bufo marinus) were especially com­mon, and often congregated in a circle around the fruit being used as bait. On all of the islands, small web-building spiders were extremely numerous and often seriously hampered our collecting. We did not record the actual numbers of Drosophila collected on the various islands. On Rongerik we were able to collect a few hundred flies in about three hours, collecting over fallen fruit and yeast bait. On Rongelap ( 1956) we col­ lected well over 6000 flies within an hour, primarily under breadfruit trees, and there seemed to be no diminution of flies as we collected. The population on Bikini was somewhat larger than that of Rongerik; in 1956 we collected approximately 2000 flies in four mornings using several collecting stations. On both Majuro and Ponape, it was possible to collect 6000 flies within an hour at one locality; on one occasion, on Ponape, we collected a large number of flies in an open room con­ taining bananas and other fruit. Studies in the Genetics of Drosophila Pipkin (1953) collected for a year (1951-52) on Moen Island, Truk Atoll. D. ananassae was the most numerous of the four species she collected. Using pro­tected trap cans baited with fruit and placed close to breadfruit and papaya plants, she observed that the ananassae population increased considerably when the breadfruit matured twice a year. The population of ananassae on Moen was neither minimum nor maximum during August, being about one-eighth the size of the population at that locality during the two months when breadfruit was available. The two other species numerous enough to score, hypocausta and anuda, were also relatively low in August, so that this seems to be a less favorable season of the year for Drosophila. Patterson (1943) and Dobzhansky and Pavan (1950) showed that in both temperate and tropical localities, various species have cycles of abundance which do not necessarily coincide with the cycles of other species. At the present we are unable to say when the peak population of ananassae occurs in the Marshall Islands. The amount of radiation to which the various tested Drosophila populations have been exposed is unknown, although certain minimum amounts can be sur­mised from other data (Cronkite, Bond and Dunham, 1956) . The Bikini Island population received considerable direct radiation during the Castle Operation in the spring of 1954 as well as in tests of previous years. It received a relatively small amount in the Red.wing test series of 1956. Following the explosion of a thermonuclear device March 1, 1954, the wind deposited radioactive materials not only on Bikini but also on Rongelap and Rongerik atolls. Control ananassae populations of Majuro and Ponape received negligible radiation from fallout as far as our measures of genetic effects on the populations are concerned. Sondhaus et al. (1956) estimated that at three feet above ground level the cumulated gamma radiation from fallout (assumed completed in about 12 hours) gave a dose of approximately 200 r in 48 hours (or about 4000 mr/ hour) on both Rongelap and Rongerik atolls. This decayed so that five days later the rate was about 375 mr/ hour and in two more days about 280 mr/ hour. On Bikini the fallout (over and above the direct radiation) cumulated to about 800 r in the first 48 hours. We cannot, however, even approximately guess the dose received by the Drosophila. Most adult and immature stages are at ground level where in­tensity is greatest, and their small size also makes beta rays effective. Further­more, they and their descendants remained on the islands and obtained their food from sources contaminated with radio-active materials. Cohn et al. (1956) state as follows: "The importance of ingestion as a continuing source of contamination is evi­denced by the level of internal contamination of the pigs from Rongelap. These animals had about ten times the body burden of the human population in the same locality. As the air-borne activity had already dropped to a low value at the time of evacuation of the humans, the contamination of the pigs during their pro­longed stay on the island necessarily derived from ingestion of radio-active food and water. "Radioanalysis of water and soil samples from Rongelap indicated high levels of contamination from the fallout at early times following detonation. "It appears that during the first month a limited amount of fission products was available to plants growing on the contaminated soil. Significant amounts of beta activity as well as small amounts of alpha activity were present on the external surface of plants at 42 days post detonation. Only very small amounts of beta The University of Texas Publication activity and no alpha activity were detected in the edible portions of fruits such as pandanus, papayas and coconuts. However, high levels of activity were found in the coconut tree sap, and the isotopic concentration was very similar to that of water." Y91 They list as biologically hazardous internally deposited fission products Sr89 , , Zr95 Ru103 Ru106 P 31 Ba140 La140 Ce144 , , , , , , Ce141, Pr143, which produce beta or beta and gamma radiations and suggest that Sr8 9 is the most hazardous. Since Dro· sophila larvae live in fruit and sap and since these are also food for the adults, such sources could keep the level of radioactivity available internally to Drosophila high even after the air level of radiation had fallen. These authors report on their studies of animals from the fallout islands. They point out that a much greater amount of the activity from internal deposition of radionuclides is removed from the body of the chicken through egg laying than the amount excreted in urine and feces during their study. They state: "Studies made on egg production of contaminated hens gave no evidence of any effect of radiation. The rate of production and the eggs produced were both normal. The extraordinary ability of fowl to mobilize calcium in shell formation resulted in the presence of very high activity in the shells of the first few eggs. The activity was associated with the fission products of the alkaline earth group. A significant amount of activity was found in the yolk, and lesser amounts in the albumen. The removal of activity from the body of chickens by egg production provides an effective natural decontamination process." If Drosophila concentrate radioactive materials in their gonads-and the storage products in their eggs have many similarities to the hen's egg yolk-then the amount of effective soft radiation to the germ cell chromosomes would be much higher than the radiation in the air around them. All these facts would seem to indicate that we can only estimate a minimum amount of radiation damage to the genetic structure of a population such as Drosophila from measurements of the radiation in air and other sources. SECTION II COMPETITION TESTS WITH DROSOPHILA ANANASSAE Florence D. Wilson, June T. Neuenschwander, and Wilson S. Stone One of the interesting problems of these island populations is why ananassae is the dominant or a major species. A second and related problem is the effective­ness of ananassae in larval competition, which must be a major factor in theit survival. Some of these factors were studied in population cages of the type de­vised by L'Heritier and Teissier (1934). In the tests the cages were started with equal numbers of each species-1000 ananassae plus 1000 of another species. Under these circumstances the population builds up so that a great excess of eggs is laid on the food soon after a new container is added. Preliminary experiments in the spring of 1955 showed that D. funebris, D. busck#, D. novamexicana could not survive long in competition with the more rapidly 'reproducing ananassae (a strain from Central America). D. virilis survived for several generations but D. melanogaster (ebony11 mutant) was a very effective competitor. Studies in the Genetics of Drosophila The data on competition in Table 1 were obtained by placing four fresh con­tainers of food in the cage and leaving them about five days. A sample was taken every thirty days approximately. Two of these food masses with many larvae and eggs were transferred to bottles and the flies allowed to develop (restricted food). Two were spread into two added bottles of the yeast-banana-agar food so that the larvae had about three times as much food available as the others (in­creased food) . These spread bottles reduced the intensity of competition and provided increased opportunity for the less effective competitors to develop. In the cages of ananassae and melanogaster, two fresh containers of food (banana, yeast, agar, propionic acid) were added Tuesdays, Thursdays and Saturdays, so that all the food in the cage was replaced every 16 days. In competition with virilis and the other longer life cycle forms, one fresh container was added Mon­day, one Wednesday and two Friday so a full food change for a cage required 28 days. ' Table 1 and Figure 1 show that ananassae can displace virilis at both temper­atures. The points on the figure represent the percent of ananassae. The replace­ment occurs more slowly at 71 ° ± 1 (tests 1-4) than at 77° ± 1 F (tests 5-8). In the figure the numbers refer to the tests given in Table 1, solid lines to changes measured on restricted food, and dotted lines to changes measured when the food in the cups was supplemented. A much smaller percent of the flies that emerge from the supplemented food are Drosophila ananassae. The data show that some­what more ananassae emerge on better food conditions but a great many more of the competitor emerge. This differential is lost near 0 and 100% ananassae. Test 7 is an exception but the food was bad in this test and spreading improved the development of all flies. The tests with melanogaster were similar except that melanogaster is a much more effective competitor. At 71 ° F it very rapidly replaces ananassae but at 77° ananassae usually displaces melanogaster. The tests at 74° ± 1 F show the two species nearly equal. Drosophila ananassae first falls, then increases. Neverthe­less there was still a considerable melanogaster population in the cages at the end of three months as indicated by the number that emerged on spread food. Both tests illustrate the tremendous effectiveness of ananassae in larval compe­tition under limited food conditions. The more strenuous competition probably approached more nearly the conditions on the islands. At temperatures close to that of the islands, they cart compete with other species most effectively. Zimmering (1948) tested competition between D. pseudoobscura and D. mel­anogaster in population cages. He showed that strains of the former of two origins (Mather and Pinon) differed in their competitive ability. Four strains of melan­ogaster, one wild type and th~ee mutant stocks, differed also. One with six second chromosome recessive mutants was displaced by pseudoobscura, but the other three displaced pseudoobscura. Timofeef-Ressovsky (1933, 1935) demonstrated that different strains of D. funebris were of different competitive abilities, which was related to the temperature of their origin. His tests differed in that a certain number of eggs from funebris and melanogaster were placed together under crowded conditions. Merrell (1951) also tested competition between funebris and melanogaster. Most of the competition was between larvae. The species could coexist in population bottles for long periods since the larvae of melanogaster MAJURO RONGERIK FIGU~E I BKIM IOOr-~~--"-""'"-'-J~~~~~~~~~~~-'o~~~~~~~~-:-""'.'::::::~~~~~~~~~~~~~~-,IOO /; 3. ' ' ' ' / ' ' ' ' '...' '-...-----· ' ' ' ' '' L~~~~=-~~~~~~~~~'­ ~~~~~~~~~~~~-:;::::====..-:::;:::;-c<.:=:;c---;;:-:c:-:-:-::-:;-.;;:;;;;;;;;;;;;;;;--.:;;;;-0 + VIRIUS 71°F ..____..... Limited Food; .. -----• Supplemented Food ~0 r-~--:::-::::=='!o-~~~~~~~-f-~o::::-::z-~~~~~~~~~~~r-~~~~~~~-:-~-::-::c:i--~--,100 ,,:• B. ,' 5. _,,. + VIRIUS 7 7° F 100 9. 10. II. 12­ \ . 0 50 + MEL ANOGAS TER 71 ° F 100 13. "'· IO. 16. 100 ' ' \ _.. ' -_.­-0­+ MELANOGAST ER 74° F .._ ----.... -­ --· \ ...­ .. --- \ .._____ ....._____ 50 0 100 100 17. 18. 1a 20. _____,_______ _ 50 '·..-­ 0 -1-MF.LANOGASTER 77° F FIG. 1. Competition between stocks of Drosophila ananassae and Drosophila virilis or Dro­sophila melanogaster in population cages. Solid line shows percent ananassae in the sample re­covered from cage on restricted food, dotted line shows percent ananassae on supplemented food, Numbers correspond to tests in Table 1. TABLE 1 Competition between Drosophila ananassae and Drosophila virilis or Drosophila melanogaster Restricted Food Increased Food Stock of Temperature Date Date ananassae ananassae ananassae Competitor o F Started Tested Total flies number % Total flies number % 1. Majuro virilis 71 ° 12/ 23/ 55 1 / 2'1 156 28.~ 173 61.1 1599 502 31.4 2/2,0/ 56 1104 1094 99.1 2449 2354 96.1 Cl) 2. 3. Rongerik Rongelap virilis virilis 71 ° 71 ° 12/ 14/ 55 12/ 9/ 55 1/ 13/ 56 2/ 20/ 56 1/ 7 / 56 3/ 2/ 56 485 582 47'5 637 331 571 452 629 68.2 98.1 95 2 98.7 2051 1146 2693 1518 101 871 858 1455 4.9 76.0 31.9 95.8 ......s:: ~-· (1) "'-.;:s ...... 4. Bikini virilis 71 ° 12/ 13/ 55 1 / 13/ 56 421 305 72.4 2700 584 21.6 ~ 2/ 20/ 56 491 407 82.9 1065 246 23 .1 ~ 5. Majuro virilis 77 0 2/ 6/ 56 31 7156 4/ 6/ 56 129'5 1638 1251 1630 96.6 99.5 1574 3280 1026 31 78 65.2 96.9 (1) ;:s (1) ......-· 6. Rongerik virilis 77 0 2/ 27/ 56 2/ 25 / 56 4/ 29/ 56 1462 1370 1462 1337 100.0 97.6 2108 2 158 1778 21 37 84.3 99.0 ~ 0- 7. Rongelap virilis 77 0 2/ 7/ 56 3/1 t/ 56 4/ 11 / 56 500 557 223 557 44 n• 100.0 2207 2407 1982 2320 89.8 96.4 tl-; ~ 8. Bikini virilis 77 0 21 2/ 56 31 715•6 1745 1737 99.5 2535 2386 94.1 'l::I 4/ 8/ 56 21 10 2110 100.0 3127 3073 98.3 ~-· .._ ~ 9. Majuro melanoaaster (ell) 71 ° 12/ 7/ 55 1/ 3/ 5156 9/ 56 313 746 1 7 0.3 0.9 1617 2606 16 16 1.0 0.6 10. Rongerik melanogaster (ell) 71° 12/ 2/ 55 1/ 5/ 56 550 79 14.4 1990 96 4.8 11. Rongelap melanogaster (ell) 71 ° 12/ 12/ 55 1/ 13/56 2/ 12/ 56 525 427 58 30 11 .5 7.0 2390 205 1 94 40 3.9 1.9 3/ 7/ 56 287 13 4.5 2483 62 2.5 - ~ O') 'l to Ol TABLE 1-Continued Co:npetition between Drosophila ananassae and Drosophila virilis or Drosophila melanogasler Restricted Food Increased Food Stock ol T emperature Date Date ananassae ananassae ananassae Competitor 'F Murted 1·ested Total flies number % Total flies number % lZ. Bikini melanogaster (e1 1) 71 ° ] '}.,/ 2/ 55 1/ 5/ '.J ti 480 9 UJ 206::i 2 0.1 13. Majuro melanogaster (e'') 74° 4/ 23/ 56 5/ 27/ 56 tJ/ 'L4/:Jli 1/ 'L-o/ oti 809 8'40 10ti4 164 oUO 1046 20.3 bl.0 98.3 3636 Hi/8 1879 205 604 483 5.6 HUi 25.7 "-j ~ (1) 14. 