BULLETIN • OF THE TEXAS MEMORIAL MUSEUM Number 13 / July 1967 Geographic Variations in Survival of Hybrids Between Etheostomatine Fishes by CLARK HUBBS Texas Memorial Museum W. W. Newcomb, Director 24TH&Trinity Sts. Austin, Texas Contents ABSTRACT 5 INTRODUCTION 5 METHODS 8 SPECIES INVOLVED 11 HYBRID FERTILITY 23 INTERPOPULATION-INTRASPECIFIC HYBRIDIZATION 24 . . INTRAGENERIC ETHEOSTOMA HYBRIDS 31 ... INTERSPECIFIC HYBRIDS OF PRIMITIVE DARTERS 36 HYBRIDS BETWEEN PRIMITIVE AND ADVANCED DARTERS 42 DISCUSSION 54 Egg Size 54 Hybrid Fertility 57 Patterns of Hybrid Survival 58 Phylogenetic Differentiation 58 Differences Between Reciprocals 58 Ecologic vs. Geographic Factors 59 Gametic Inhibition 60 Reinforcement 62 Introgression 65 Mutual Coexistence 65 Proximate Inhibition 66 ACKNOWLEDGMENTS 68 LITERATURE CITED 68 List of Tables 1. Relative survival of interpopulation crosses of Etheostoma spectabile 25 2. Relative survival of interpopulation crosses of Etheostoma lepidum 26 3. Relative survival of interpopulation crosses of Etheostoma caeruleum 28 4. Relative survival of interpopulation crosses of Hadropterus scierus 28 5. Relative survival of interpopulation crosses of Percina caprodes 29 6. Relative survival of Etheostoma spectabile9 and E. lepidum d hybrids 31 7. Relative survival of Etheostoma lepidum9 and E. spectabile d hybrids 32 8. Survival of Etheostoma lepidum 9 and E. caerulem d hybrids 34 9. Survival ofEtheostoma caeruleum $ and E, lepidum d hybrids 34 10. Survival of Etheostoma caeruleum ? X E. spectabile d hybrids 35 11. Survival of Etheostoma spectabile $ X E. caeruleum d hybrids 36 ... 12. Survival of various intrageneric Etheostoma hybrids 37 13. Survival of Hadropterus scierus $ X Percina caprodes d hybrids 40 14. Survival of Percina caprodes $ X Hadropterus scierus d hybrids 40 15. Survival of hybrids using H. shumardi as a parental species 41 . . 16. Relative survival of Etheostoma spectabile ? and Percina caprodes d hybrids 43 17. Relative survivalofPercinacaprodes ? X Etheostoma spectabile d hybrids 45 18. Relative survivalofEtheostoma lepidum $ X Percina caprodes d hybrids 47 19. Relative survival ofPercina caprodes $ X Etheostoma lepidum d hybrids 48 20. Averagerankingsofsurvivalindicesof greenthroat X logperch hybrids 49 21. Survival of various intergroup darter hybrids 49 ... 22. Medianindexofsuccessofvariousintergrouphybrids 53 23. Comparisonofhybridizationsuccesswithlevel of phylogenetic differentiation 60 GeographicVariationsin Survival ofHybrids Between Etheostomatine Fishes * Clark Hubbs Abstract Comparative analyses of more than 500,000 darter eggs variously tested forfertilityandviabilityfrom 1954to1965,andgatheredfromwidelysepa­rated geographic locations, showed that darter hybrids were as viable as or more viable than the controls. Both reciprocals of 70 hybrid combinations were reared and more than 60 additional combinations had one reciprocalreared. Eggs were able to inhibit the activity of heterospecific sperm, es­pecially after it had expended about one half its normal activity period; an inhibition obviously designed to reduce the effect of chance meeting of gametes in the water. Several patterns of differential survival were noted, all associated with decreased hybridization potential in sympatry. Included were the first ex­ample of postmating reinforcement of isolation mechanisms, and an exampleof reinforcement being masked by more significant behavioral isolation. Several hybrid combinations had more viable hybrids in one reciprocal than in the other, always occurring where one parent had a high laboratory sur­vival and the other was difficult to rear. All examples were maternally in­fluenced. Relativefecunditystudies showthatthedartersinareaswithmanyspecieshave more and smaller eggs than equal sized females in the peripheral areas where few darter species occur. Introduction It has long been known that hybrids can be produced between relativelyclosely related species (Hertwig, 1936), and itis suggested that hybridiza­tion may affect selection of the parental taxa. Since such selection might be expected to affect interspecies’ interactions, various explanations have been proposed for differences in responses to other species. Suggested causes have been arbitrarily classified into three major groups: (1)Thefirstisintrogression assuggestedbyAnderson (1953)andothers. That is, hybridization is selectively advantageous because it increases varia­ * ProfessorofZoology, The UniversityofTexas atAustin. tion. Therefore, those individuals that hybridize produce more vigorous off­spring (the hybrids) than the others, and subsequent generations (of both species)containrelatively greaterfractionsofhybridgenotypesthanmightbe expected otherwise. The hybridization results with a number of plant groupshave beenexplainedthroughintrogression (AlstonandTurner, 1963; Brown and Pratt, 1960; Hall, et al., 1962; Haller, 1962; Raven, 1962; and numerous others); however, few examples are known of animals. Most studies such as those of Raney (1957) or McCarley (1954), provide evidence that the morphologic attributes of the parental taxa approach each other in sympatry but do not test the expected change in hybridization potential. If introgression isadvantageousthesympatricpopulationsshouldbemorelikely to produce hybrids than their allopatric relatives. Even Svardson’s (1965)excellent account of introgression in Coregonus which shows the increased survival probability of Fi hybrids does not demonstrate differences between the hybridization potential of sympatric and allopatric populations. Investi­ gations designed to determine how often this happens are needed far more than further proof of morphologic similarity in sympatry. (2) The second arbitrary classification of geographic variation in hybridpotential is reinforcement or character displacement (Dobzhansky, 1940). That is, hybridization is selectively disadvantageous to the parental species.Any individual that produced hybrids could not produce its own species with those gametes. Consequently those individuals would contribute less genetic material to subsequent generations. In contrast to introgression, this theory suggeststhatthe hybrids donot contribute significant quantitiesofchromatin to subsequent generations and may displace one or both parental speciesecologically. Therefore, hybrids between sympatric parents would be less likelytooccurthan wouldthosebetweentheirallopatriccounterparts,ifthey were placed together. Variations in the hybridization potential of several animal groups seem to fit the reinforcement model (Blair, 1955 and 1958; Hubbs and Delco, 1962; Koopman, 1950; Mecham, 1961; Sibley, 1961; and Vaurie, 1957). Similarly greater morphologic dissimilarity of sympatricspecies compared with allopatric species is in accord with the same model (Brown and Wilson, 1957, and citations). (3) The third suggested result of hybridization interactions between re­lated species is mutual coexistence outlined by J. A. Moore (1957). That is,hybridization doesnotplayasignificantroleinspeciation.Accordingtothis theory hybridization barriers are formed while the parental taxa are diverg­ing to form distinct species in allopatry, and when sympatry occurs at a later date, hybridization is effectively impossible. The failure to produce intra­specifichybrids(Moore, 1946;Minamori, 1955;DuyvenedeWit,1964)sup­ports this theory. One ofthe difficulties associated withevaluating someofthehybridization results is ascertaining the species criteria of the respective authors. That is, Hall (1952) considers that Juniperus ashei and /. virginiana are allopatricspecies that exchange genetic material wherever their ranges contact. It is equally plausible to consider them morphologically distinct geographic races of the same species, and therefore the use of this example as