15. Rongerik Rongelap melanogasler le"·) melanogaster (el") 74° 74° 4/ 23/ :;6 4/ 25/ 56 5/ 27/ 56 'O/ 'L'+/ ciO I / 'LV/ :.JV 5/ 27/ 56 b/ 'L4/ oti / / 'L-uhti 1295 'o'Li Y'tJ 1843 487 7'46 263 '1-:J 1 8oti 71 277 579 20.3 '.J 4.o Yl.8 3.9 56.9 7Y.8 2803 'l.,'L,~ti '4ouo 3868 2o13 2805 825 b'41 196 141 245 b88 29.4 '}.,/ .0 66. ? 3.6 9.7 24.5 ~ ;:i-. ~ (1).., "'-.... · ~ 0- 16. Bikini melanogaster (ell) 74° 4/ 25/ 56 5/ 27/ 56 b/'L,<~/:.Jti I / 'L-u/oti 485 781 btJ9 53 402 58ti 10.9 5 1.5 8/ .ti 3982 '4vU2 'L-104 127 211 8ti 3.2 8.1 4.0 ~ ~ 17. M ajuro melanogaster (ell) 77 0 2/ 6/ 56 4/ 11/ 56 5/ 11/ oti 564 12o2 160 814 28.4 b4.::i 2099 'l.,'L,/1 341 1o~1 16.2 / 4.o 'ti i.:: \:!"'..._-· 18. Rongerik melanogaster (ell) 77 0 3/ 7/ 56 4/ 8/ 56 :.i / 1U; ou V/ O/:;O 1095 i6b 884 575 4JY b95 52.5 :.iu.9 /8.ti 3077 '2112 2o91 728 :;·o/ 10/ ti 23.7 '40.5 41.5 (") I::)....-· 0 ;:i 19. Rongelap melanogaster (ei") 77 ° 2/ 1/56 3/ 't/ 5/ 7/06 tJ / :.Jli b/Jti 995 2JY2 Lti08 758 lotJ4 1071 76.2 I t.4 tiD.ti 1756 j (J'L,/ 2/'L4 667 2UU8 1499 38.0 tJu.6 5!.>.0 20. Bik:ni melanogaster (el') 77 ° 3/ 12/:)6 4/ 12/ 56 5/ lu/:.iti l>/ 9/oti 1156 l:J8 l'l.,J[ 62:j 56:i 18!.i 54.l mu 14./ 2968 2u46 '4itli 1455 ·v29 86/ 49.0 26.8 6U.2 •Bad food. developed more effectively on fresh food whereas funebris larvae could develop in old food which was not usable by melanogaster. L'Heritier and Teissier ( 1935) had noted that mixed populations of funebris and melanogaster came to equilibrium with a small proportion of funebris. Merrell showed that the balance between the competitors was precarious since two populations became all funebris, another all melanogaster in addition to the group which ended as funebris because no fresh food was added. Moore (1952) reported a series of studies of competition between the sibling species D. melanogaster and D. simulans. He showed that at 25° C with rapid food replacement (up to 8 cups per week) melanogaster displaced simulans but that at 15° C with slow food replacement (2 or 3 cups per week) simulans re­placed melanogaster. In addition simulans will utilize an "undesirable" food sur­face for laying much more effectively. Drosophila ananassae is in the melan­ogaster species group, but is not as closely related as simulans. In our tests, which usually stopped short of complete replacement, melanogaster displaced anancJsae rapidly at 71° F but ananassae had the advantage at 77° F (25° C). Even at 74° the superior competitive ability of ananassae larvae gives the latter species an advantage. This factor and greater temperature tolerance must account for the dominant role of ananassae in the Marshalls and even at Ponape. SECTION III CYTOLOGICAL ANALYSIS Robert L. Seecof INTRODUCTION This report describes the results of sampling the chromosome' arrangements of populations of Drosophila ananassae from the Marshall Islands. ·Populations were sampled from three of the islands that had been exposed to radiation from atomic weapons, Bikini, Rongerik and Rongelap, and from two of the islands that received no irradiation, Majuro and Ponape. Previous studies on Drosophila have already established the kinds of chromo­some rearrangements which are successfully transmitted after irradiation. The variety and incidence of X-ray induced aberrations have been used: as a basis for estimating breakability among parts of chromosomes, physical arrangements of chromosomes during cell processes, and efficiency of the radiation-employed. Su€h studies were made by Bauer et al. (1938), Helfer (1941), Koller and Ahmed (1942), Kaufmann (1946) and Haldane and Lea (1947). Later experiments on D. melanogaster by Liining (1952) and Auerbach (1954) demonstrated a·pat­tern of X-ray sensitivity through the stages of spermatogenesis which was con­firmed for D. virilis by Alexander and Stone ( 1955). ·In a recent article Stone ( 1955) has reviewed the extensive changes in chromo­some pattern that have accompanied Drosophila speciation. Evidence is sum­marized to show that gene sequences, as preserved by chromosome rearrange­ments, have adaptive value which is dependent on the nature ,of the ·rearrange­ment, the gene pool, and the habitat of the species. In order to establish perspective for evaluating the rearrangements recovered from irradiated wild populations, a control experiment relating the ra_diation sensitivity of D. ananassae to other Drosophila has been performed. Information concerning the distribution of chromosome breaks and the differential sensitivity of stages of spermatogenesis was gathered from analyses of the salivary gland chromosomes of F1 larvae from X-rayed males. The results of the laboratory control and the wild population survey are presented together as subsections A and B respectively. MATERIALS AND METHODS A. The Control X-ray Induced Rearrangements All X-ray treatment was applied with a Westinghouse "Quadrocondex" ma­chine through a filter of 1 mm. of aluminum and ¥2 mm. of copper. Dosage was calibrated with a Victoreen-made Condenser r-Meter, model 70. Two sixty second ·r-meter readings were taken before each irradiation and one afterwards. The three readings fell within a 15 r range to be considered acceptable. In all tests, X-rays were delivered at 250 kv., 15 ma., at about 800 r per minute and at 24° C. D. ananassae shows four metacentric chromosomes at mitosis and six chromo­some arms in salivary nuclei. In larval ganglion cells the three autosomes are of different sizes and the sex chromosome is intermediate in length (Kaufman, 1937; Kikkawa, 1938). In salivary nuclei the largely heterochromatic fourth chromosome is buried in the chromocenter. The few euchromatic bands repre­senting the fourth are described by Kikkawa (1938) and Kaufman (1937). These bands are indistinct and have been ignored in the present study. The base of 1R (numbered 12 on the included map, Chart 2) is often indistinguishable and breaks were not localized within it. The flies used in this experiment were derived from a stock collected on the Pacific island of Majuro. They were selected, by the author, to be homozygous for a "standard" chromosome arrangement. Kaufmann (1939) showed that a homozygous inversion did not influence the chromosome breakage pattern in D. melarwgaster. Iri common with all other D. ananassae strains examined, several extra chromatin bands were present heterozygously in the salivary chromosome set. These give no detectable phenotypic effect. All of the tests were made by X-raying adult males, mating them to virgin females, and examining male and female F1 larvae for chromosome aberrations which could be detected in the salivary gland nuclei. The salivary glands were prepared as paraffin sealed aceto-orcein squashes and then examined within twelve hours. A salivary chromosome map was drawn, using a camera lucida, from these preparations (Chart 2) . Series 1500. Males received 1500 r 12 to 24 hours after eclosion. After irradi­ ation, a mass mating was set up of 20 to 30 males and an equal number of mature virgin females. New virgins were supplied to these males, at 48 hour intervals, for twelve days. The number of pairs per vial was adjusted to the expected egg hatch in an effort to keep the number of larvae at an optimum. The ratio of males to virgin females was established at 1: 1 in every 48 hour interval tested. Males were etherized once before and once after irradiation, and then again at 48 hour intervals when being transferred. Matings were made and larvae were ' raised on heavily yeasted banana food; This sequence, from X-raying through · raising larvae, was repeated eight times during a period of seven months. The ,.. :m: v x = , L i 1111t1111~1111fH'j11 L1111i11: ~ 1.•111 ui:1 Ji 1111r~111 11 di 111:~· , :i,11111 t-i I 1 J 2 I3 I4 / 5 I6 I1 Ia 9 I10 I11 I12 I13 I14 , 15 I16 I11 I1a I1~ l20 : >?;« >>~<< x~ y d' x ~ x = I R ro}LT/I L/llfl I r1j;jff,JIW.1·· h,1'11,i;ftli,i Ji1111~ !umt-J I I I2 3 I4 I5 6 7 8 I 9 10 I11 12 2 L !i\itr1.11n1m11· 11;., u11 :m111 11 1''\''' J1 1nim1tll'fl i 11rn 11 1. 111 1111 11 :11.11 1111 1 H1/~' '·\'t t 1i1 ~1111~111u1 1r: <11 :tJJ.Hrn.\.i1r1rn :111;11l1rn1u ~ 1 1 12 l3"1 15 [6 11 a 9 1of 11 l12!13l14 2R lf\t111::~ri111L '':l''"'~111T1111 1 I I2 3 14 5 16· I7 15l16l11l1a l19l2ol21 22l23 24 25 26f21l2al29f30 131132/33 34 135 3sf,1l3a139 1 .:( 11r i1 111 1:. r~ 1 11 nr 11 1 r 11.,lll '1u 1ur~n :1 1m 111u1 Lir·t :1 111m1u 1_1htJ' lll\11 1.1mm1 _J I r 8 9 I10 I,, I12 I13 I14 I15 116117 118, 19 I20 I21 I22 I23 124 125 1 26 ;i11 111 3L f.~Jll.1: UH :t Jl'il !JlilL..lli::1n.;. : llll/ 1111il 11 1illll'l jll/!l •• l,ll:j1)1 ll·lll\\..:11 1111,,;/111/ 11 0. ANANASSAE I 1 I 2 13 14 15 16] 1 J a I9 !10 [,, l12 l13 l14 115 116 11 1e 19 20!21 L 2 /23 24,25 21 f 2e 129 30 I31 jll ' 1l1nJ~~r)Pl»1 26 21f2a/ 29/30 : 3R ; ~11 11llllil~ l \'A\l'i Htllllil lli:lli/ll.l·lliri. 111:1111 1, llUl~J . Jill :!'.Lllrn~;i"'ll!Hltti l;;iH 'llU~ ,))l· it 11 IUl.J11\l~1\i. MAJURO II 3 r4 6 17Ia I9 I10 II I12 13 I14 I15 i 16 I17 I1a 19 20 I21 I22 ) 23 I24 I25 I26 127 2 5 r28 T29 CHART 2: Chromosome map of Drosophila ananassae. i . "' ~ ~ ;::3 ~· .Q. r ~ >:l to '1 ...... The University of Texas Publication combined score of one hundred larvae for each 48 hour interval of series 1500 is made up by contributions of from four to seven of these sub-series. Series 1000. Series 1000 was similar to series 1500 in all respects except that the dose given to the males was 1000 r. Series 1000 and series 1500 were run concur­rently. Series 1570. In series 1570, seven-day-old virgin males were given 1500 r, placed with virgin females for the following 24 hours, and then discarded. Other details were similar to those in series 1500 and 1000. The larvae derived from the successive 48 hour periods of series 1500 and 1000 represent sperm which were in successively earlier stages of maturation when irradiated. The larvae from series 1570 represent irradiated mature sperm from seven-day-old males and are compared to the first offspring of the 12-24 hour old males irradiated in series 1500. The aberrations were scored for type of rearrangement, identity of chromo­somes involved, and location of each break. Very small rearrangements and de­letions were not detected. Breaks were located with an accuracy of two numbered sub-divisions on the included map. Five to ten percent of the squashes were un­analyzable because too few nuclei were well spread, and such slides were not included in any of the results. Catcheside (1938), in a similar study, found no indication that greater doses of X-rays induce more unanalyzable glands. B. The Analysis of Wild Populations Island populations were sampled in the summers of 1955 and 1956. The 1955 sample males were taken in the wild or were F1 offspring from females insemi­nated in the wild, one male from each female parent. The 1956 sample males were either captured in the wild or were taken from a large population of flies, from the respective island, maintained in the laboratory during four months of sampling. These males were crossed individually to females homozygous for an arbitrary standard chromosome arrangement. The salivary chromosomes of four female F1 larvae from each male were analyzed for chromosome abnormalities. Six chromosome arms were inspected, the dot fourth ignored. RESULTS A. The Control X-ray Induced Rearrangements Over-all break distribution. In the three series, a total of 1100 sperm were analyzed, of which 137 showed some chromosomal aberration. In all, 321 breaks were detected among six chromosome arms and the chromocenter. Break fre­quencies are given in Table 2. The distribution of 284 breaks within the numbered divisions of the salivary chromosome map is shown. These represent all of the breaks from the three series which were located with a precision of two numbered divisions. Total breakage for each chromosome arm is seen to be roughly proportional to the relative salivary arm lengths as given in Table 2. The expected number of breaks for each arm was calculated from the lengths of the arm as shown on the salivary chromosome map. Again, 1R interval 12 was not considered. This cal­ TABLE 2 (Ill) Chromosome Arm Map Interval 1L 1R 2L 2R 3L 3R 1-2 3-4 5-6 7-8 9-10 11-12 13-14 15-16 17-18 19-20 21-22 23-24· 25-26 27-28 29-30 31-32 33-34· 35-36 37-38 39-40 Total number of observed breaks Expected number of breaks Arm length ratio from salivary map 3 4 2 2 2 3 1 0 2 2 c 21 x 2 42 39 1.7 3 4 2 1 3 2 c 15 x 2 30 23 3 6 4 3 1 1 5 3 6 7 9 7 4 3 4 2 0 4 3 1 c 76 78 3.4 3 8 2 2 6 3 1 6 0 1 2 3 2 1 4 7 c 51 62 2.7 3 15 6 2 5 2 6 5 1 0 3 2 8 4 1 4 5 2 3 4 3 3 5 5 1 0 1 7 1Z 3 c c 63 58 60 58 2.6 2.5 Table 2 lists the number of breaks recovered and their locations for six chromosome arms. The intervals are identical to those shown on the included salivary chromosome map. C means centromere. culation was made after doubling the total number of observed sex chromosome (1L and 1R) breaks, an irradiated sex chromosome having been present in only half the larvae examined (in female larvae only). A small error is caused by this procedure. Table 6 gives the ratios of males and females examined in each in­terval. ' It should be remembered, when considering salivary chromosome measure­ment, that some distortion is caused by stretching of interband regions. Mitotic arm lengths have not been established for D. ananassae but the arms of the second and third chromosomes are roughly equal. See the metaphase drawings on the salivary chromosome map. The distribution of breaks within arms is shown in Table 2. A chi-square test indicates that breaks are distributed to chromosome arms in proportion to their relative salivary lengths (.5 > p > .4). The low number of breaks per map di­vision necessitates arbitrary grouping to test the within-arm break distribution. Chromosome lengths of 6 map divisions, from tip toward centromere with extra divisions included in the proximal grouping, were compared. Breaks totals within the resultant 25 chromosome segments, tested by chi-square against the overall average number of breaks expected per segment, indicate that within-arm dis­tribution is not other than random ( .05 > p > .01). Relative frequencies of multiple break rearrangements. Table 3 gives the pro­portion of intra-arm to inter-arm rearrangements for chromosomes 1, 2, and 3. The University of Texas Publication Two-break rearrangements confined to one arm are listed as 2. Two-break re­arrangements between different arms are listed as 1, 1. Higher break rearrange­ments are listed accordingly. The ratios of "2" to "1, 1" breaks recovered is 1:5 to 1. These are comparable to the values obtained by Bauer et al. (1938), Helfer (1941), and Koller and Ahmed ( 1942) which are listed in Table 3 along with data from the present study. Bauer's figures were obtained from D. melanogaster at 1000 to 5000 r. Helfer and Koll~r worked with D. pseudoobscura using 4500 to 5000 r. TABLE 3 (III) Ratio A. Bauer (1938) 1000-SOOOr No. of No. of B. Helfer ( 1941) 5000r Breaks Distribution Cases Ratio C. Koller (1942) 4500r A B C 2 1, 1 65 1.5 1.8 1.9 1.2 2 42 1 1 1 1 3 1, 1, 1 1 2, 1 3 3 4 2, 2 5 2, 1,1, 1 Alterations involving heterochromatin 26 Kind of alteration not determined 6 Table 3 lists the recovered rearrangements by number of breaks and number of chromosome arms involved. The first four columns show data from the present study, the last three from previous studies. The 107 two-break rearrangements shown in Table 3 are made up of 41 para­centric inversions, 23 pericentric inversions, 42 translocations and one deletion. Six of the paracentric inversions include more than half of their chromosome arm. This is not significantly different from the 10.25 expected on the basis of random breakage and fusion. Chi-square test gives p > .05. Because breaks in the heterochromatic fourth chromosome could not be dis­tinguished from breaks in the Y, no direct measure of Y breakability is available. However, among the total of 26 two-break rearrangements involving heterochro­matin, 17 occurred in males and 9 in females. A 17 .3 to 8. 7 ratio is expected from equal numbers of male and female larvae if the fourth and Y chromosomes are considered equal in length and were breaking and recombining at random. This is because an irradiated fourth chromosome was present in all examined larvae (ignoring non-disjunction) , but an irradiated Y was present only in male larvae. Variation of damage with the course of spermatogenesis. Table 5 shows the numbers of damaged sperm and the total chromosome breakage. Periods B-F represent successive 48-hour intervals during which males inseminated fresh virgins. In all three series, 100 sperm were analyzed for each 24-hour interval. Figure 2 illustrates the information in Table 5. The stages of spermatogenesis are inferred from studies on D. melanogaster which are discussed later. Comparison of mature sperm to the most sensitive stage of spermatogenesis. Because of the similarity in number of breaks and number of larvae showing aberrations (Table 5 and Figure 2), both series 1500 interval B, and series 1570 Studies in th.e Genetics of Drosophila are considered to be derived from mature sperm. Similar results were obtained by Khishin ( 1955) who found that the sensitivity of maturing spermatozoa in newly emerged D. melanogaster imagines is the same as that in mature sperm. Too few breaks were scored to compare period B against period D for distri­bution of breaks along chromosome arms. Table 4 gives the relation between FIGURE 2 80 70 60 50 40 SERIES 1570 -A 30 20 SERIES 1570 ­ 10 12 13 G 1415 H 16 POSTMEIOTIC IMEIOSISI SPERMATOGONIA SPERM SPERMOGENESl81 Fie. Z. Variation in recovered X-ray damage from successive brood intervals. The abscissa is marked off in two-day intervals A-H. A is the first two days after eclosion. The stages of spermatogenesis are inferred from work on D. melanogaster which is discussed above. Ordinates for the triangle and the solid lines are to be read as "number of breaks." Ordinates for the dia­mond and the dotted lines are to be read as "% of sperm showing a rearrangement." TABLE 4 (III) No. of Times Recovered Ratio No. of Interval Interval Breaks Distribution B D B-F B D B-F 2 1, 1 19 20 65 1.58 1.33 1.55 2 12 15 42 1 1 1 Table 4 lists the recovered two-break rearrangements among chromosomes 1, 2 and 3. TABLE 5 (III) Day 3,4 5, 6 7,8 9, 10 11, 12 Interval B c D E F total B-F Series 1500, no. of larvae with rearrangements 16 21 30 5 0 72 No. of breaks 40 48 79 10 0 177 Series 1000, no. of larvae with rearrangements 7 16 21 6 0 50 No. of breaks 14 38 44 12 0 108 Series 1570, no. of larvae with rearrangements 15 15 No. of breaks 36 36 Table 5 shows the chromosome damage recovered from 100 larvae sampled in each 48-hour period. TABLE 6 (III ) B c D E F Total B-F Interval 6 'i' 6 'i' 6 'i' 6 'i' 6 'i' 6 'i' Series 1500 Series 1000 Series 1570 49 56 55 51 44 45 48 61 52 39 55 43 45 57 44 48 55 52 44 48 5G 52 240 256 55 2CO 244· 45 551 549 Table 6 shows the numbers of male and female larvae examined in each 48-hour interval. kinds of two-break rearrangements in intervals B, D, and B through F. This com­parison fails to show any qualitative differences for interval B (mature sperm) versus interval D (interval showing most damage.) B. The Analysis of Wild Populations Ten paracentric inversions represent all the rearrangements discovered in the sampled populations. Two of these were presumed identical to those described as CII L-0 and CIII L by Kikkawa ( 1938). The frequency and distribution of these two and the eight previously undescribed inversions, a through h, are listed in Table 7. The approximate break points associated with each inversion are shown in Figure 3. Inversions a, b, and c were observed in 1955 before the chromo­some map was completed and are not accurately located. Studies in the Genetics of Drosophila FIGURE 3 h h cllL-o c II L-o 2L I I I I \ 2.6 12.8 30.3 34.4 I I I d f f d I I l l I I 2R I 3.4 4.9 7.2 13.6 3L cllll l c I I g g I l f cllll I a I I I a I I 0 3.1 6.2 8.2 12.5 19.3 22.4 26.5 I b b I I 3R I I 8 : I 'e :1 I I I : 6.2 : 11.5 I I I I 5J 10.9 Frc. 3. An illu9tration, drawn to scale, of the inversions recovered from the wild population Gamples, each break being located with a number. Digits to the left of the decimal point give the map interval. Digits to the right give the band closest to the break, counting bands from left LO right within the interval. All break points are located to within ± two map bands except the breaks for inversions a, b and c, shown dotted, which are located within ± one map interval. DISCUSSION A. The Control X -ray Induced Rearrangements There are a number of disadvantages in using the salivary analysis technique in radiation studies. Primarily, radiation is administered to adult flies, but the scoring of damage is postponed to the following generation of larvae. During this interval, chromosome breaks may restitute or reunite in new combinations. Developing germ cells in male Drosophila are protected from germinal selec­tion after the spermatogonia enter the synchronously dividing cyst stage (Ponte­corvo, 1944; Auerbach, 1954). Breaks in mature sperm do not heal and rearrange­ments are not effected until after fertilization (Kaufmann, 1941, and others). Recovered aberrations are restricted to those which prove viable in the developing larva and, therefore, may not reflect accurately the radiation-induced break fre­quency and distribution. However, the salivary analysis technique does permit a comparison of chromo­some arms with regard to recovered breakage. Further, the relative frequencies of recovered rearrangements indicate the kinds of chromosome damage that are successfully transmitted after irradiation. The apparently random break distribution recovered in D. ananassae is in agreement with that found by Bauer et al. (1938) among D. melanogaster sal­ivary chromosomes. In both studies the number of breaks per chromosome was roughly proportional to salivary and mitotic lengths. Bauer's data, although too scant to be statistically significant, indicated preferred breakage at the proximal and distal ends of certain chromosome arms. More data are required in order to resolve these speculations for either species. A more detailed study of the D. melanogaster X-chromosome was made by Kaufmann (1946). He was able to show that recovered breakage is not random in this chromosome but is found most frequently in the heterochromatin. Data presented above indicate that the heterochromatic 4th and Y chromosomes are probably being broken in proportion to mitotic lengths. It is not possible to deter­mine absolute break frequencies in the two chromosomes. Theoretical expectations for the distribution of multiple-break rearrangements have been advanced by Bauer et al. (1938), Haldane and Lea (1947) and Baker (1949). Bauer and his collaborators compared inter-arm to intra-arm rearrange­ments by assuming random breakage and recombination. For five chromosome arms, a 4: 1 ratio was expected for "1, 1" and "2" rearrangements, respectively. TABLE 7 (Ill) No. 1L Chromosome Arm Stock males 1R 2L 2R 3L 3R Bikini 171 + en L-0= 22/89,30/82 f= 0,2/82 a= l/89,0 e=0,2/ 82 1955, 1956 g=0,1/82 Majuro 93 e n L-O= 7 / 47,o d= 1/47,3/46 CnI L=l/47,0 b= 1/ 47,0 + 1955, 1956 c=l/47,0 Ponape 8 cu L-0= 3/8 d= 4/8 +. + + 1956 h=4/8 Rongerik 7 1956 + + + + + Rongelap 5 1956 + + + + + Table 7 lists the frequency of each rearrangement (all inversions) detected in the Marshall Island samples. The expression: a= 1!89,0 means that inversion a was found in 1 of 89 larvae examined from the 1955 Bikini sample, but was not detected in the 1956 sample. Their results and similar results by Helfer (1941) and Koller and Ahmed (1942) are listed in the last three columns of Table 3. The expected ratio for six arms of chromosomes 1, 2 and 3 in D. ananassae would be 5: 1. These theoretical ratios have been criticized by Pontecorvo in Koller and Ah­med (1942) and by Baker (1949) because contributions from dominant lethal chromosome arrangements to the F1 generation were not considered. Preliminary calculations by L. S. Lockingen (personal communication) indicate that the ex­pected proportion of "1, 1" to "2" rearrangements is greater than 2: 1 for doses inducing a mean of 2 breaks per recovered rearrangement, and increases rapidly with dosage. It is impossible to calculate the true expected ratio at this time be­cause the efficiency of X-ray induced breakage in sperm is unknown. Information presented above and inspection of Table 3, however, show that the ratios recovered in this and previous studies for "1, 1" versus "2" rearrange­ments are consistently below 2: 1. As would be expected from the delay in healing­of spermatid chromosome breaks, recovered rearrangements from different sper­matogenic stages show the same low ratio (Table 4). Thus, D. ananassaR behaves like other Drosophila in that the recovered rearrangements after X-raying sperm Studies in the Genetics of Drosophila do not follow a random expectation. This may be due to preferred patterns of breakage and recombination, or to differential viability conferred by rearrange­ment types. Variability in the rate of sperm emission causes uncertainty in interpretation of induced damage versus germ-cell-cycle curves. This was overcome in part by Khishin (1955) who irradiated larvae and pupae of D. melanogaster. He deter­mined cytologically the most advanced stage in spermatogenesis at irradiation, and correlated this information with the variation of mutation rate from sperm recovered in future test intervals. Premeiotic stages were further identified by recovered bunches of like mutations. He found that mutation rate is low up to the onset of meiosis and increases sud­denly in meiosis. It rises to a peak in late spermatids and then drops to a level below meiosis in mature sperm. This is in close agreement with the results of Auerbach (1954) who also mentioned that rearrangements reach their peak simultaneously with recessive lethals. Due to variation of sexual activity among the males, all of the intervals shown in Figure 2 may have received contributions from more than one stage of sperm development. For these experimental conditions, however, Figure 2 does show contributions in each post-irradiation interval from stages of widely differing sensitivity. The 48-hour interval following first sperm elimination from young males already contains contributions from more sensitive stages. By the twelfth day of adult life chromosome damage was not recovered in larval samples. These curves are in close agreement to those obtained by Alexander and Stone (1955) who measured recovered translocation frequency from D. virilis sperma­ togenesis. The male germ cell cycle is more rapid in the shorter lived D. ananas­ sae, but the variation of sensitivity within stages is similar in both species. B. The Analysis of Wild Populations From a study of natural populations of D. an.anassae near Japan, Kikkawa (1938) ·described one reciprocal translocation and five paracentric inversions. Each arm of the second and third chromosomes showed at least one inversion and two of the inversions involved more than half of their chromosome arm. Ac­cording to Freire-Maia et al. (reported in the discussion following the article by Stone, 1955), Brazilian populations have yielded at least 19 different paracentric inversions, one translocation, one deletion and two transpositions. This shows D. ananassae to contain a highly polymorphic chromosome pool but not un­usually so. See Stone (1955) for comparisons. In addition, Freire-Maia reported finding five different pericentric inversions, a very high frequency. We cannot accurately estimate the levels of direct radiation and subsequent fall-out radiation to which Drosophila populations in the Marshalls were sub­jected. Bikini has received radiation from an unknown number of "atomic" tests and from the thermonuclear test in March 1954 and other tests in 1956. Figure 2 indicates that numerous rearrangements must have been