introgression may be challenged. In contrast Minamori (1955) has shown that the two morphs of Cobitis taenia are unable to produce viable hybrids. Biologically the morphs are acting as distinct species, and if so they are not a valid ex­ample ofhybridizationinhibitionwithinracesofasingle species. Despite the occasional difficulty in ascertaining the phylogenetic differ­ences between the taxa studied, all three of these interspecies interactions undoubtedly occur. It is also likely that the three theoretical models gradetoward each other. That is, the selection toward introgression may occur between entitiesthatpreviouslyhadlost mostoftheirpotentialtohybridize in allopatry, or that reinforcement may similarly act with previous existingreductions inhybridizationpotential.Likewise,theapparentlycontradicting introgression and reinforcement may differentially affect the same speciespair, especially at different points of secondary contact. Therefore, a varietyofpatternsmight bepredicted. Teleost fishes are among the animal groups best known for production of natural hybrids (C. L. Hubbs, 1955), and would be expected to show the effects of interspecies interactions through hybridization. The members of the subfamily Etheostomatinae previously have been shown to be amenable to hybridization experiments (Hubbs and Strawn, 1957a, c; Hubbs, 1959) and it is probable that any intrasubfamilial hybrid combination can be reared to mature size; however, hybrids between etheostomatines and related tele­osts die before hatching (Hubbs, 1967), showing a correlation of hybridpotential with phylogeny. Although most or all etheostomatine hybrids can be produced, only one of the numerous natural hybrids (C. L. Hubbs, 1955) is between taxa representing the major subdivision of the subfamily (Hubbsand Laritz, 1961a). It is therefore apparent that hybrid inviability is only one of the many factors involved in preventing gene exchange. It is likely that habitat preference (Hubbs, etal, 1953), breeding site and seasonal restric­tions(Winn, 1958a),gameticcompatability(Hubbs,1959),matepreference(HubbsandMartin, 1965),and othersallmutuallyservetomaintaingenetic stability. Etheostomatine fishes are useful for studies of geographic variation of hybridization potential. The local populations can be distinguished morpho­logically (Strawn, 1961) so that the various races are likely to reflect the circumstances ofthatlocalityandnot be extensivelydistortedby geneflow from distant populations subject to different selective factors. Adults are seldom used for bait or raised commercially so that human dispersal is nil. Adults seem to be more or less sedentary. Although certain etheostomatines will breed in one environment and ripe individuals will move there only to move away between spawns, extensive collecting at one locality throughoutthebreedingseasonmightdepopulatethatlocality,indicatingthatadults do not move extensively during the breeding season. Developmental, probablyfree swimming larval, stages are likely to be the major source for dispersal.An over-worked collecting site will soon be populated with young fish, indi­cating that dispersal occurs during ontogeny. Likewise, there is some evi­dencethatnonreproductiveadultswillmoveabout. Most ofthe above notes ondarterhomeranges arebased onobservationsofE.lepidumandE. specta­bile; however, more limited observations on the other species are not dis­cordant with the outline. Hubbs and Strawn (1957b) showed that one (andprobably most) etheostomatine species breeds several times during a repro­ ductive season. A long reproductive season permits numerous tests of hy­bridization. Etheostomatines are also useful for hybridization experiments because they are relatively easily maintained in the laboratory (Strawn, 1955). They will spawn under a variety of circumstances and are not subject to a number of typical aquarium diseases; i.e., we have lost none to ich. Methods Eggs were stripped into enamel pans or glass finger bowls following tech­niques describedbyStrawnandHubbs(1956). Eachgroupofeggswasthen placed in a location where temperatures were known to be relatively con­sistent. The eggs were examined daily, development recorded, dead eggsremoved, and temperaturesrecorded. Aftercompletionofthe experiment the temperatureswere averaged androunded tothe nearest °C. for comparisons.The thermal survival curves were then smoothed by a running three-point average. Most of the thermal variations in tire experiments were less than ± I.o° C. The magnitude of thermal variation is known to affect the survival during etheostomatine ontogeny (Hubbs, 1964a); however, the variations during the experiments reported here were typically less extreme and equiva­lent among the tests so that comparisons ofrelative survival are considered valid. The numbers of eggs and resulting larvae were carefully checked five times, providing survival figures for four intervals. The first counts were of the number of extruded eggs. The second counts were of the number of eggscontaining embryos withpigmented eyes. This stage is easilyrecognized and apparently seldom if ever achieved by gynogenetic eggs (see Hubbs and Drewry, 1960, for a discussion of the problem). Many hybrid larvae were from parents with distinctly different larval stages; all such examples pos­ sessed morphologicfeatures ofboth oftheparental species showingthat they were hybrids. At optimal temperatures the fraction of eggs containing em­bryos withpigmentedeyes isthoughttoapproximate thepercent offertilized mature eggs. As this figure varies widely between tests (probably because of variations in the efficiency of the investigator), further developmental success is based on the percentage of eyed eggs to achieve the appropriate stage. The third stage is hatching; most darters hatch at approximately the same stage of ontogenv after an interval approximately twice that required to develop pigmented eyes. Darter organogenesis is affected differentially by temperature,butnoreversalintimeofappearance ofstructures,suchasthose reported by Hayes, et al. (1953) for salmonids, has been noted. The fourth stage recorded was the number of young that completed prolarval life. Pro-larval life span is approximately equal to that preceding hatching, and is arbitrarily designated to be so. The last stage recorded was survival throughthe postlarval stage. Because this stage contains individuals from prolarvalstages to small juveniles, its length was arbitrarily set as equal to the sum of prehatching and prolarval stages. Survivals through four developmental stages, individually or in various combinations,arethereforeavailableforcomparisons. Similarresultsprevailintestswithlargenumbers ofeggssothat eachanalysisundoubtedlyreflects the same relative survival. Two figures are presented in this report. The first is the sum of percent fertilization, percentage of fertilized eggs to hatch, percentage of fertilized eggs to complete prolarval stages, and percentage of fertilized eggs to live through postlarval life. Greater emphasis is placed on earlystagesbecause avarietyofunknowncircumstancesmightaffectsurvival in later stages. Fewer of these are effective during the early stages. Micro­organisms in rearing pans would be likely to increase after the experimentshad been maintained for weeks. The second figure analyzed is the sum per­centage of survival of the two stages of posthatching. This figure is con­sideredtorepresent onlyhybridsurvival,becausematernal influencetypical­lyslowsat gastrulationandisnotlikelytobepreponderantbeyondhatching.The temperaturerange for thermal survival varies among species at a singlelocality(Hubbs, 1961a) andbetweendifferentpopulationsofasinglespecies(Hubbs and Armstrong, 1962; Hubbs and Strawn, 1963). Therefore, com­parisons ofrelative survival at a single temperature are hazardous. The