induced in the germ cells of contemporary D. ananassae populations on Bikini. However, the number of recovered new rearrangements, peculiar to the island, was not appreciably higher from Bikini populations (4/ 171) than from control Majuro populations (2/ 93). It is unlikely that test explosions ever destroyed the Bikini D. ananassae popu­lation so that our samples were taken from an immigrant population. Bikini is about 100 miles from Rongelap, the closest D. ananassae source and, moreover, an apparently equivalent fauna was observed on the island at each collection. It is possible that the D. ananassae population was regenerated mainly from the primary sex cells of larvae and pupae, blast exposed adults being largely sterilized. Figure 2 shows that spermatogonia are comparatively resistant and transmit few rearrangements at the X-ray levels of the control. However, post­irradiation flies were certainly subjected to beta, gamma and alpha radiation from fallout deposited on vegetation and from contaminated food. The elimination of induced rearrangements could be effected by chance and selection pressure. Translocations, deletions and other changes that lead to future aneuploid gametes would be lost rapidly. Paracentric and small pericentric in­versions, which this control study indicates were induced in high proportion, do not have this mechanical disadvantage. Such inversions may have been eliminated due to harmful effects associated with the rearrangement per se, that is, with break points and position effects. In addition, through crossing over suppression, inversions may have become dis­advantageous by preserving groups of radiation induced deleterious mutations. Ordinarily, fortuitously grouped detrimental genes are separated by meiotic crossing over to yield combinations of differing selective value. The variability of the population is thus enhanced and individual mutations proven against various genetic backgrounds. Newly formed inversions or any that had been present in low frequency in the pre-irradiation population would rarely become homo­zygous so as to allow this recombination. Chromatid exchange on either side of a heterozygous inversion will usually be reduced (Sturtevant and Beadle, 1936), '"1· adding to the effect for small inversions. Selection against such rearrangements would represent a mechanism, in Drosophila and all other forms exhibiting crossing over, for the elimination of inferior gene associations. Certainly, very few if any rearrangements capable of establishment.through positive selective value have been recovered from these irradiated populations. SUMMARY A. The Control X-ray Induced Rearrangements Drosophila ananassae males were X-rayed at dosage levels of 1000 and 1500 r. Recovered salivary chromosome aberrations were scored in successive broods of F1 larvae. Analysis of 321 breaks indicated that recovered break points were distributed at random along chromosome arms in rough proportion to salivary and mitotic arm lengths. . A higher proportion of intra-arm to inter-arm rearrangements was recover~d than would be expected from a hypothesis of random chromosome breakage and reunion. The susceptibility of D. ananassae germ cell chromosomes to X-ray damage was shown to vary during the course of spermatogenesis. Very few breaks were recovered from premeiotic spermatogon~a, more from mature sperm and the greatest number from stages of spermiogenesl.s. A map of the salivary chromosomes of D. ananassae is included. B. The Analysis of Wild Populations Populations of Drosophila ananassae from radiation exposed and non-exposed Marshall Islands were sampled for chromosome rearrangements. Four F1 female larvae were examined from each of 284 sample males. The number of previously undescribed rearrangements recovered from both irradiated and control islands was low. Examination of laboratory conducted experiments and field conditions indi­cates that numerous rearrangements were probably induced in the radiation ex­posed populations and subsequently eliminated. Factors contributing to the nega­tive selective value of chromosome aberrations are mentioned. A possible role for inversions in trapping unfavorable gene sequences through crossing-over suppres­sion, thus leading to their elimination, is suggested. SECTION IV VISIBLE MUTATIONS IN POPULATION SAMPLES OF DROSOPHILA ANANASSAE FROM BIKINI, MAJURO, AND PONAPE Warren P. Spencer Various methods have been employed by different workers in the analysis of population samples of Drosophila for both visible and lethal mutations. These have been reviewed by the present author (Spencer, 1947). For autosomal lethals the best method is to cross the wild fly to a testor stock containing a genetic marker and crossover suppressor in the chromosome which is being studied for the inci­dence of lethals in natural populations. By the proper breeding technique, which will not be outlined here, all lethals present in that particular chromosome in the population sample can be determined. At first glance this might seem to be the best way to analyze populations for visibles. However, many wild flies will contain both a lethal and a visible in the same chromosome; therefore the visible will never be observed by the above method, as all flies carrying the visible in homozygous form will die because of the lethal present. On the other hand, if one uses either of the two methods outlined below visibles may be recovered even if there is a lethal in the same chromosome, unless this lethal is sufficiently close to the visible so that few crossovers occur. The methods depend upon the recovery of a certain percent of the visibles and an estimate of the remaining visibles present by simple statistical analysis. Method 1. A pair of wild flies may be mated and from the F1 of this mating a mass culture may be made and the F2 generation inspected for visibles. Theo­retically, if one of the P1 parents carried a recessive autosomal visible in hetero­zygous form then in the F2 one out of 16 of the flies should show this visible. Un­fortunately the author has found from considerable experience that the F1 mass mating method is not very reliable. The F1 flies may not form an actual random mating population; furthermore some visible mutant types may have a viability much lower than wild type or may be linked to a iethal or semi-lethal. Method 2. A pair of wild flies may be mated and from the F1 any number of pair matings may be made. The F2 flies emerging may be examined for visible mutations. Theoretically these F2 visibles should appear in one-fourth of the flies in any culture in which both parents are heterozygous. But even if such a visible is linked to a lethal or has low viability it will still show up in a larger proportion of the F2 flies than with the mass mating method, and therefore will be less likely to be overlooked in scoring visibles. The number of visibles present in the popu­lation sample can be estimated rather accurately for comparative purposes, if 5ome consistent method of scoring is used. The author has arbitrarily set up the criterion of the appearance of three flies in an F2 culture, showing the same marked deviation from the wild-type as evidence that a visible was present in one of the P1 parents. Ifthis criterion is used consistently, then samples from different populations may be compared objectively. Some of the visibles which appear are sterile in one or both sexes, and even though further test of such mutants would be possible, the comparison of population samples may be made with some ac­curacy without this laborious check. In the analysis below such elaborate tests were not run on sterile mutant types. In any case the estimates for the several populations are minimum estimates. The actual number of visibles present was probably somewhat higher than that reported. In those F2 cultures where only a few flies appeared no correction factor was introduced for the chance of a visible not appearing even if present in heterozygous form in both F1 parents. As the criterion of analysis remained the same for the three population samples it would seem that the comparison of these populations by the method used presented a consistent and fairly accurate picture. Difficulties in the Analysis of Drosophiia ananassae Populations This author has analyzed population samples of D. melanogaster, simulans, busckii, hydei, mulleri, immigrans, robusta, subquinaria, suboccidentalis, and montana. In none of these has he found the problems of culturing the flies as difficult as in D. ananassae. But unfortunately for laboratory study this is the species which is found in largest number in the Marshall and Caroline Islands, and therefore the species which must be studied in this investigation. D. ananassae is a fragile fly, easily mired in the food medium. We have found that it can be cultured better in pair matings in small vials, but even so it has a lower fecundity than any of the above mentioned species. Flies collected in the Marshall and Caroline Islands in the summers of 1955 and 1956 were sent first to the Texas laboratory and then shipped on to the Wooster laboratory, either as F1 cultures from pair matings or as the original wild flies. Many of these F1 cultures gave no offspring at all. Small pupae were formed, but no adult flies emerged. Well over half of the P1 pair matings made from wild flies sent to our laboratory gave no offspring. Many of these became mired in the food or were apparently sterile. In the tables below the varying numbers of F1 cultures reported from different P1 parents were due to the failure of many F1 pair matings to reproduce, and not to varying the number of these Studies in the Genetics of Drosophila cultures set up. The failures of P1 and F1 cultures did not appear to be markedly different in the three population samples. If only one F1 pair mating is made from each P1 pair of flies, then one-fourth of the autosomal visible recessives present in the flies should be recovered; for two F1 pairs 44%. of the mutant types should be recovered; for three pairs 58%; for four pairs 68%; for five pairs 76%; for six pairs 82%; and for seven pairs 87%. In the tables below the number of visibles present in the population sample analyzed has been estimated, on the basis of the above figures, from the visibles actually recovered. Results of the Analysis of Population Samples In Tables 8, 9, and 10 the results of the analysis of population samples are given respectively for Bikini, Majuro, and Ponape. The Bikini population in the test area had been subjected to radiation, while the populations from Majuro in the Southern Marshalls and from Ponape in the Eastern Carolines serve as con­trols, hundreds of miles from the test area and therefore subject to very little radiation. Table 8 shows an estimated 128 visible autosomal recessive mutations present in the population sample of 113 pairs or 226 flies from Bikini, an average of .566 mutants per fly tested. Table 9 shows an estimated 35 visibles present in the sample of 22 pairs or 44 flies from Majuro, an average of . 795 mutants per fly tested. Table 10 shows an estimated 40 visibles present in the sample of 38 pairs or 76 flies from Ponape, an average of .529 mutants per fly tested. When the two small control samples are combined, out of a population sample of 120 flies it is estimated that 75 autosomal recessives were present, an average of .625 mutants per fly tested. TABLE 8 (IV) Number of Visibles Recovered and Estimated Number Present in Sample of Flies from Bikini No.Mutants No. Mutants P, Pairs of F, Pairs per Recovered Estimated Wild Flies P, Pair in F, Present 48 3 25 43 35 2 18 41 30 1 11 44 Totals 113 pairs 54 1Z8 TABLE 9 (IV) Number of Visibles Recovered and Estimated Number Present in Sample of Flies from Majuro No.Mutants No. Mutants P, Pairs of F, Pairs per Recovered Estimated Wild Flies P, Pair inF, Present 18 3 14 24 3 2 3 7 1 1 1 4, Totals 22 pairs 18 35 The University of Texas Publication T ABLE 10 (IV) Number of Visibles Recovered and Estimated Number Present in Sample of Flies from Ponape No.Mutants No. Mutants P, Pairs of F, Pairs per Recovered Estimated Wild Flies P, Pair in F, Present 7 7 10 11.47 9 6 7 8.54 8 5 6 7.90 3 4 1 1.47 3 3 0 0.00 5 2 3 6.82 3 1 1 4.00 Totals 38 pairs 28 40.20 Discussion of the Results of Analysis While the samples analyzed are disappointingly small, they are almost cer­tainly of sufficient size to have shown large differences in the visible mutation load carried by the populations of control and test areas, had such large differ­ences been present. Actually the visible mutation load in the population sample from Ponape was slightly but not significantly smaller than that from the Bikini sample, but in the other control population sample from Majuro the visible mutation load was higher, though possibly not significantly higher, than that from Bikini. The combined population samples from control areas give .625 mutants per fly tested, while that from Bikini gives slightly less, .566 mutants per fly tested. Since it has been well established by a very large number of laboratory investi­gations on various plant and animal forms that radiation produces mutations, the apparently anomalous finding here reported on visible mutation loads car­ried by a natural population subjected to radiation and those in co:i.trol areas requires some explanation. In a very extensive study of mutation rate in controls and in flies subjected to low dosages of radiation Spencer and Stern (1948) report a ratio of 5.2 lethals: 1 visible in the X chromosome of D. melanogaster. Muller (1954) reviews the work on semi-lethal and deleterious mutations and gives a ratio of 3 to 5 semi­lethals and deleterious : 1 lethal. The overall ratio would seem to be approxi­mately 1 visible : 5 lethals : 20 semi-lethals and deleterious. In addition there are a large number of mutations, invisible morphologically, but which reduce fertil­ity and fecundity. While visible mutations have advantages for certain types of study, when one is investigating samples of control populations and populations subjected to low dosages of radiation, at least low in comparison to standard lab­oratory experiments on mutation rate, one may