data presented here are the sum of survivals at comparable temperatures. That is, comparisons are made only at those temperatures at which data are available for both the hybrid and the parental species. Most parental species had equivalent survival rates, and the survival rates of the hybrids were con­trasted with the combined parental rates. When the parental survivals are very different from the hybrid survival, both relative figures are presented.In order to determine different effects of temperature on developmental survival, most species were raised at a variety of temperatures. The rangesof survival are listed under the various species accounts. The percentage of eggs fertilized with heterospecific sperm approximatesthat of the controls when the tests are made in a damp pan. Tests also have been carried out to determine the duration of sperm function in a largevolume of water. There is a gradual reduction in sperm vitality with time and this reduction is greater in hybridization experiments than in the con­trols. The tests follow the techniques presented by Hubbs (1957, 1960, and 1961b). Briefly, the experiments were carried out by placing one gallon of agedAustintapwater, at 15± 22°C.inan enamelpan.Approximately one pinhead of semen was stripped into the pan and the water stirred. After an interval timed by a stop watch, a set of eggs was scattered in the pan. The number of eggs developing pigmented eyes was considered to be the percentfertilized. The survival numbers were lumped by seconds (10.0-10.9 etc.)and then three-point running averages calculated. The resulting figures were compared with those of the paternal controls. Temperature differences of 5 to 10°C. were shown to change sperm vitality. Preliminary tests at 20°C. showed that sperm lost their fertilizing capacity after less time in the water than they did at 15°C.; however, no significant differences were noted within the temperature range utilized. Moreover, more than 90 percent of the tests were at 15° ± O.5°C. Thus, if experimental circumstances were equivalent,thepercentageofeggstobefertilizedwould dependuponbiologicalfactors. Most of the extensively tested controls had more or less equivalent fertiliza­tionpercentagesuptoabouttenseconds delayandthen agradualdecrease. It seems likely that experimental circumstances seriously affect the tests at less than ten seconds delay and biologic factors are more significant with longer delay. Therefore, the comparisons listed below are limited to those withmorethantenseconds delayat15±2°C. Any differences in survival rates of hybrids and controls in experiments maybe causedbyavarietyofextrinsicfactors;therefore,statisticaltreatment has beenconservative.Obviously,thegeneticsurvivalpotentialwouldvary among siblings, permitting differential survival within experiments. Super­imposed onthisintrinsicsurvivalpatternareanumberofextrinsicfactors.If the biotic and/or chemical environment happens to be adverse, a greaterfraction of fish die than would those in a favorable environment. Therefore, the survival of eggs is not necessarily independent. Moreover, if a series of fisharecollectedfromanenvironmentwithreduced quantitiesofanessential nutrient, this adverse factor might affect all experiments based on the fish. Therefore, the survival of experiments is not necessarily independent. As a consequence, statistical comparisons usually are not presented; however, the 0.001 level is used in this paper as an index of significance in comparingsurvival ofasinglehybridcombinationwiththoseofitscontrols. Therelative survival of a variety of combinations is more likely to be independent and more normal levels of significance are used when contrasting relative success of different combinations. Racial variations in egg sizes have been reported occasionally. Because the numbers of eggs available relate to the necessity to conserve eggs, and be­cause size inversely reflects egg number, an analysis of variations in egg size should reflect reproductive potential. The eggs from a single female were permitted to water harden and five were measured by use of an ocular mi­crometer. The median size was recorded and these averaged. This technique is modified from that proposed by Clark (1925), Species Involved The listing of species follows that of Bailey and Gosline (1955) and the genericrankingfollowsthatofBailey(1951).Thecommonnamesand geo­graphic ranges were taken from G. A. Moore (1957). Semicolons separateriver systems. Hadropterus scierus Swain. The species ranges from the Guadalupe River System, Texas, to central Indiana. The stocks used came from the GuadalupeRiver at Gonzales and San Marcos River at various localities within three miles ofSan Marcos; theColoradoRiver andOnionCreekinAustin;the San Gabriel River at Georgetown and the Lampasas River four miles southwest of Belton; various tributaries to the Neches River in Rusk, Nacogdoches,Polk, andTylercounties;andvarioustributariestotheSabineRiverinSabine County, The Guadalupe River stock has been named as a distinct subspecies(Hubbs, 1954),andHubbsandJohnson(1961)showedthatthefemalesfrom the Guadalupe River had fewer and larger (1.78 mm.) eggs than those from the Colorado River (1.64 mm.). Brazos River System and Neches River Systemeggsaverageevensmaller,being1.58mm.and 1.45mm.respectively.The duskydarterhasbeenshowntohaveanarrowdevelopmentaltemper­ature tolerance, primarily between 22° C, and 27° C. (Hubbs, 1961a). Ad­ditional experiments confirm the range and the relatively low survival (up to 25 percent) at any single temperature. Hubbs (1959) reported that five of eleven attempted intrafamilial hybrids based on H. scierus eggs had been reared through larval stages—P. caprodes, E. radiosum, E. spectabile, E. caeruleum, and E. lepidum being the successful paternal species. Subsequent tests show survival through larval stages using H. shumardi, E. grahami, E. chlorosomum, E. tetrazona, E. punctulatum, and E. nianguae sperm. One of the above, E. chlorosomum, had previously been tested unsuccessfully. Usingless refined techniques, hybrids based on E. proeliare, E. fonticola, E. micro­perca, and E. asprigene sperm also have been reared. The first two had been tried unsuccessfully so that three failures are retained. Two additional fail­ ureshavebeenrecorded—H.copelandiandE. zonule—bothbased ononetry.Ofthefivefailures nowonrecord, nonehasbeentriedmorethanthreetimes. It is not surprising to have occasional failures of hybrids derived from the eggs of a species with up to 25 percent survival. Hadropterusphoxocephalus(Nelson). ThisspeciesrangesfromOklahoma and Arkansas to Minnesota and Pennsylvania. The specimens were obtained from the Big Maries River near Westphalia, Missouri. The eggs averagedapproximately 1.21mm. The single attempt at a slenderhead darter control died before hatching.Eggs from one test using E. caeruleum sperm were successfully reared through the larval stages. Those based on P. caprodes and E. tetrazona died at the same stages as the maternal controls. Failure of two of three hybridswhosematernalcontroldiedisnotunexpected. Usinglessrefined techniques,hybridsbasedonP. caprodesspermwerereared,furtherindicatingthehighsurvival potential of hybrids. Hadropterus evides (Jordan and Copeland). This species ranges from Ar­kansas to lowa to New York. The specimens were obtained from Richland and War Eagle creeks, Washington County, Arkansas. The eggs averaged ap­proximately 1.76mm. Nocontrols havebeenattemptedonthisspeciesandallthreehybridizationcombinations were unsuccessful. The males were P. caprodes, H. phoxo­cephalus,and E.lepidum.Thisisamongtheleastsuccessful seriesofhybridi­zationexperimentsavailable;however,thefailuresarebased onminimaltests andrelated speciesaredifficulttorearinthelaboratory. Hadropterus shumardi Girard. This species ranges from Canada to Texas. The