well expect to find significant differences in the mutation load of lethals, semi-lethals, deleterious mutants, and mutants affecting fertility and fecundity, even when the visible mutation picture shows no significant differences. It must be remembered that by the best esti­mates not more than one visible mutation occurs to 30-50 of the other types mentioned. These facts need to be kept in mind in any apparent discrepancy be­tween the data on visible mutations and the data in other sections of this report. Studies in the Genetics of Drosophila However, in any investigation of the mutation loads carried in natural popula­tions some attention must be given to the breeding structure of these populations, their size at any given time, and possible fluctuations in population size. It might be thought that on the atoll islands of the Marshalls, if a population of some species of Drosophila became established, it would show little fluctuation in population size from season to season and from year to year, owing to the uniform climate throughout the year, with some food plants bearing fruits at all seasons. This is not necessarily the case. In the summer of 1955 a small, but widespread, population of D. ananassae was found on one of the larger islands of Rongerik Atoll. The fly was collected on the ripe and rotting fruits of Guettarda speciosa, a large shrub quite abundant on this island. These fruits were white berries about two centimeters in diameter. At approximately the same time in the summer of 1956 four collectors, working intensively, were unable to collect a single fly of this species, although the fruits were again quite abundant. Pandanus and Marinda, two other plants whose fruits were found on other islands to attract these flies, were also present on this island, but in very small numbers. Thus the Drosophila population of this island had undergone marked fluctuation in size in a year's time. On Bikini D. ananassae were collected on fruits of Papaya, Marinda, and Pan­danus. However, the population of this fly was quite small here, probably only a few thousand individuals, a rough estimate on the basis of several day's collecting and the difficulty of securing an adequate sample for analysis. Such a small popu­lation, particularly if it fluctuated in size from season to season, might be ex­pected to lose by chance many of the visible mutant factors which had arisen either from radiation or spontaneously. Conversely one might expect a high inci­dence of a few visibles. Of the 54 visibles actually recovered from the Bikini sample by inbreeding in the F2 generation 11 were rough eye mutants and 11 were small bristle mutants. It seems likely that some of these were actually the same mutant, spread through the population by random drift, although no tests for allelism were made. Thus for a given small population, particularly one fluctuating in size, the visible mutation load might be increased or decreased markedly by random drift. On the other hand, for the 30-50 fold more abundant load of lethals, semi­lethals, lowered viability and sterility mutants in the same population, the effect of random drift on overall numbers of these mutants would be much less marked. Random drift would, of course, be expected to have much the same effect on a sample of these mutant types comparable in size to that of the total number of visibles arising in the population. Furthermore, if exposure of different segments of the population to radiation or other mutating factors has been uneven, then one might expect the visibles to be more abundant in chromosomes carrying lethal, semi-lethal and sterility factors, and therefore often not be recovered even when present. In any apparent discrepancy in the findings on visibles and lethals, semi­lethals and other deleterious factors in the populations the a hove facts must be borne in mind. Certainly the control populations on Majuro and Ponape were many times the size of the Bikini population and would presumably not be sub­ject so markedly to the effects of random drift. However, of the 28 mutants actu­ The University of Texas Publication ally recovered from the small Ponape sample six were a distinct type showing tiny scutellar bristles, and probably representing a widespread mutant which at some time in the past had become spread through the populations of this island by random drift. This particular mutant was not recovered from either Bikini or Majuro and therefore is probably not a species specific type such as has been recorded for certain other species of the genus. The Visible Mutants Actually Recovered The primary purpose of this investigation was to make a comparative quanti­tative study of the numbers of visible mutations carried in the population sam­ples. However, it seems worthwhile to.put on record a brief description of all the visibles recovered and a somewhat more elaborate description of certain of these visibles. These data may prove of value to others working on population samples of D. ananassae from these islands or on other populations of this and other species of Drosophila. Table 11 includes a check list of all visibles recovered in the F2 generation from the Bikini, Majuro, and Ponape samples. Mr. Richard Dickerman, a senior stu- TABLE 11 Check List of Mutants Recovered from Bikini, Majuro, and Ponape Samples in F, Generation Bikini Mutants Affecting the Eye bronze-eye color brown--eye color distinct brown, accompanied by very minute bristles brownish-eye color slight brown burnt-an area in the eye has a burnt appearance peach--eye color recovered three times, not necessarily alleles purplish-eye color rough-facets of eye irregular, recovered seven times very rough--extremely rough eyes accompanied by small bristles, recovered three times rough-coarse-eye rough; wings coarse textured, margin nicked mosaic-eyes with mosaic spotting; wings coarse, L4-5 short Bikini Mutants Affecting the Wings abrupt-L4 and L5 short short-L3 and L4 short extreme net-see text for description crossveinless-crossveins missing broken-posterior crossveins broken; recovered four times arch-wings convex dorsally, good expression club-wings club-like, very abnormal crumpled-wings crumpled and folded eagle--wings held out at sides and elevated folded-wing edge folded up nick-cut in margin of wing pads-wings not unfolded shredded-wings light textured, wrinkled and shredded warped-wings warped and dark in color Bikini Mutants Affecting the Bristles erect-post scutellars stand erect fuzzy-see text for description ruffled-hairs on thorax ruffled Studies in the Genetics of Drosophila silver-some dorso-centrals and scutellars silvery; variable small-bristles reduced in size; ten recoveries, probably not all alleles, though tests were not made spineless-bristles very tiny; eyes rough stubby-,--bristles short and stubby Bikini Mutants Affecting the Legs twisted-legs twisted and misshapen Majuro Mutants Affecting the Eye brownish-slight eye color deviation from wild; recovered twice dubonnet-similar to mutant eye colors in simulans and immigrans dull-eye color slightly darker than normal peach-eye color sepia-very dark eye color Majuro Mutants Affecting the Wings blister-blisters on one or both wings roofed-wings held roof-like, sloping branch-a branch on posterior crossvein extra-small bit of vein between ZL and 3L near wing margin net-medium expression of net veins; recovered twice triangle-small vein connecting ZL to margin near wing tip; recovered four times Majuro Mutants Affecting the Bristles small-bristles reduced in size vortex-hairs on thorax in whorls Ponape Mutants Affecting the Eye brownish-slight brownish eye color cinnabar-brilliant red eye color, recovered twice dark-eye color slightly darker than normal mahogany-dark reddish eye color, recovered twice peach-eye color, recovered twice sepia-very dark eye color translucent brown-very easily classified eye color rough-facets of eye irregular rough-short-eyes rough textured; all bristles short rough-shrunken-small rough eyes; wings shrunken Ponape Mutants Affecting the Wings net-extra venation, slight to medium expression roof-wings held roof-like, sloping roof-inturned-wings held roof-like; hairs on abdomen turned in squatty-see text for description warped-wings small and dark, warped downward Ponape Mutants Affecting the Bristles fuzzy-see text for descriptiontiny scutellars-scutellar bristles very much reduced in size; recovered six times Ponape Mutants Affecting Body Color ebony-dark body color, recovered three times; see text for description Note: The names applied here are meant to be descriptive and are not to be considered as implying homologies with mutants of the same name in other species. No symbols have been assigned as the stocks are not being maintained. This check list is included to give an overall picture of mutants recovered from Bikini, Majuro, and Ponape. The University of Texas Publication dent at the College of Wooster was assigned the project of extracting mutants from the F2 Bikini cultures by further inbreeding. By small mass matings he reared F3, F4, and in some cases still later generations of many of these Bikini flies and picked up many of the mutants which by the nature of the F analysis 2 were not then recovered. In Table 12 the mutants which Dickerman recovered in this way are recorded, 34 in all of the 7 4 mutants estimated to be present in the P1 Bikini flies and not picked up in the F2 analysis. It should be noted that Dicker­man did not work on all of the F2 cultures from Bikini and that by the mass mat­ing technique he would not be expected to uncover all mutants present in the TABLE 12 Check List of Mutants Recovered from Bikini Sample in Fa, F,, and Subsequent Generations Mutants Affecting the Eye maroon-reddish-brown eye color difficult to classify brown-eye color garnet brown rough-1-facets of compound eye irregular rough-2--facets of compound eye irregular rough-3-facets of compound eye irregular rough-4--facets of compound eye irregular Mutants Affecting the Wings roof-wings sloping in position, normal overlaps eagle-rotated-see text for description thickened-wings heavy and coarse textured opaque--wings dull coloi:ed and opaque . . . inflated--two plates of wmg separated and con.tammg flmd blistered-fluid filled blister on one or both wmgs incised-wing margin notched between L2-L3 and L4-L5 scalloped-wing margin has cut to beaded appearance xasta-like-see text for description curled-see text for description lance-wings about one half normal width and convex dorsally upturned-see text for description drooping-wings extended at nght angles, down curved; small body rumpled-wings rumpled bent-tip of wing bent pinched-wings slightly rumpled crumploid-wings not fully expanded rippled-wings slightly rumpled rumploid-wings rumpled; normal overlaps abrupt-1-L4 and L5 short radius incompletus-L2 short veinlet-see text for description . . delta-L2 divides into two veins before reachmg margm Mutants Affecting Bristles barbed-tips of posterior scutellars bent . . broken posterior scutellar-post:sc~tellars broken or m1ssmg missing-posterior scutellars m1ssmg ~~~:=..:.-_:__~~~~~~~~~~~~~---­ Mutants Affecting the Legs bent tarsi-tarsi bent; expr~ss~on variable no-joint-see text for descnption Note: The names applied here are meant to be desc.riptive and a.re not to be considered as im 1 in homologies with mutants of t1!e s~me na~e m othe~ sI?ec_1es. No symb.ols have been I? y dg the stocks are not being mamtamed. This check hst is mcluded to give an overall ass1gne as h By · 1 picture of mutants recovered from t e I m1 samp e. Studies in the Genetics of Drosophila material on which he did work. Of course a _very few of the mutants which he' extracted may have arisen in the laboratory, but almost certainly the vast majority of them were present in the original population sample ·collected on Bikini. Descriptions of Some of the More Interesting Mutants Recovered no-joint-Bikini-(Dickerman)-autosomal recessive. This was certainly the most interesting and unique mutant recovered. All five tarsal joints are miss­ing from all legs. The terminal claws present normally at the distal end of the fifth tarsal joint are present on the distal end of the tibia. Males are apparently completely sterile, but from females a few offspring were secured by mating them to wild-type males. No mutant of this type has been recorded for any other species of Drosophila. It is certainly not a homologue but may be compared to four-jointed in D. melanogaster, two alleles of which were found by Schultz and Ives and described by Bridges and Brehme (1944), and to rotund in the same species, alleles of which have been found by Glass and reported in Bridges and Brehme ( 1944) and by Spencer (unpublished) . In rotund there are three joints in the tarsus. This author has found a mutant, two-joint, in D. mulleri, in which the three middle joints of the tarsus are missing, and Krivshenko (personal com­munication) reports the same type of mutant in D. busckii. ebony-Ponaµe-(Spencer)-autosomal recessive. Recovered from three dif­ferent pairs of Ponape flies, this mutant darkens the spiracle sheaths of the larva; pupa .case is dark; in adult flies body, legs, and wings are very dark. There seems little doubt but that this is a homologue of ebony in chromosome III of D. me­lanogaster, many alleles of which have been reported in Bridges and Brehme (1944). Ebony has also been reported by Spencer (1949) in D. hydei and by Chino (1936a) in D. virilis. Moriwaki (unpublished) also reports finding ebony in the second chromosome of D. ananassae, but as Sturtevant and Novitski (1941) have pointed out this chromosome is actually homologous to chromosome III of D. melanogaster. · eagle-rotated-Bikini-(Dickerman )-autosomal recessive. The wings are held out at an angle of 90 degrees from the body and rotated through 90 degrees on the long axis. Thus, seen from above, the wings appear as two narrow bars held at right angles to the body. No such mutant has been been reported in any other species. curled-Bikini-(Dickerman)-autosomal recessive. The wings show the posterior one-third curled upward and are held somewhat divergent, with pos­ terior scutellars sharply crossed. Apparent