specimens were obtained from the Guadalupe River at Gonzales. The eggs averaged 1.67 mm. The river darter has not been reported previously to have been reared in thelaboratory.Controlsurvivalsarelowbutoccurredbetween 13°and26°C. Successful survival of some hybrids through larval stages were those using H. scierus, P. caprodes, E. spectahile, E. lepidum, E. euzona sperm. Four combinations, all based on one test, were not successfully reared, E. caeru­leum, E. chlorosomumE. proeliare, and A. vivax providing the sperm. The , failures offourofthe combinations arenotsurprisingbecause morethanhalf of the control experiments also failed. The first two listed failures as well as hybrids with E, radiosum, E. fonticola, E. punctulatum, E. asprigene, and £.gracilespermhavebeensubsequentlyreared usinglessrefinedtechniques.Hadropterus copelandi (Jordan). This species ranges from Oklahoma to Canada. The stock of the channel darter came from the Little River near Nashoba, Oklahoma. The single control died after hatching as did the hy­bridsbasedonP. caprodesandE.radiosum sperm.Thefailuresofthehybrids arenot surprisingbecausethematernalcontrolsalsofailed.Usinglessrefined techniques hybrids have been reared using sperm of E. radiosum, E. specta­bile and E. asprigene. Percinacaprodes(Rafinesque).This speciesrangesfromCanadatoTexas. The specimens wereobtainedfrom severallocalities inthe GuadalupeRiver between Hunt and Waring, the Guadalupe River at Gonzales, and the San MarcosRiverwithinthreemilesofSanMarcos; theColoradoRiverinAustin,Cummins CreeknorthofFayettevilleinFayette County,PedemalesRiverin Hays County, Llano River at Junction, San Saba River at Menard and Ft. McKavett, and the S. Concho River four miles south of Christoval; San Ga­briel River at Georgetown, Salado Creek at Salado, Lampasas River four miles southwest of Belton, the Brazos River in Falls County, Texas; an un­named creek just north of Wilbarton, Oklahoma; Clear Creek and MuddyFork near Savoy, Arkansas; the White River near Durham, Arkansas, Rich­land Creek and War Eagle Creek east of Fayetteville, Arkansas; and the Little and Big Maries rivers near Westphalia, Missouri. Racialvariationhas beenrecordedforP. caprodesbyanumberofauthors,and at least three subspecies are often recognized. All of the material here discussed is traditionally placed in the subspecies carhonaria. Within this group Hubbs and Strawn (1963) have shown racial differences in develop­mental tolerance, and Hubbs (1958b) has shown that females from the Colo­radoRiverinTravisCountycontainmoreeggsthanfemalesfromthe Guada­lupe River in Kerr County. This is substantiated by studies of egg size. Ken-Countyeggsaverage 1.74mm.andTravisCountyeggsaverage1.63mm.The difference could involve river systems or west longitude. The latter seems likely because western Colorado System eggs average 1.71 mm. and eastern Guadalupe system eggs average 1.60 mm. There seems little question that females collected from the edge of the Edwards Plateau have more and smaller eggs than those collected to the west in the middle of the plateau.Eggsfrom femalesfromthe Brazos systematthe edgeofthePlateau average1.49mm.ThosefromtheIllinoissysteminArkansasaverage 1.44,thosefrom the White system in Arkansas average 1.59, and those from the Big Maries inMissouriaverage 1.35mm.Winn(1958a)reportedthataMichigansampleaveraged 1.31 mm. The trend toward smaller eggs is, therefore, both west to east and south to north. TheP. caprodesvariationiscomplicatedbythepresenceofasecondmorphin Central Texas. It has been collected from the Guadalupe River six miles east of Kerrville, the Guadalupe River at Gonzales, the Colorado River in Austin, and the Pedemales River in Hays County. This morph differs from “typical” P. caprodes of Central Texas by having finer features; i.e., the color marks are thinner, the head structure is more delicate, the fin spines are less stout, etc. Similarly, the eggs are smaller, averaging 1.32mm. A multitude of taxonomic questions concerning the two sympatric morphs of P. caprodes are not resolved. It is, of course, possible that the differences reflect some varia­tion in developmental circumstances; however, it is more probable that the morphs represent distinct species because intergrades have not been found, andtheyare sympatricoveraratherlargearea.Whichofthetwomorphs ac­tually represents P. caprodes is difficult to determine. Traditionally the coarse morph has been called P. caprodes; however, the color patterns and egg size ofthefine morphmorecloselyresembletheattributesofMissourispecimens.In contrast, stocks from intermediate localities tend to form a cline between Missouri and the coarse morph of Central Texas. An additional complicationis the allocation of the name carbonaria. Girard’s (1859) description and Evermann and Kendall’s (1894) discussion do not serve to determine which morph was used and the best distinguishing characters are not those that would be present on one hundred year-old specimens. The types are from the sympatric area. In this paper the coarse morph is called P. caprodes and the fine morph P. species. Up to 25 percent of the logperch controls can be reared at temperaturesbetween 22° C. and 25° C. Hubbs (1959) has shown that the eggs can be crossed successfully with H. sciems, A. vivax, E. hlennioides, E. radiosum, E. lepidum, and E. spectabile sperm, and that the test with an E. fonticola male failed. Additional successful hybridization experiments with P. caprodes eggs occurred with P. species, H. shumardi, E. tetrazona, E. grahami, E. caeruleum, E. punctulatum, and E. flahellare males. Single unsuccessful tests were also made with H. copelandi, H. phoxocephalus, E. zonale, and E. euzona sperm. The five failures are not surprising considering that control eggs have a relatively low survival. Moreover, E. fonticola, E. chlorosomum, and E. asprigene sperm now have produced viable hybrids in subsequentless refined tests. Up to 25 percent of the controls of Percina species develop at temperaturesbetween 19°C.and25°C.Successfulhybridizationhasbeencarriedoutwith P. caprodes, H. scierus, H. shumardi, E. lepidum, and E. spectabile males. Ammocrypta vivax (Hay). This species ranges from Texas to Missouri. The stocks come from Horse Pen Creek west of Woodville, Texas. Hubbs (1959)reported failure of both hybrids and controls. The only subsequent tests on the southwestern sand darter are based only on males so that the results will belisted under thematernalparent. Etheostoma chlorosomum (Hay). This species ranges from Minnesota to Texas. The stocks were obtained from Cummins Creek north of Fayetteville,Texas; various Neches River tributaries in Nacogdoches, Polk, Tyler, and Rusk counties; and Sabine River tributaries in Sabine County. The eggs aver­aged 1.05mm.indiameter. Oneofsixcontrolexperiments withthe bluntnosedarterwassuccessful.It was done at 26° C. Hubbs (1959) reported failure of the only attempted hybrid with E. lepidum sperm. Succesful hybridization is here noted using E. radiosum, E. lepidum, E. spectabile, and H. scierus males. The few tests withE.gracile,H.shumardi,andP.caprodesspermallfailed.Failure ofthree ofsevencombinationsusingeggsofaspeciesinwhichoneofsixcontrols sur­vived is not unexpected. Using less refined techniques, hybrids based on E. nigrum, E. punctulatum, E. asprigene, and E. gracile sperm have also been reared. Etheostoma nigrum (Rafinesque). This species ranges from Oklahoma to Canada. The single male used came from the Big Maries River near West­phalia, Missouri. Hybrids have been reared based on E. punctulatum, E. blennioides, E. radiosum, and E. lepidum sperm after this manuscript was completed. Etheostoma stigmaeum (Jordan). This species ranges from Oklahoma to Florida. The stocks were obtained from Clear Creek near Savoy, Arkansas; and the White River near Durham and