homologues of this mutant have been found in D. melanogaster, simulans, psuedoobscura, subobscura, montium. llnd mulleri. up-turned-Bikirii-(Dickerman)-autosomal recessive. The posterior fourth of the wing is upturned. The wings are held out at a 30 to 45 degree angle. Acrostichal hairs are brownish, giving the thoax an ashen appearance. Scutellars are often disarranged; deep depressions between tergites of abdomen; fertility greatly reduced. A mutant with marked manifold or pleiotropic effects. veinlet-Bikini-(Dickerman)-autosomal recessive. Longitudinal veins 2, 4 and 5 quite short; posterior cross-vein often broken or missing. Apparently a homologue of mutants of the same name and description in D. melanogaster (Bridges and Brehme, 1944), in D. pseudoobscura, affinis, and willistoni (Sturte­vant and Novitski, 1941), and in D. virilis (Chino, 1936b). fuzzy-Bikini and Ponape-(Spencer)-autosomal recessive. Hairs on the wing margin stand out at a wide angle; hairs on the abdomen are irregularly ar­ranged; dorso-central bristles tend to turn in. A mutant of this description was discovered once from the Bikini sample and once from the Ponape sample'and is similar in phenotype to fuzzy in Chromosome II of D. melanogaster and to frizzled and inturned in Chromosome III of this species. Mutants of similar type have also been described under the name irregular in D. virilis by Chino (1929) and in hydei by Spencer ( 1949), and have been found in mulleri and simulans by Spencer (unpublished). extreme net-Bikini-(Spencer)-autosomal recessive. Wings show an ex­treme net venation in homozygous form; some flies show slight net venation when heterozygous. Similar mutants under the name of plexus or net have been described in D. melanogaster, simulans, peseudoobscura, affinis, virilis, robusta, subobscura, ananassae and immigrans. In fact this is one of the commonest paral­lel or homologous autosomal recessives to be found in various species of the genus. A net vein mutant of medium to slight expression was also found twice in the Majuro population sample. squatty-Ponape--(Spencer)-autosomal recessive, Wings are broad and short; cross-veins very close together; legs short and thick; thorax short. This is a good parallel of dachsous, a common second chromosome mutant in D. me­lanogaster (Bridges and Brehme, 1944), dachsous in the fourth chromosome of D. virilis (Chino, 1929), and squatty in the fourth chromosome of D. hydei (Spencer, 1949). xasta-/ike-Bikini-(Dickerman)-autosomal recessive. Wings shortened and very deeply notched in region of second longitudinal; this mutant is similar in appearance to the dominant, Xasta, associated with a II-III translocation in D. melanogaster (Bridges and Brehme, 1944). The Autosomal Eye Colors In a list of 71 autosomal visibles in '[). ananassae compiled from reports of Moriwaki, Kikkawa and others in Drosophila Information Service and from the published papers of Moriwaki (1935, 1938) only three autosomal eye colors are reported, cardinal in Chromosome II, Plum in Chromosome III and purploid, chromosome undetermined. But from the small Majuro sample of 18 autosomals recovered five distinguishable eye colors were found: brownish, dubonnet, dull, peach and sepia. From the small Ponape sample of 30 autosomals recovered six distinguishable eye colors were found: translucent brown, brownish, cinnabar, dark, mahogany, and peach. From the 88 autosomals recovered from the Bikini sample seven distinguishable eye colors were found: bronze, brown, brownish, burnt, maroon, peach, and purplish. The translucent brown and cinnabar of Ponape and the dubonnet and sepia of Majuro were sufficiently different from the Bikini eye colors to be distinguishable, so that in all at least eleven distinguishable eye colors were found. It seems likely that some of the other cases were mutants at different loci, though not easily distinguished phenotypically. Tests for allelism were not run, nor were the linkage groups determined. With the number of known eye color mimics, where different loci give pheno­typically identical mutant eye colors in D. melanogaster, it is useless to suggest possible homologies of these eye colors found in the samples analyzed. The names have been used as descriptive and are not to imply supposed homologies with mutants of the same name in D. melanogaster or other species. The overall pheno­typic picture of the mutants recovered in this study is similar to that for numer­ous other species of this genus. SUMMARY Wild Drosophila ananassae, collected on Bikini in the summer of 1955, were inbred for two generation? by pair matings. On the basis of autosomal visible mutants recovered it was estimated that there were 128 such mutants present in the population sample of 226 flies, an average of .566 mutants per fly tested. Similar procedure for flies collected on Majuro Atoll in the summer of 1955 gave an estimate of 35 such mutants present in the population sample of 44 flies, an average of .795 mutants per fly. The sample of 76 flies collected on Ponape in the summer of 1956 and tested carried an estimated 40 such mutants, an average of .529 mutants per fly tested. Many of the visibles in the Bikini sample, not recovered in the F2, were sub­sequently found by further inbreeding the stocks. Factors which might explain the fact that the apparent mutation load of visibles from the Bikini test area was no greater than that from the control areas were considered: such factors as population structure, random drift, and the masking effect of lethals and sterility mutants carried in the same chromosome with visibles. -Check lists of visibles recovered and.fuller descriptions of some of the more striking visibles were included. SECTION v FACTORS AFFECTING VIABILITY IN DROSOPHILA ANANASSAE POPULATIONS FROM THE MARSHALL ISLANDS Wilson S. Stone, Florence D. Wilson, June T. Neuenschwander and Thomas G. Gregg The study of the genetic composition of populations of any species is always difficult, particularly when one wishes to measure factors which modify the evolutionary fitness of these populations. There exist in crossbreeding populations a number of genetic differences which influence their fertility, fecundity, and viability. Many of them are recessive, others cause such characteristics as hetero­sis. Some are lethal, others reduce the viability and in combination may act as lethal equivalents, while still others increase the viability singly or in combi­nation. We tested for such factors by comparing results from crossbreeding and inbreeding. PROCEDURES .. The females captured on the islands were placed in individual food vials and allowed to lay. Usually they were fertilized in nature and produced progeny but sometimes no offspring were produced within a few days and a male captured at the same locality was added to the vial. Ordinarily a Drosophila female will produce offspring from one male but some mate more than once and a few of the offspring from th.ese matings may have been half sibs although most were full sibs. [In the following tests we often use an abbreviation to represent a locality: Bikini= B, Rongelap =A, Rongerik = K, Majuro= M, Ponape = P.] The progeny of these isolated females were tested in pair matings in the following ways: (a) random matings between individuals from different parents in the same population, (b) several brother-sister matings between the sibs of those mated at random, ( c) crosses between individuals from populations on different islands (for example, B X M) . The progeny of (c) were inbred in brother-sister matings and their sibs were crossed to the progeny from other crosses in three­way (eg. MP X BP) or four-way crosses (eg. BA X MP). Finally the progeny of these three~ or four-way crosses were tested by random matings. All of these tests were used to get information on the effect of the several genotypes and their heterozygous and homozygous combinations on fertility measured as percentage of pairs fertile; fecundity measured as average number of eggs laid per day per female, and viability measured as percentage of eggs laid that developed into adults. This is an absolute measure of viability against the upper limit of 100 per cerit rather than a comparison of classes so it can be used to compare any tests. Ordinarily the egg count for the first day that eggs developed was not used in order to insure that the females were inseminated. Most of the tests were run at 77° F. (76°-78° F.) but a few in 1965 were run at 71 ° F. (70°-72° F.). The in­breeding and crossbreeding tests compare the range of effects of the different homozygous and heterozygous genetic combinations on characteristics vital to the species. As Spencer pointed out in Section IV, we miss some of the possible genotypes by the limited size of our tests but this is unavoidable.' The nature of the tests involves a succession of generations for each year's collections. General experience has demonstrated that an appreciable part of the culture-to-culture variation in viability within any test will be environmental and due to variations in micro-flor.a, consistency of the medium and other factors. Some evidence on this point was provided by obtaining two or more cultures of eggs from the in­dividual females on successive days; the pooled variation between such replicates will arise chiefly from such uncontrollable environmental differences. RESULTS The results of the tests are summarized in Tables 13 and 14 and Figures 4 through 13. Table 15 and Figures 14 through 16 show data from two other species, Drosophila novamexicana and Drosophila hydei, which are included for comparison. As part of the routine, the eggs laid per day per female were recorded for the period during the egg development tests. There is no evidence for consistent or general differences for the different series for the two years. The heterozygous females from population crosses did not lay more than females from the parent populations. There is no evidence for heterosis in egg laying. This agrees with the tests with novamexicana and hydei (Stone, Alexander, and Clayton, 1954). There is a considerable variation in fertility, the proportion of pairs which pro­ duced offspring, in the populations of ananassae and in heterozygotes between them. Table 16 shows that the average fertility of all stocks available both years (A, B, K, M) was low in 1955 and improved slightly by 1956. The heterozygotes between populations were significantly more.fertile than their parents and were alike for the two years. The F2 averaged higher in fertility than the F10 which must be due to the heterozygosity of their female parents. The differences imply a genetic basis for part of the sterility. In setting up pair matings between indi­ viduals within populations or their heterozygotes, such as three-way crosses, ap­ proximately equal numbers of parents were chosen from different parent cultures to avoid as far as possible any bias, that is, the more fertile matings did not con­ tribute disproportionately to the flies chosen for the next series of matings. The tests for factors influencing egg development proved satisfactory under laboratory conditions. In such a test it is not possible to separate point lethals from lethal combinations due to interaction or to an accumulation of genes which reduce viability. The data on egg counts and development are not given in detail. The zero egg hatch is not included since a great many of the females in pairs which produced no offspring laid eggs. All the pairs which produced no offspring are included in the sterile classes, Table 13. Table 14 gives the egg development results grouped in classes. These are one to nine per cent of the eggs developed, 10 through 19, etc. These are diagrammed by twenties in Figures 4 through 13 which give viability class profiles of the populations and their different combina­tions. In the figures, the per cent of females having from 1 to 19 per cent of their eggs develop are plotted as 10, those with 20 to 39 per cent as 30, etc. In these tests egg development from 80 to 100 per cent is considered normal. When less than 80 per cent of the eggs laid by the female develop, the flies are considered to carry one or more lethals or lethal equivalents. With a maximum upper limit of 100 per cent development, we cannot demonstrate supervital factors even though they may influence the viability of many gene combinations. When brother-sister matings were made, we could determine how often their parents carried lethals. These were detected by the fact that at least one of the one to four brother-sister matings had one or more lethals or lethal equivalents; that is, less than 80 per cent of the eggs laid developed into progeny. These tests indi­cate that the original females, usually fertilized in nature but sometimes mated to males from the same lot, almost always produced offspring that carried one or more lethal equivalents. In 1955, over 90 per cent of the original pairs carried lethals, with Rongerik the exception (75.6% detected). In 1956, about 75 per cent of the pairs from Rongelap and Rongerik carried lethals, about 85 per cent of those from Ponape and Majuro, while only Bikini was extremely high with 96 per cen): of the original pairs carrying lethals. Because of the small number of F 1 pairs inbred, the frequency of lethals and lethal combinations was underesti­mated (see Spencer, Section IV). The brother-sister matings provided tests for all autosomal lethals, lethal combinations, and cumulative subvital forces. Sex linked lethal factors would have been rapidly eliminated by natural selection. l'O <.O TABLE 13 ..... Egg Development on Inbreeding and Crossbreeding Drosophila ananassae • Crosses in wh·ich values are given in this column are those in which F, brother-sister matings were made from pairs. All others were mated at random. P, Females T ested* Fertile Pairs T ested Egg Development % Based Cross 2 xg 1955 Tests Total Pairs T ested % Sterile Number % with lethal Number % with lethal Days eggs Counted Eggs/ day 2 Number on Angular % Transfor-Developed mations >-.:] ~ 1. 2. 3. 4. 5. 6. Rongerik (K ) Bikini (B) Rongelap (A ) Majuro (M ) B X M A X M 170 500 216 288 101 96 43 .5 27.2 57.4 46.5 51.5 54.2 4 1 117 44 61 75.6 98 .3 90.9 91.8 93 297 90 139 43 48 49.5 91.2 83.3 82.7 69.8 64.6 219 716 227 359 79 91 17.2 29.9 17.9 24. 1 29.9 31.4 3760 21464 4058 8640 2361 2856 71.3 46 .4 40.8 56.6 67.8 64.3 75.4 46.1 45.0 56.3 65.7 65 .3 ~ ;:s-·~ (I) ""j ""-...... 7. 8. B X A BM X BM 91 75 48.4 25.3 20 90.0 39 52 64.1 67.3 77 142 33 .3 34.2 2563 4853 70.2 62.5 69.0 65.0 ~ 0- 9. 10. AM X AM BA X BA 79 77 31.6 10.7 23 22 95.7 100.0 51 58 76.5 81.0 137 155 31.8 22.9 4351 3560 59.8 52.4 61.9 56.6 >-.:] 11. BA X BM 87 25 .3 94 31.3 162 32.8 5320 81.1 84.7 ~ 12. AM X BA 101 41.6 59 30.5 151 31.1 4703 84.3 86.4 13. BM X AM 102 49.0 48 14.6 125 31.8 3970 89.4 93 .4 "'ti 14. 15. 16. (BA X BM ) 2 (AM X BA) 2 (BM X AM) 2 112 94 106 33.9 26.6 20.8 73 69 83 40.5 34.8 47.6 188 165 196 22.3 28.3 32.8 4194 4668 6429 78.7 80.8 77 .8 83.8 83.5 82.8 i::: ~ ......-· ~ ~ .....-· 1956 Tests 0;:s 17. 18. 19. 20. 21. 22. 23. 24. 25. Ponape (P ) P (different P ,'s) Majuro (M ) M (different P,'s) Bikini (B) B (different P ,'s) Rongerik (K ) Rpngelap (A ) M x P 6 15 91 495 110 421 45 458 403 123 32.2 28 .6 45.1 40.0 34.0 17.8 49.3 27.0 49.6 145 106 101 84 98 85.5 85.8 96.0 75 .0 74.5 339 47 2 17 55 256 34 173 263 52 61.7 21.3 70.5 38.2 69.5 41.2 58.4 42.2 59.6 748 128 569 146 653 83 393 651 118 27 .5 21.8 20.9 23.7 22.9 27.7 26.3 28.2 13.6 20532 2791 11880 3463 14953 2303 10343 18349 1603 67.6 88.3 66.4 80.8 60.3 75.4 68.2 78.9 58.2 72.0 89.8 67.8 82.6 63.4 79.6 73.5 83.7 64.0 26. A X M 125 76.0 26 19.2 60 22.2 1329 84.6 88.5 27. M X B 142 36.6 76 50.0 188 23.9 4489 75.4 79.7 28. P X A 104 62.5 .. 32 21.9 73 26.3 1918 84.9 90.0 29. BX P 138 43.5 74 52.7 184 33 .9 6237 72.7 75.3 30. B X A 112 41.1 .. . . 66 45 .5 168 30.3 5091 76.5 78.6 31. AM X BP 21 1 36.5 .. 124 17.7 366 23.6 8638 89.7 93 .3 32. BAX MP 143 55.2 50 24.0 11 2 17.7 1987 87 .6 89.0 33 . MB X PA 70 61.4 23 4.3 45 16.6 748 91.7 95.4 34. AM X AM 90 44.4 21 81 .0 47 50 6 133 21.1 2811 71.1 72.4 35 . BA X BA 177 31.1 42 76.2 116 60.3 285 27.8 7930 70.3 72.8 36. MB X MB 215 32.6 54 92.6 143 81.8 415 21.0 8·695 58.6 60.1 37. BP X BP 177 54.2 3 1 83.9 79 62.0 23) 18.9 4442 68.4 38. 39. 40. 41. MP X MP PA X PA MB X BA BA X PA . 96 38 86 85 37.5 31.6 12.8 38.8 24 11 75.0 72.7 50 24 73 39 60.0 62.5 27.4 33 .3 136 66 208 98 18 .0 20.9 22. 1 16.8 2454 1379 4587 1645 67.4 74.7 85 2 80.5 90.7 Cl'.).... ;:: !:I. ~­ 42. 43. 44. 45. MP X AM AM X MB PA X AM PA X MP 68 75 83 38 36.8 54.7 30.1 57.9 43 29 56 14 27 .9 3 1.0 12.5 2 1.4 109 72 171 37 17 .4 23.2 19.1 10.1 190 1 1668 3261 375 83.7 83 .7 89.1 89.9 85.6 .... ;::i .... ~ 4'5. AM X BA 75 12.0 .. fi5 24.fi 194 20.1 3904 82 8 89.5 47 . PA X BP 65 47 .7 29 3.4 73 15.4 1124 91.7 ~ 48. 49. 50. 51. 52. MP X BP BP X BA (AM X BP ) 2 (BA X MP ) 2 (MB X PA ) 2 48 87 80 85 63 70.8 39.1 37.5 34.1 19.0 12 50 50 50 45 0 20.0 54.0 44.0 24.5 27 124 143 123 109 14.6 20.8 23.6 25.6 14.6 395 2580 3381 3145 1591 93 .9 90.8 67.4 73.9 84.2 ;::i 11)....... . ~ "' c- 53. 54. 55. 56. 57. 58. 59. 60. 61. (AM X BA ) 2 (PA X AM ) 2 (MP X BP) 2 (BP X PA)" (P AX MP) 2 (AM X MB ) 2 (BP X BA ) 2 (AM X BP ) 2 (BP X MB ) 2 100 88 50 60 40 86 120 12 41 30.0 18.2 24.0 26.7 40.0 22.1 15.8 00.0 26.8 .. 59 64 35 42 33 60 94 12 22 55.9 20. 3 37.1 42.8 30. 3 33.3 30.8 66.7 40.9 120 178 80 124 80 171 249 3 1 60 16.3 17.6 27 .2 27.5 18.1 13.8 21.9 12.8 21.1 196 1 3141 2177 341 3 1445 2362 5453 396 1267 73.9 84.9 78.8 80.9 78.8 81.0 78.8 70.7 77.4 73.5 83.2 tl.., ~ .g;:::.. ~ I:) 62. 63. (MB X MP ) 2 (BA X PA ) 2 39 11 3 35.9 16.8 19 89 15.8 33 .7 45 216 16.6 24.3 748 5259 83.8 82.4 64. (MP X AM ) 2 99 15.2 81 43.2 199 14.4 28 75 79.8 65. (MB X BA)2 108 29 .6 .. 71 36.6 184 14.9 2755 81 .6 85.0 to <.O °' TABLE 14 Frequency of Classes with Different Egg Development Ranges on Inbreeding or Crossbreeding Drosophila ananassae Cross Number of Pairs with Indicated Percentage Range of Progeny Developing 'i' x c! 1-10-20-30-40-50-60-70-80-90-100 1955 Tests 1. Rongerik (K) 1 0 4 6 4 6 15 10 24 23 2. Bikini (B) 9 25 45 43 52 36 34 27 17 9 3. Rongelap (A) 8 13 13 6 9 8 9 9 12 3 4. Majuro (M ) 4 6 12 11 20 21 30 11 12 12 5. BX M 0 4 0 2 4 5 1 10 8 5 6. AXM 3 3 2 1 4 7 5 7 7 10 7. BXA 0 2 2 1 4 3 7 6 7 7 8. BM X BM 0 1 6 5 5 9 5 4 8 9 9. AMXAM 0 1 2 6 8 8 7 7 8 4 10. BAX BA 1 3 4 8 10 10 3 8 7 4 11.· BAX BM 0 2 0 2 2 4 8 2 16 28 12. AMX BA 1 1 2 2 1 3 5 3 7 34 13. BMXAM 0 0 0 1 1 1 2 2 5 36 14. (BAX BM)2 0 0 1 1 5 5 10 8 11 33 15. (AM X BA)2 0 0 1 1 3 7 6 6 14 31 16. (BM X AM)2 1 1 0 2 4 8 11 13 9 35 1956 Tests 17. Ponape (P) 2 4 17 30 24 38 50 44 50 80 18. P (different P,'s) 0 0 0 1 1 2 4 2 7 30 19. Majuro (M) 1 4 10 19 20 31 33 35 23 41 20. M (different P,'s) 1 0 3 0 2 4 4 7 10 24 21. Bikini (B) 13 11 16 15 26 36 30 31 36 42 22. B (different P,'s) 0 2 0 0 2 3 5 2 7 13 23. Rongerik (K) 1 6 3 8 12 25 20 26 28 44 24. Rongelap (A) 2 2 2 8 14 17 23 43 43 109 25. M X P 3 9 4 3 4 3 1 4 3 18 1 0 1 1 0 2 6 15 26. AXM 0 0 27. M X B 0 0 2 4 4 11 6 11 11 27 2 19 28. PxA 0 0 0 1 1 2 1 6 14 22 29. BXP 2 1 2 4 5 5 6 13 4 4 7 11 25 30. BXA 0 3 3 1 8 31. AM X BP 0 1 2 1 4 1 5 8 15 87 0 0 0 0 0 3 5 4 11 27 32. BA X MP 0 0 0 0 0 0 0 1 5 17 33. MB X PA 6 13 13 19 15 14 32 34. AMXAM 0 1 2 3 6 4 6 6 35. BAX BA 1 1 5 7 9 7 15 21 35 23 13 15 11 36. MB X MB 1 2 1 2 6 9 9 11 11 15 15 0 3 4 0 6 6 3 8 8 12 37. BP X BP 0 38. MP X MP 4 4 1 8 39. PAX PA 0 0 1 1 1 5 1 2 5 4 7 9 44 1 1 0 6 5 11 15 40. MB x BA 0 0 1 41. BAX PA 0 0 0 1 0 0 1 1 0 3 6 10 21 42. MPXAM 0 1 1 1 0 1 2 3 7 13 0 1 1 0 0 0 1 4 9 40 43. AMxMB 44. PAX AM 0 0 2 1 4 7 45. PAX MP 0 0 0 0 2 1 2 3 2 5 6 43 46. AMX BA 0 1 47. PAX BP 0 0 0 0 0 0 1 0 6 22 0 0 0 0 0 0 0 0 3 9 1 2 0 6 4 36 48. MP X BP 49. BP X BA 0 0 0 1 2 6 2 3 8 10 13 50. (AM X BP)2 0 5 1 51. (BAX MP)2 1 1 3 2 2 4 4 5 11 17 1 0 0 0 3 1 6 13 21 52. (MB X PA)2 0 2 0 2 1 4 6 8 10 11 15 53. (AM X BA)2 0 0 1 3 0 1 5 3 13 38 54. (PAX AM)2 1 2 0 5 4 8 14 55. (MP X BP)2 0 0 1 0 1 3 6 8 13 11 56. (BP X PA)2 0 0 0 1 1 2 1 0 1 4 9 14 57. (PA X MP)2 0 0 2 2 2 1 8 5 15 28 58. (AM X MB)2 0 0 3 2 2 1 3 9 9 28 37 59. (BP X BA)2 0 4 0 4 2 2 60. (AM X BP)2 0 0 0 0 0 1 1 1 3 2 3 10 61. (BP X MB)2 0 1 0 0 1 0 2 1 6 9 0 2 3 4 4 7 10 27 32 62. (MB X MP)2 0 0 63. (BA X PA)2 0 0 1 1 2 4 4 8 9 10 36 64. (MP X AM)2 1 1 2 5 16 20 25 65. (MB X BA)2 0 0 1 TABLE 1S Frequency of Classes with Different Egg Development Ranges on Inbreeding or Crossbreeding Drosophila novamexicana and Drosophila hydei Average+ Number of Pairs with Indicated Percentage Range of Progeny Developing % show % % of Eggs Cross 1-9 10-19 ZO-Z9 30-39 4-0-49 SO-S9 60-69 70-79 80-89 90-100 Total Lethal Sterile Sterile Developed D. novamexicana 1. Z3S1.1 * 0 0 0 0 0 3 z 0 3 1 9 S6 3 ZS 67.7 z. Z3S4.4* 0 0 0 z 0 4 s s z 10 Z8 S7 1 3 73.1 3. Z3S8.6* 0 0 0 0 0 1 z 8 10 zo 41 Z7 39 49 86.7 V)..... 4. 8.6 X 8.6 random S. 4 x 1 6. 4 x 6 0 0 0 0 z 0 0 0 0 0 0 3 0 0 0 1 4 1 z 3 3 1 z 1 z 4 s 17 3 10 Z3 18 Z3 17 61 3S Z7 7 9 S4 Z8 Z7 91.7 69.Z 79.8 i;;;· ·"> 7. 1 x 6 0 0 0 0 1 0 0 0 7 16 Z4 4 6 zo 90.9 ~· 8. (4 X 1) F, * 9. (4 X 6) F,* 10. (1 X6)F,* 11. (4 X 1) F, a x b 12. (4 X 6) F, a X b 13. (1 X 6) F, a X b 14. (4 X 1) F2 X6 0 0 0 0 0 0 1 0 1 0 0 0 0 z z 1 0 z 0 0 0 0 6 0 1 0 0 0 s 6 1 0 1 0 0 6 1 3 1 0 0 1 8 s 7 I 1 1 0 4 7 8 1 4 3 1 s 6 s z z s 9 3 s 18 z 3 8 3 33 38 4Z 10 11 17 17 76 71 4S t>O SS Z4 z9 16 37 13 s 6 10 4 33 48 Z4 33 3S 37 19 66.0 67.S 8Z.8 70.7 77.8 8S.S 64.3 ...... ~ 0 (':);:x (':) .....-. three-way cross ~ D. hydei 1S. Z3S4.5* 16. 23S7.S* 17. Z3S8.9* 18. 2360.3* 19. 4.S x 8.9 zo. 7.S x 8.9 Z1. 7.S X 0.3 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 z 0 0 1 z 0 0 0 0 0 0 z s 0 0 0 0 0 4 z z 1 3 0 1 3 3 I 3 z 1 s 4 8 s 7 3 6 3 3 9 s 11 8 z 11 18 Z7 13 Z4 16 9 Z7 61 37 Z3 ZS 31 11 0 1 4 8 4 14 11 0 s 13 38 17 47 SS 78 .S 74.3 77.0 83.0 8Z.Z 8Z. Z 84.8 .Q.. ~ "I 0 "' 0 ~ ;:i-­-. ~ zz. 8.9 x 0.3 Z3. (4.S X 8.9 ) F,* Z4. (7.S X 8.9) F,• ZS . (7.S X 0. 3) F,• 36. (8.9 X 0.3) F1• Z7. (4.S x 8.9 ) x (7.S x 0.3) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 1 1 1 z 0 1 4 z 1 1 1 1 10 7 3 3 z 6 11 11 z 13 1S 9 8 1S 4 1S 19 17 3S 36 11 34 37 1Z 46 z9 46 18 8 6 10 11 6 10 37 Z6 zz 2,4 36 Z3 so 87.1 80.1 83.4 80. Z 87.6 88.0 four-way cross Stocks used in crosses are indicated by the last one or two numbers. • I nbred. to t Based on 1370 to 10,8ZS eggs in a test. co 'I The University of Texas Publication In view of the uncertainty of the origin of lowered viability which may be due to the presence of one or more completely or partially recessive lethals, semi­lethals, or effects arising from interaction of a number of genes, we cannot evalu­ate lethal progeny except in very general terms. Therefore, we have calculated the mean viability on inbreeding and crossbreeding, in addition to the egg develop­ment classes for each test. We wish to measure the reproductive efficiency of these populations. Inbreeding shows the lower limit and crossbreeding the upper limit measured as egg viability within the sampling limits set by our procedure. Since the data are expressed in percentages, the arc sine transformation has been applied to all records before analysis; variance analysis has been used to test for differences between contrasted groups, samples tested on successive years or at different temperatures, etc. (Means have been restored to percentages in the tables.) The variance of viability between pair matings is compounded of ge­netic and environmental difference, but the status of both sorts of effect may be expected to differ in different categories. Thus the genetic variance between sib matings might be expected to be lower than in matings between unrelated indi­viduals, although this may not be the case if there is a high frequency of recessive lethals or semi-lethals in the population. Also inbreeding, which lowers the chance of survival, probably increases the sensitivity to environmental variation so that environmental effects may be relatively more important than is true of the random matings from a population and especially of three-way and four-way crosses, which are particularly vigorous. Some tests were run at 71 °F. in 1955; the rest of the tests in 1955 and all tests thereafter were run at 77° F. Samples from the untreated Majuro population, which is used as control, were run at both temperatures although at different times; viability was 10.6% lower at 77° F.-a difference which is significant at the .05 level (p > .01 < .05, Table 17). We might infer that higher temperature is adverse, but we cannot be certain that flies from different populations will behave in the same way with respect to such a temperature difference. In fact two collections were made at Bikini in 1955, one at the end of July and the second at the end of August. The first was tested at 71 ° F., the second at 77° F. The change is in the opposite direction from that with the Majuro population so no conclusion can be reached on the effect of temperature on egg development. The Bikini population may have been improving during the month between col­lections but it is improbable that it improved so markedly. The two samples at different temperatures were combined in other tests. The differences of this origin will be reflected in higher Standard Errors. In order to see if the inbred populations differ, the others were compared to Majuro as the control available each year. Table 18 shows the comparisons. In 1955 Rongerik was significantly higher than Majuro while Rongelap and Bikini were significantly lower in per cent development. In 1956 only Rongelap differed significantly from Majuro. Table 19 shows the reduction in egg viability on inbreeding. In each case in­breeding significantly reduced the number of eggs that developed. The random mated individuals show that egg development is good despite the appreciable number of lethal and lethal combinations demonstrated on inbreeding. However, it may be noted that the differences between Bikini and Ponape random-mated Studies in the Genetics of Drosophila TABLE 16 Average Fertility of Stocks and their Heterozygotes Percent Fertile 1955 1956 53.0 55.8 69.4 68.7 72.9 72.8 TABLE 17 Comparisons Between Temperatures Difference Viability N (71-77) p % 1955 Majuro 71 ° 63.1 44 10.6* >.01 <.05 1955 Majuro 770 52.5 95 1955 Bikini 71 ° 40.9 179 -13.2• >.01 <.05 1955 Bikini 770 54.1 117 TABLE 18 Comparison of Different Inbred Populations with Majuro 1955 Deviation from Majuro 1956 Deviation from Majuro Population in% viability p Population in % viability p Rongerik +19.1 * <.05 Ponape + 4.2 >.1 Rongelap -11.3* <.05 Rongerik + 5.7 >.05 Bn + n -10.2* <.05 Rongelap +15.9* <.05 Bikini -4.4 >.05 TABLE 19 Showing Effects of Inbreeding with Individual Populations (1956). Percentage development Majuro Ponape Bikini Random mated 82.6 89.8 79.6 Inbred 67.8 72.0 63.4 Decline with inbreeding - 14.8* - 17.8' - 16.2' ' Indicates significance at .05 level. and also between Bikini and Ponape inbred are statistically significant, so, if Ponape were our standard, Bikini was still low in 1956. One simple demonstration of the existence of genetic differences within the stocks is a comparison of the variability between sib matings within families with that between families. This is shown in Table 20 for pooled data for 1955 and 1956. In addition to the tests of brother-sister matings within the populations, the brother-sister matings of the F1 heterozygotes between the stocks is included. There is a significantly greater variation between families in all cases but one. Table 21 was made to show what kind of factors accounted for the differences between similar tests in 1955 and 1956. The starred differences are all significant at the .05 level. The sexes in the cross between stocks from Majuro and Bikini were reversed for the two years. This should make little difference as sex-linked detrimental factors should be eliminated very rapidly and therefore would have been eliminated from the populations. Therefore, most reduction in egg develop­ment must result from autosomal factors. The equivalent crosses are placed to­gether with test values given in the appropriate year. Rongerik is not included in the averages for the populations as it was not tested in crosses. The inbreeding tests showed that egg development improved in 1956 compared with 1955 in the brother-sister matings from