Greenland, Arkansas. The Illinois Systemeggsaveraged 1.07mm.andtheWhiteSystemeggs 1.38mm. All four control experiments with the speckled darter failed. Hubbs (1959)reported no hybrids reared using males of five species with these eggs. Suc­cessful hybrids were subsequently produced using E. juliae, E. caeruleum, and E. spectabile males, and one more failure with E. euzona sperm was noted. Three successful and six unsuccessful combinations are not surprising using eggs of a species not yet reared experimentally. The successful combi­nations often were tried more times than the failures. Using less refined techniques hybrids with E. nigrum and E. blennioides males were reared. Etheostoma tetrazona (Hubbs and Black). This species is found in Mis­souri River tributaries in Central Missouri. The stocks were obtained from theBigMariesRivernearWestphalia,Missouri.Theeggsaveraged 1.63mm. Up to 20 percent survival of Missouri saddled darters was noted between 18° C. and 28° C. No previous hybridization has been reported. Hybridiza­tion was successful when E. caeruleum, E. spectabile, E. punctulatum, E. lepidum, E. juliae, and P. caprodes males were used, and the one attemptwith E. stigmaeum sperm failed. Again the survival of hybrids was equal to orgreaterthanthatofthe controls. Thesubsequentrearing ofhybridsbased on sperm of E. flabellare, E. euzona, E. zonale, E. blennioides, and E. radio- sum, isinaccord withanhypothesisofhybridvigor. Etheostomaeuzona (HubbsandBlack).ThisspeciesisfoundintheWhite River system of Arkansas and Missouri and associated areas. The stocks were obtained from the Buffalo River, War Eagle Creek, and the White River near Durham, Arkansas. The single control of the Arkansas saddled darter failed; however, it is probable that survival approximates that of its near relative, the Missouri saddleddarter.Hubbs (1959)reported survivalofhybridswithE. caeruleum sperm. Additional hybrids were produced successfully with E. lepidum sperm, and the single test with E. punctulatum sperm failed. Two of three hybridcombinationswereraised andthesinglecontrolfailed. Etheostoma zonale (Cope). Thisspecies isfound fromArkansas toGeorgia to the Lake Michigan drainage. The stocks were obtained from Muddy Fork near Savoy, and Clear Creek at Savoy and Johnson, Arkansas; the White River at Durham and Greenland and a tributary, Richland Creek; and the Big Maries River near Westphalia, Missouri. The Illinois and White system eggsaveraged 1.56mm.,andthosefromtheBigMariesweresmaller,averag­ing 1.42 mm. Control experiments on the banded darter have been tried fourteen times. None was reared through the larval stages. The best success was at 26° C. and 27° C. One of six subsequent less refined control experiments was suc­cessful at 20° C. It was the result of a Big Maries X Richland Creek inter- population test. Hubbs (1959) reported rearing hybrids with E. caeruleum males and failures with E. euzona and H. nigrofasciatus sperm. Additional successfultests werecarried outwithE. spectabile,E. lepidum,E. stigmaeum, E. blennioides, E. tetrazona, E. punctulatum, E. nigrum, and P. caprodesmales. Two additional failures were noted, all based on three or fewer tests withE. juliaeandH.phoxocephalussperm.Successfulrearingofnineof 13 combinations mustbe considered tobehighsurvivalbecause theintrapopu­lation maternal controls have not yet been reared. Moreover, the maximum number of tests with an unsuccessful hybrid combination was three, and all fourteen intrapopulation control tests failed. Subsequent experiments have resulted in successfulrearing of hybrids with H. copelandi, E. fiabellare, and E. radiosum sperm. Etheostoma blennioides (Rafinesque). This species ranges from the Ozarks tothe GreatLakes. Thespecimens werefromClearCreekatJohnson’sand GreathouseSpringsnorthofFayetteville, MuddyForknearSavoy,Arkansas; theWhite River at Greenland andDurham, Brush Creek eastofFayetteville,Arkansas; and the Big Maries River at Westphalia, Missouri. The Illinois (1.81) and the White River (1.76) eggs were larger than those from the BigMaries (1.52). Northern eggs are much larger. Winn (1958a) obtained an averageof 1.85mm.forMichiganeggs;andFahy (1954) obtained,preserved(and presumably shrunk) mature eggs between 1.83 and 1.89 mm. from Salmon Creek, New York, females. Only six control experiments have been run on the greenside darter, and one at 24° C. was successful. Fahy (1954) showed that a New York popula­tion spawnedfrom 13° C. to 22° C. Apparently there is some geographic vari­ation in temperature adaptations. The two controls run at 14 and 16° C. failed. No previous hybridization has been recorded; and success was at­tained with E. spectabile, E. lepidum, E. punctulatum, E. caeruleum, and P. caprodessperm. No survival occurredinexperimentswithE. tetrazona males. The failure of one of six combinations is not surprising considering that one of sixcontrol experiments was successfuland that subsequenttestswithmales of six other species, E. chlorosomum, E. nigrum, E. stigmaeum, E. zonale, E.tetrazona,andE.jlabellare,all succeeded. Etheostoma nianguae (Gilbert and Meek). This species is restricted to central Missouri. The fish were obtained from the Big Maries River near Westphalia, Missouri. Allindividuals ofthe singlecontroltest onthe Nianguadarterdiedbefore completion oflarval stages. Hybridization was successful withE. blennioides and P. caprodes sperm, and unsuccessful with E. zonale males. Hybrid sur­vival compares favorably with control survival. Etheostoma juliae (Meek). This species occupies the White and James River systems in Arkansas and Missouri. The stocks were obtained from the Buffalo River, War Eagle Creek, and the White River from near Durham, Arkansas.Theeggsaveraged 1.68mm.indiameter. Theyolkdarterhasbeenrearedtwiceinsevenattempts,onceat20° C.and onceat 28°C.Thethermalrangefordevelopmentalsurvivalisunknownbut is likely to have an upper limit near 30° C. and a lower limit near 20° C. No hybridization has been reported previously, and success was attained using E. caeruleum, E. spectabile, E. tetrazona, E. lepidum, and E. punctulatum sperm. No survival through the larval stages occurred in the tests with E. zonale and P. caprodes males. The survival of five of seven hybrid combina­tions contrasts favorably with the survival of two of seven controls. Etheostoma punctulatum (Agassiz). This species is restricted to Ozark streams. Most fish were collected from Little Wildcat Creek and Greathouse Spring north of Fayetteville, Arkansas. Others were taken from Brush Creek near its mouth into the White River and Muddy Fork of the Illinois near Savoy.TheGreathousefemaleshadeggsaveraging 1.50mm. The stippled darter has been reared from 19° C. to 24° C. Survivals mayexceed 75 percent although the sample sizes are often minimal. No hybridiza­tion has been reported previously, and successes are here recorded with E. spectabile, E. lepidum, E. caeruleum, and E. tetrazona sperm; and a single experiment with one E. juliae male failed. Hybrid survival again comparesfavorably with that of the maternal controls. Using less refined techniques,hybridsbasedonsperm ofE.zonaleandE.blennioideshavealsobeenreared. Etheostoma asprigene (Forbes). Occurs in tire Mississippi lowlands. The stocks were obtained from Gibbons Creek 4 miles west of Douglass, Nacog­doches County, Texas. No controls were attempted in the tests, all of which were donewiththe lessrefinedtechniques. Successfulhybridswereproducedwith E. blennioides, E. radiosum, E. caeruleum, E. spectabile, and E. gracile sperm. Etheostoma radiosum ( Hubbs and Black) occurs in the Red River tribu­taries in Oklahoma, Arkansas, and Texas. The stocks were obtained from the Little River and its tributaries in Pushmataha County, Oklahoma; Neches River tributaries in Nacogdoches County; and Sabine River tributaries in Sabine County, Texas. Upto50percentofthecontrols oftheorangebellydartercanbereared at temperatures between 19° C. and 23° C. Hubbs (1959) reported hybrid sur­vival through the larval stages with males of E. spectabile, E. lepidum, H. scierus, P. caprodes, and A. vivax, and a failure with E. proeliare sperm. Ad­ditional hybrids reared were fertilized by H. copelandi sperm, and the single test with E. chlorosomum sperm failed. The failures of the two combinations are not considered of major importance because only a single test was run witheach ofthe speciesinwhichmalesproduceminimal quantitiesofsperm.Moreover, one of them, E. chlorosomum, has subsequently produced viable hybrids as have males of H. shumardi, P. species, E. stigmaeum, E. euzona, E. zonale, E. fuliae, E, gracile, E, microperca, E. nigrum, E. tetrazona, E. blennioides, E. punctulatum, E. caeruleum, and £. tohipplei. The reciprocalofthelast hasalsobeenreared. Etheostmna caeruleum (Storer). This species ranges from the Ozarks to Canada, ThestockswereobtainedfromtheBuffaloRiver, theWhiteRiverat Greenland, Durham, and its tributaries, Richland, Brush, and War EaglecreeksnorthandeastofFayetteville,Arkansas;andtheLittle MariesRiver, Big Maries River, and Loose Creek near Westphalia, Missouri. The White Rivereggsaveraged 1.78mm.(Brush),1.79mm.(Greenland),and1.82mm. (Durham).TheBigMarieseggsweresomewhatsmaller,averaging 1.72mm. The difference is supported by the much smaller average of the small Little Maries sample (1.47 mm.). Winn (1958a) reported that small females (yearlings) lay smaller (1.56 mm.) eggs than do larger, two year-old females (1.78 mm.); however, his sample size was small. Only occasionally have small, ripe eggs been noted in these experiments. All have involved small females, but more than 90 percent of the small females contain normal sized eggs- The rainbow darter has been reared at temperatures between 11° C. and 29° C. Survivals were up to50 percent, showing that this is a good laboratoryanimal. Hubbs (1959) reported survival of E. caeruleum eggs fertilized with E. lepidum, E. euzona, E. juliae, and P. caprodes sperm, and failures of one and two tests with E. fonticoh and H. scierus sperm. Additional successfullyreared hybrids are with E. spectahile, E. tetrazona, E. zonale, E. punctulatum, E. flahellare, E. stigmaeum, E. nianguae, H. shumardi, and H. phoxocephalusmales. A single test with E. hlennioides sperm failed before completion of larvalstages.Onlythreeof 16combinationsfailed,andthethreewerebased on a total of four tests. Again hybrid survival compares favorably with that of the controls. Moreover, two of the three combinations that failed, H. scierus and E. hlennioides, have been subsequently reared as have those with males of A. vivax, E. chlorosomurn, E. radiosum, and E. microperca. Etheostoma spectahile (Agassiz). This species ranges from Texas to Michi­gan. The stocks were obtained from the Guadalupe River just west of Mo Ranch, at Hunt, west of Ingram, five miles west ofKerrville, three miles east of Comfort, and near Kendalia, the San Marcos River, within four miles of San Marcos, and the Blanco River five miles east of Blanco; Cole Creek, Gil­lespie County, the Llano River at Junction, the San Saba River at Ft. Mc-Kavett, the South Concho River four miles south of Christoval, and the Colo­rado River in Austin; the San Gabriel River at Georgetown, Berry Creek three miles east of Georgetown, Salado Creek at Salado, the Lampasas River fivemiles southofBelton,andthe BrazosRiverinFallsCounty;anunnamed creek north of Wilbarton, Oklahoma; Mountain Creek, Franklin County, Ar­kansas; Muddy Fork and Clear Creek near Savoy, Greathouse Springs and Little Wildcat Creek near Fayetteville, and Clear Creek near Johnson, Ar­kansas; the White River near Winslow, Durham, and Greenland, and Brush Creek east of Fayetteville, Arkansas; and The Big Maries and Loose Creek near Westphalia, Missouri. The Guadalupe River eggs tend tobe the largest,averaging 1.48mm.to1.57mm.forfoursampleswithtenormorefemales studied. The Kerr County samples are distinctly larger, averaging 1.52, 1.55 and 1.57mm., than the San Marcos sample, averaging 1.48mm. The Colorado systemeggstendtobeslightlysmaller,rangingbetween1.43and 1.52mm. in diameter.ThelowerfigureisforAustin andtheupperforJunctionsamples.Similartotheresults ofP.caprodes,thewestern samplesareinvariablylargerthanthosefrom theeasternedgeoftheEdwardsPlateau.TheBrazossamples,allfromalongtheedgeofthePlateau,average1.46mm.(Georgetown), 1.41 mm. (Salado), and 1.36 mm. (Lampasas) in diameter. The Illinois System eggsarerelativelysmall,averaging 1.31to1.41mm.indiameter.WhiteRiver eggs are slightly larger, both samples based on ten or more females averaging 1.46mm.,thesmallsamplefromBrushCreekdeviatesbybeingmerely 1.36 mm. The Missouri eggs are the smallest, the large sample from Loose Creek averaging 1.22 mm. and that from the Big Maries, 1.32 mm. Winn (1958a)reported small eggs (1.24 mm.) from Michigan. There is a distinctly smaller averageeggsizeinnorthernsamples.Apparentlyanorth-south aswellasan east-west dine prevails. Hubbs and Armstrong (1952) have shown that northern eggs and larvae tend to survive better at warmer temperatures than do southern samples.They suggested that the difference was due to rapid seasonal wanning in the north and similar thermally induced termination to the reproductive season. The larger samples now available substantiate the hypothesis. The survival until hatching is nearly equivalent, whereas the maximum temperature for optimal postlarval survival is 26° C. to 27° C. in Texas populations and near 30° C. for stocks obtained in Loose Creek. The orangethroat darter adapts well to laboratory conditions. Up to 75 percent of fertilized eggs at temperatures between 10° C. and 27° C. survive through the larval stages (Hubbs 1961a). Hubbs (1959) reported survival through larval stages of eggs fertilized with E. lepidum, E. grahami, E. cae­ruleum, E. gracile, E. parvipinne, E. hlennioides, E. fonticola, H. scierus, and P. caprodes sperm, and failure of the single experiment using E. radiosum sperm. Additional hybrid combinations are with E. radiosum, E. zonale, E. stigmaeum, E. jiahellare, E. tetrazona, E. euzona, E. punctulatum, E. juliae, E. proeliare, H. phoxocephalus, H. shumardi, H. copelandi, and Percina spe­cies males. The eggs of this good laboratory animal have been exposed to sperm of 22 other members of the same family and in all combinations indi­viduals have passed through the larval stages. Occasional examples have failed as have some controls, and none can be considered to indicate hybridinviability. Three additional combinations have subsequently been reared based on sperm of E. microperca, E. whipplei, E. chlorosomum, and E. nigrum. Etheostoma grahami (Girard). This species is found in clear spring-fedtributaries of the Rio Grande. The specimens were obtained from Dolan Creekand SanFelipeCreekinDelRio, TheRioGrandedarterhasbeensuccessfullyreared (Strawn, 1961),but controls have not been attempted in this investigation. Hubbs (1959) re­ported one successful hybrid combination with E. spectabile sperm and a failure with P. caprodes sperm. A single test with E. radiosum has also failed. The two failures are based on a total of six fertilized eggs and are not to be considered to demonstrate hybrid inviability. Moreover, hybrids with P. caprodes males have subsequently been reared as have hybrids based on H. scierus, H. shumardi, E. caeruleum, and E. microperca sperm. Etheostoma lepidum (Baird and Girard). This species occupies clear spring-fedwatersoftheNueces, Guadalupe,andColoradoriversystems.The stocks were obtained from the Nueces River five miles south of Camp Wood, andatBarksdale,theWestFrioRiver andKent Creekattheirjunction,and the Frio River at Leakey and Garner Park; the South Guadalupe River