Bikini, Majuro and Rongelap (which were all low in the first test), although in Rongerik there was no differ­ence between the years. The heterozygotes from crosses between different popu­lations also show a higher level in 1956, and there is a similar trend in the effects of inbreeding the heterozygotes from crosses between different populations, except for the MB X MB tests. Such a trend from one year to the next naturally raises the question as to whether it is due to genetic differences in composition of the stocks in successive years or whether it really reflects improved environmental conditions in 1956. There are several indications that environmental differences cannot be regarded as primarily responsible. Firstly, technique and general treat­ment of the flies was the same on the two years, secondly the Rongerik population when inbred gave the same value on the two years, and thirdly the performance of three-way crosses and also their crossbred offspring was the same in 1955 and 1956. Thus the average egg development for the former was 88.2 and 88.6% in 1955 and 1956, while for the latter the corresponding figures were 83.4 and 80.6%. While is it quite possible that such crosses may be generally more re­sistant to environmental variation than either random mated individuals derived from individual population or especially the inbreds, it is unlikely that such a difference in sensitivity could be reflected so dramatically in a hypothetical gen­eral environmental difference between 1955 and 1956. Therefore, it is reasonable to conclude that composition of all the populations, other than Rongerik, differed in the two years, presumably due to a different level of recessive genes or gene combinations . . The evidence from three-way crosses suggests that if there are semi-dominant detrimental factors present they are about equal in effect the two years. Crosses between stocks produce all heterozygous progeny but egg developement in 1955 was below that in 1956. As there is no difference in semi-dominant detrimental factors, then the factors responsible for the low egg development in 1955 must have been due to recessives or more probably to combination effects. A difference TABLE 20 Environmental and Genetic Variation. ~ Between Sib Matings within Families Between Families ;:: Data Pooled from Sum of Degrees of Mean Sum of Degrees of Mean i:i.. 1955 and 1956 Squares freedom Square Squares freedom Square F test ~· Populations Inbred s· Majuro 43,787 .93 193 226.88 40,185.40 164 245.03 1.08 ~ Bikini 72,672.45 335 216.93 80,848.18 218 370.86 1.71:j: Rongerik 20,003.07 Rongelap 45,262.60 144 214 138.91 213.50 46,877.29 50,141.23 122 140 384.24 35 8.15 2.77:j: 1.68:j: ~ ;:s (I)..... Ponape 43,11 8.80 184 234.34 59,870.90 153 391.31 1.67:j: ~· Sum 224,844.85 1070 210.14 277,923.00 797 348.71 1.66:j: .Q.. F, of.Crosses between Populations Inbred tl..., ~ AM X AM 10,835.32 BM X BM or MB X MB 22,234.49 56 122 193.49 182.25 1 l,720.49 18,763.29 42 73 279.06 25 7.03 1.44. 1.41t .g ~ BA X BA 20,692.03 109 189.84 24,691.91 63 391.94 2.06:j: ~ Sum 53.85 1.84 287 187.64 55,175.69 178 309.98 1.69:j: *Indica tes significa nce .at .05, t at .01 , :tat .001 level. ,, :~~-. w ...... TABLE 21 Inbreeding Versus Crossbreeding. Cross Difference Type of T est '? x (; 1955 N 1956 N 1956--1955 Inbreeding, Rongerik 75.4 93 73.5 173 - 1.9 brother-sister Bikini 46.1 297 63.4 256 + 17.3* matings from Rongelap 45.0 90 83.7 263 +38.7* population stocks Majuro 56.3 139 67.8 217 + 11 .5 * Average, A, B, M 49.1 71.6 +22.5 Crossbreeding, B X M 65.7 43 initial crosses M X B 79.7 76 + 14.0* between A X M 65.3 48 88.5 26 + 23.2* populations B x A 69.0 39 78.6 66 + 9.6 Average 66.7 82.3 + 15.6 Inbreeding, BM x BM 65.0 52 brother-sister MB xMB 60.1 143 -4.9 matings of F, from AM X AM 61.9 51 72.4 47 + 10.5 crosses between BA X BA 56.6 58 72.8 116 + 16.2* populations Average 61.2 68.4 + 7.2 Crossbreeding, BA X BM 84.7 64 F, of crosses MB x BA 90.7 73 + 6.0* between popula-AM X BA 86.4 59 89.5 65 + 3.1 tions inter-mated BM XAM 93.4 48 (three-way AM x MB 85.6 29 -7.8" crosses) Average 88.2 88.6 + 0.4 Crossbreeding, (BAX BM) 2 83.8 73 random matings (MB X BA) 2 85.0 71 + 1.2 between non-sib (AM X BA) 2 83.5 69 73.5 59 -10.0 progeny of (BM X AM) 2 82.8 83 three-way (AM X MB)2 83.2 60 + 0.4 crosses Average 83.4 80.6 -2.8 • Indicates significance at .05 level. between adverse sex-linked characters for the two years should have been de­tected in differences between the three-way crosses. It would seem that the amount of heterosis present in three-way crosses was about the same for the two years. Seecof showed that gross chromosomal abnormalities that would produce inviable gametes at meiosis were absent or very infrequent both years. As an ad­ditional test for induced chromosomal abnormalities, Ward examined the testes of grasshoppers collected on Bikini in 1956. He found no inversions, bridges or fragments, translocations or supernumerary chromosomes in this material. Figures 4 to 13 give profiles of egg development with inbreeding ( ~7) and crossbreeding (8-13). Similar tests for the two years are together. These are plotted for 20% intervals of egg development so only the class at 90 (80-100% range) may be regarded as free of lethals or summed lethal equivalents. The classes plotted at 70 may be regarded as having one lethal or lethal equivalent, those at 50, 30 and 10 as having two or more lethals or their equivalents. Classes with such values as those plotted at 10 must be regarded as having six or eight lethals or lethal equivalents. As the number of lethal equivalents increases, the actual numerical reduction becomes smaller for each factor reduces the residual survivors by some fraction. Alternately, a gene or genes which caused few func­tional sperm to be present or shortened the life of the sperm or produced eggs Studies in the Genetics of Drosophila 60 !50 "' "' :3 040 :J: !:: 3: "'~ 30 ~ ::i: ... IL ;' 20 10 60 50 :3 "' './) 0 40 :J: 1­ j "' ~ 30 ~ ... IL ;' 20 10 FIGURE 4 60 0----A 45.0 0---·-·-·-· B 46.1 • All!:r------K 75.4 I 5 M 56.3 I I I I ~ 1955 ~ Inbred I ..J I 0 40 I I j!:I I j I I "' P-·---·-I I ~ 30 i ~ ' ::i: ... i ' I IL ' ..., 0--...i i -0­i ---0-_:.. , ·, -a. ~ 20 I · , -_ 4 i , . , 3 i I ' i ' ,fo------1!. I ' IO ,, ' "02 , , ,' 10 30 50 70 90 % DEVELOPMENT IN CLASS FIGURE 6 60 0-·-·-·-· BAXBA 56.6 tr------AMXAM 61.9 BMXBM 650 50 "' ~ 1955 F1 Inbred "' ..J 0 40 :J: 1­ j 8 "'... 30 <1 ~ IL ~ 20 10 90 % DEVELOPMENT IN CLASS 10 FIGURE 5 p24 M 67.8 I ... p 72.0 I 0----A 83.7 I <:>-·-·-·-· B 63.4 I l!ir-----K 73.5 I I 1956 P1 Inbred I a23 I I I I 17 I I I I 10 30 50 70 90 % OEVELOPMENT IN CLASS FIGURE 7 0-·-·-·-·-BAXBA 72.8 l::r ------AMXAM 72.4 0----MBXMB 60.1 -----BPXBP 6a4 .t. MPXMP 67.4 • PAX PA 74.7 34 38 1956 ~ Inbred fl. 35 ·39 1\ 37 I \ I I I ' I ;.f!f ' 'a3G p; ,/ / , . 10 30 !50 70 90 •t. DEVELOPMENT IN CLASS FIGs. 4-7. Profiles showing the capacity of eggs to develop using brother-sister matings. Pro­geny of wild females collected in 1955 inbred (4), and 1956 inbred (5); offspring of crosses be­tween the stocks collected in 1955 inbred (6), and 1956 inbred (7). The numbers for each profile correspond to test numbers in Tables 13 and 14; the mean is given with the symbol representing each profile. which usually failed to develop could give such a result. It is better to regard the mean (which is included on the figu~es) and the distribution of egg development classes (the profile of the test) as giving us measures of the viability of the breeding population. · The inbred tests show that the stocks in 1955 (Figure 4) produced fewer viable eggs and had much worse profiles of breeding classes than in 1956 (Figure 5), Rongerik excepted. This is shown again on inbreeding the F1 from crosses be­tween these populations (compare Figures 6 and 7). In the latter, the MB X MB cross gives a very different profile from the other five tests. The initial crosses between the stock did not lead to a good egg development, especially in 1955 which was significantly below 1956 (compare Figures 8 and 9). This demon­strates that there existed in 1955 a greater number or more effective concentra­. tion of (homozygous or adversely interacting) genes which reduce egg develop­ment. These acted on the parents or their gametes since the zygotes were heterozygous. The similarity of the three-way crosses and the four-way crosses (Figures 10, 11 and 12) rules out semi-dominant factors as the cause of the difference in viability shown by comparing Figures 8 and 9. Even the hetero­zygous three-and four-way crosses have a residue of pairs which have a low egg development. These classes with low productive viability are present in random crosses between progeny of the original females, which simulate the crosses under natural conditions (Figure 13). Table 15 and Figures 14 through 16 give data on the viability and fertility characteristics of some populations of novamexicana and hydei (Stone, Alex­ ander and Clayton, 1954). The former lives in very small linear populations along desertstreams and the latter occurs as a small part of the desert Drosop!Ula. The population studied came from widely separated localities in the Southwest. The novamexicana came from smaller populations than any of the ananflSsae. Comparisons between the results with ananflSsae and those with novamexicana and hydei are very enlightening. Because of the small numbers we combined similar crosses of hydei in Figure 14. All the hydei tests show these populations have a much more favorable structure in terms of egg viability than even the best counterparts in ananassae, except perhaps Rongelap in 1956. None of the very low egg development classes appeared and very few with 20-39% development. Two of the novamexicana stocks (2351.1and2358.6) resembled hydei and were as good or better than Rongelap 1956, Figure 15. No pair with egg development below 40 per cent occurred in tests with these stocks and only six per cent had less than 60 per cent of the eggs laid develop. (Data in Table 15. The average egg development in this table was taken directly from the data.) Stock 2354.4 and its crosses showed a different condition, Figure 16, for these tests resembled tests of a'nanflSsae stocks and crosses in 1956. This stock carried a seemingly semi­dominant factor(s) which caused death of the gametes or zygotes in crosses­contrast 1 X 6 in Figure 15 with 4 X 1 plus 4 X 6 in Figure 16. Even with this : ~ ! :• J.l :'..; . '!. : .. .,:;.,F1cs. 8-1L Profiles showing the capacity of eggs to develop after crossbreeding. Progeny of wildJemales.col~cted in 1955 (8) and 1956 (9) crossed to other stocks; offspring of crosses be­tween stocks collected in 1955 (10) and 1956 (11) tested in three-way crosses. The numbers for each profile correspond to test numbers in Tables 13 and 14; the mean is given with the symbol representing each profile. Studies in the Genetics of Drosophila 50 Ill Ill cl _J 0 40 :I: .... i Ill ~3 cl 2 IL "' at 20 10 80 70 60 50 gi '.) 0 :I: 40 .... j Ill ~ 30 cl 2 IL"' 10 FIGURE 8 D----BXM 65.7 A-·-----· AX M 65.3 <:>-------BXA 69.0 1955 ~ Crosses 1 6 5 ,, , -!--_1!1.' 10 30 50 10 90. % DEVELOPMENT IN CLASS 13 FIGURE 10 0--------BAXBM 84.7 80 70 60 50 Ill Ill cl _J 0 :I: 40 .... i Ill ~3 cl 2 "' IL 10 80 1 60 50 Ill Ill cl _J 0 :I:40 .... i Ill ~ 30 cl ::;; IL "' at 20 10 FIGURE 9 ~ 26,,,, 0---MXB 79.9 £.------AXM 88.5 0-­--­---BXA 78.6 1956 fl Crosses I I I I I I I ' _A.._ --_.J.. 0-·-·­ 10 % 30 50 DEVELOPMENT IN 70 CLASS · 90 FIGURE ·11 O-­----­MBX BA 90.7 l!r ­----AM X BA 89.5 e AM X MB 85.6 1956 3 Woy Crosses 10 30 50 70 90 10 30 50 70 90 'r. DEVELOPMENT IN CLASS % DEVELOPMENT IN CLASS 4' 33 FIGURE 12 I 31 80 80 FIGURE 13 18 32 70 0---BAXMP 89.0 70 0-·-·-·-B 79.6 AMXBP 93.3 -M82.6 l!r---­ - MBXPA 95.4 p 89.8 • 2060 1956 4 Woy Crosses 60 1956 Pi Random Matings . 22 50 50 f/) f/) f/) V> <( ...J <( ...J u u I­ i i V> f/) ~ 30 ~ 30 <( <( :E :E .... .... ... ... ~20 ~ 20 10 10 I ~A. ~ 10 30 50 70 90 50 70 90 o/o DEVELOPMENT IN CLASS DEVELOPMENT IN CLASS "· FrGs. 12--13. Comparison of profiles of egg development after crossbreeding (12) and after random matings within the populations (13). This contrasts forced heterozygosity with random (i .e., natural) matings within a population. The contrasts between inbreeding and crossbreeding or random mating are shown in Figures 5 versus 13, and in Figures 7 versus 12. seriously detrimental factor, crosses involving this novamexicana stock were better than those involving ananassae in 1955. DISCUSSION Many types of studies have been made on the variability of populations. This has been measured for a number of categories: gross chromosomal abnormalities, visible mutations, fertility, and factors affecting viability or heterosis such as lethals, semi-lethals, subvitals, normals and supervitals. Sometimes the varia­bility of all chromosomes has been measured, often only one chromosome has been tested. These Drosophila ananassae populations have been analyzed for most of these factors. Spencer (Section IV) showed that the frequency of visible mutations was in the frequency range found in other Drosophila. As he points out, the large num­ber of lethals present must have reduced the probability of obtaining a recessive visible mutation homozygous. This would cause an underestimate in frequency 60 Cl) Cl) j u :i:: 50 t: "" ID ~40 ~ ... i... FIGURE 16 5+6 8+9 20 10 10 "• 30 50 DEVELOPMENT IN 70 CLASS 90 10 30 50 70 90 10 30 0!. DEVELOPMENT IN CLASS % DEVELOPMENT IN CLASS FIG. 14. Profiles of average egg development on inbreeding or crossbreeding Drosophila hydei. Each line is the average of tests with several stocks (see Table 15). The mean is given with the symbols representing each profile. Zero points are not plotted unless necessary. Fms. 15-16. Profiles of average egg development on inbreeding or crossbreeding Drosophila novamexicana. Figure 15 shows tests involving only stocks 2351.1 and 2368.6 and their crosses. Figure 16 shows tests involving 2354.4 and its crosses to 2351.1 or 2358.6 (See Table 15). The mean is given with the symbol representing each profile. Zero points are not plotted unless necessary. 90 80 70 60 ~30 20 10 FIGURE 14 • e ~ 27 I I 9 I I I I I 80 70 60 D. "' "'