at Lynxhaven approximately nine miles west of Hunt, the North GuadalupeRiver at a spring one half mile west of Mo Ranch, the head spring and asso­ciated concrete ditches supplying the State Fish Hatchery at Mountain Home onJohnsonCreek, theGuadalupeRiveratHunt,threemileswestofIngram,five miles east of Kerrville, and three miles east of Comfort; the Colorado River and tributaries in Austin, the Llano River at Junction, the San Saba River at Ft. McKavett, and the S. Concho River four miles south of Christo­val. Hubbs and Delco (1960) reported that Nueces River females had fewer and larger eggs than any other population of E. lepidum analyzed. They also reported that Frio River females had relatively numerous(small) eggs.Additional information shows that the apparently large numbers of Frio River eggs was erroneous, probably based on a small sample size—l 7 females—because the much more extensive Garner Park sample here reported—44females—have egg sizes nearly identical with those from the Nueces System. Theaverageeggsizesforsampleswithmorethan 10femalesare1.52mm. (Gamer Park), 1.53 mm. (Barksdale), and 1.56 mm. (Camp Wood). The egg size varies among the Guadalupe and Colorado populations with ten or more females analyzed, averaging 1.30 mm. nine miles west of Hunt, 1.34 mm. at Hunt, 1.39 mm. one half mile west of Mo Ranch, 1.31 mm. at the Mountain Home State Fish Hatchery, 1.34mm. at Austin, 1.38mm. at Junc­tion, 1.28 mm. at Ft. McKavett, and 1.30 mm. four miles south of Christoval. The samples closer geographically to the Nueces system are no more likely tohave large eggsthan are thosemore distant. Thisspeciescanberearedattemperaturesbetween 11°C.and27°C.with survivals up to 80 percent (Hubbs, 1961a) based on the sample from the South Concho River, and subsequent experiments have extended the rangeto 8° C. to 29° C. The Nueces River sample deviates slightly by having its survival between 7° C. and 28° C. Hubbs (1959) reported that eggs of this species were successfully crossed with sperm of E. spectahile, E. radiosum, H. scierus, and P. caprodes. In addition the following males have successfullyproducedviablepostlarvaewithE.lepidum eggs:E. chlorosomum,E.gracile, E. punctulatum, E. caeruleum, E. juliae, E. tetrazona, E. nianguae, E. hlen­nioides, P. species, H. shumardi, and H. copelandi. Single tests, each with E. nigrum and E. proeliare sperm failed. The latter has subsequently been reared as have hybrids with E. zonale and £. asprigene males. Etheostomaflabellare (Rafinesque).Thisspeciesisfoundfromthe Ozarks to Canada. The specimens were collected from Little Wildcat Creek, Great-house Spring,and Clear Creekat Johnson,allnorth ofFayetteville, Arkansas; and from Big Maries River and Loose Creek near Westphalia, Missouri, The Loose Creek eggs average 2.06 mm. and those from the Big Maries 2.23 mm. Comparable sized eggs (2.3 mm. and 2.22 mm.) were noted by Lake (1936)and Winn (1958a) for New York and Michigan populations respectively. This species was not successfully reared in these experiments. The large eggs are difficult to expel without damage, and sperm quantities are minimal. The successful combinations are those with E. spectahile, E. lepidum, E. cae­ruleum,E.tetrazona,E.punctulatum,andP. caprodesmales;andthefailures with E. zonale, E. hlennioides, E. juliae, and H. phoxocephalus males. Six out of ten hybrid combinations were successful despite the failures of the con­trols. Most of the unsuccessful combinations (including the controls) were attemptedfewertimesthanthe successfulones.Moreover,thefirsttwolisted failures have been reared subsequently using other techniques. Etheostoma gracile (Girard). This species ranges from Texas to the Great Lakes. The specimens were from Cummins Creek north of Fayetteville,Texas,BrazosRiverinFalls Countyand atributaryinBrazos County,and in HardinCounty,Texas.Theeggsaveraged 1.01mm. This darter has not been reared using this technique, although naturallylaid eggs have been reared. No hybrid combinations have previously been recorded. The hybrids have been successfully reared using E. lepidum and E. spectabile males, while the two tests with E. punctulatum males failed. The failure is not surprising considering the failure of the controls. Etheostoma proeliare (Hay). This species ranges from Texas to Illinois. The specimens were obtained from Cummins Creek, Fayette County, Texas; various tributaries of the Neches River in Polk and Tyler counties. This darter has not been reared in these experiments. A relatively large fraction of naturally laid eggs have been raised by other techniques. Hubbs (1959) reported that two males, E. radiosum and A. vivax, had produced hy­brids that survived through the larval stages and that four combinations, with E.stigmaeum,E.gracile,H.scierus,and 11.nigrofasciatus,didnot.Fourmore tests failed using E. lepidum, E. spectabile, H. shumardi, and P. caprodes sperm. Failure of eight out of ten hybrid combinations is not as discordant as appears at first glance because maternal controls all failed under comparablecircumstances. Moreover, the first listed new unsuccessful combination has been subsequently reared as have hybrids with E. microperca. Etheostoma fonticola (Jordan and Gilbert). This species is found only in Central Texas. Stocks were obtained from the San Marcos River in San Marcosand ComalCreekinNewBraunfels. This species has been raised at temperatures between 20° C. and 24° C. The fertilization rate is exceedingly low with 24 fertilized eggs present in 57 control experiments, and only ten of these were reared. Hubbs (1959) re­ported success of hybrids with E. spectabile, H. scierus, and P. caprodesmales; and failures with E. lepidum, E. caeruleum, E. euzona, and E. juliae sperm. No additional tests have been run, and the low hybrid survival is not surprising considering the exceedingly low survival of the controls. Since this manuscript was finished hybrids with E. asprigene, E. radiosum, and E. microperca have been reared. Etheostoma microperca JordanandGilbertoccursinthe upperMississippiValley. The stocks used in less refined experiments came from Little SpringCreek, Mayes County, Oklahoma. Hybrids were successfully reared based on spermofE. caeruleumandE.spectabile. Previously it has been concluded that any darter hybrid combination can berearediftechniqueproblemsaresolved.A summarizationofthesuccessful combinations supports this hypothesis. Fourteen of the 25 (56 percent) con­trols have been successfully reared; 92 of 144 (69 percent) of the within-group (intrageneric hybrids or those between Hadropterus and Percina, two closely related genera) combinations have been successfully reared; and 53 of 85 (62 percent) between-group hybrids have been successfully reared. This crude summarization clearly shows that darter hybrids are no more dif­ficulttorear thanarethe controls. Some darters are much more easily reared than are others. It is very ap­parentthattherelative survivaloftheparentaltypesismuchmoresignificant to darterhybridsurvival thanis the degreeofphylogeneticrelationship. The within-group hybrid combinations in which both parents have been success­fully reared have been successful in 85 percent of the combinations, 59 per­cent of the combinations in which only one parent has been reared under comparable conditions have been successful, and only 20 percent of those in which neither parental type has been reared. Comparable figures for inter­group hybrids are 80, 53, and 0 percent. The differences in survival per­centageassociatedwithparentallaboratorysuccess arefargreaterthanthose associated with phylogenetic differentiation. There is a slight indication of a phylogenetic effect in that all intergroup combination survival figures are lower than comparable intragroup figures. None of the differences are of sta­tistical significance, but may indicate an effect of evolutionary differentiation on hybrid survival. Theabovecalculationsareonlyroughapproximations becauseacombina­tion tried a single time may have failed by chance alone. For instance, the single E. euzona control failed and less than 10 percent of the E. fonticola controls were successful. Obviously the latter is difficult to rear; and most of its hybrid combinations failed, yet the species has been arbitrarily categorized as successful. Etheostoma euzona might be expected to have a laboratorysurvival approximating that of its allopatric sibling, E. tetrazona, and would therefore be far more successful in the laboratory than E. fonticola. The rela­tivelylargenumber ofreared hybrid combinations withE. euzona supports this hypothesis. Eighteen of the combinations that failed were subsequently reared in less refined experiments that were primarily designed to obtain stocks for hybridfertility tests. The high frequency of success with previous failures was in part based on experience in avoiding errors and on extra effort to remove failures. Hybrid Fertility Hubbs (1958a) reported that female orangethroat-greenthroat darter hy­brids were fertile, and that the males were sterile. A number of putative hy­brids that have been obtained in the field were also tested for fertility; no males produced visible milt, and all eggs exposed to “stripped” hybrid males failed to develop. Hybrid females often produced eggs, and two from Junc­tionproduced eggsthataveraged 1.43mm.indiameter. A few laboratory E. grahami San Felipe x E. spectabile Lampasas have been reared to adult size. Eggs from these hybrids have been fertilized by sperm of E. spectabile, E. lepidum, P. caprodes, and H. scierus. The males seem to be sterile and produce no visible milt. Eggs of five hybrid females were tested with “stripped” hybrid males. None was fertilized. Only four of 18 sets of eggs tested against other species also failed, showing that the failures did not involve the eggs. Hybrids between the E. grahami San Felipe and E. lepidum West Frio River are fertile, and females can be back-crossed to their parental species.Similarly, E. lepidum Ft. McKavett and E. lepidum West Frio River hybrids canproduceF2’s(allextractedfromStrawn, 1961).ApparentlyE.lepidumand E. grahami are mutually interfertile and when tested for hybrid fertilitywithE. spectabileresultinfertilefemaleand sterilemaleFi’s. Lindner (1958) presented evidence that hybrid females of E. radiosum X E. spectabile (reciprocal not stated) produce apparently normal eggs and that a male did not father young when isolated with a ripe E. spectabilefemale. Apparently darterhybridfertilityismostlikely in females. Interpopulation-Intraspecific Hybridization The relative success of allopatric vs. sympatric hybrids may result from selection relative to the interaction of the two species, or it may result from differential adaptation to the specific environments. If the latter is exceed­ingly important it should affect the intraspecific hybrids which would then provide a type of control for the interspecific hybridization experiments. As shown below, intraspecific hybrids are often hererotic; however, inhibition ismore common thanininterspecifichybrids. Etheostoma spectabile. A large number of hybrid survival tests have been carried out, contrasting different populations of the orangethroat darter (Table1).Morethan 1,000eggswereusedinmostofthecomparisons,with theexceptionofLooseCreekXWhiteRiver,andWhiteRiverX GuadalupeRiver. In general the relative success of hybrid development is high with only two readings below 95 (both in total comparisons) and 21 over 105. Neitherofthelowreadings isrepeated inthe othermethod ofanalyzingthe data so that they may be considered to be of dubious significance, whereas ten ofthe highreadings wererepeated. Therefore,heterosis occursfrequentlyin interpopulations crosses. The results of the time lapse fertilization experiments are distinctly differ-ent.Halfofthefigures arelow,theotherhalfhigh.Theextremevariationin Table 1 Relative survival of interpopulation crosses of Etheostoma spectabile. A figure of one hundred (100) would be equal to that of control (intrapopulation) experi­ments. Crosses involving a single river system are based on parents from two localities within that system and a least five miles apart. White R. Illinois R. Brazos R. Colorado R. GuadalupeR. (Posthatch) Loose Creek White R. 100.7 134.5 135.1 115.1 235.5 Illinois R. 144.4 149.6 112.8 Brazos R. 124.6 125.6 116.6 Colorado R. Guadalupe R. 117.6 97.0 101.7 Loose Cr. White R. Illinois R. Brazos R. Colorado R. Guadalupe R. 95.4 (Total)108.7 115.8 122.1 118.3 101.8 118.7 109.7 109.3 153.9 120.2 107.6 94.4 92.9 Loose Cr. (Time Lapse Fertilization)27.8 203.4 White R. Brazos R. Colorado R. 137.0 58.9 190.9 35.8 92.8 173.1 relative success may involve the somewhat smaller samples available, but is alsoprobably dueinparttodifferentialselectivefactors. Therelative fertili­zation success is not likely to be enhanced by heterotic phenomena. More-over,timelapse fertilization is somewhatof apremating isolation mechanism and therefore likely to reflect incipient isolation. The patterns available do not provide data to show intraspecific subgroupings because they are incon­sistent. For instance, the White River and Loose Creek systems have similar fish; however, they have an extremely low fertilization rate. On the other handtheyboth seemtobehighlycompatiblewithIllinoisstocks. Iftheywere different, the results with Illinois fishes should also differ. The figures do indicate, however, that some of the low successes of the hybrids survival tests may be artifacts because one of the most successful fertilization test series is between the same populations that did not show heterosis (Guada­lupe and Colorado). Hubbs (1960) reported that Brazos River sperm was more vigorous than that ofany otherorangethroat darterpopulation.It islikelythatthis involves selective phenomena because no other species with which orangethroatdarters might exchange genes is present in the appropriate segment of the Brazos. All of the time lapse experiments confirm this conclusion. Tests at comparable time delays show that Brazos River controls do better than those of the other stream systems. A summary of the results between six and 20 seconds’ delay shows the fertilization rate of the Brazos controls to be one andonehalftimesthatoftheWhitecontrols(the secondbest). Etheostoma lepidum. All of the tests are based on large samples so that deviations from the expected index of 100 are statistically significant. The relative survival of the “interpopulation” hybrids is notably lower than that of the E. spectabile complex. Except for those based on the San Saba stocks (see below for an analysis of the San Saba results) only three figures are above 105, and five are below 95 (Table 2). The low figures involve threecombinations—differentpopulationswithinthe Guadalupe System,and interstream system hybrids involving the Nueces System. The reason for low survivalofhybridsfromwithinthe GuadalupeSystemisnotreadilyapparent.Those interstream system results involving the Nueces System population are thought to show incipient allopatric speciation. Time lapse fertilization studies indicate extensive speciation. Only the within-Colorado and within-Nueces comparisons even approach equalitywith the controls. Because large samples were used for all but the within-Nueces tests the reduced survivals are highly significant statistically. There­fore, the greenthroat darter seems to be in the process of dividing into at Table 2 Relative survival of interpopulation crosses of Etheostoma lepidum. Other circumstances as described in Table 1. Colorado R. Guadalupe R. Nueces R. Colorado R. (Posthatch)116.2 106.3 85.6 GuadalupeR. Nueces R. San Saba R. 151.2 81.3 77.8 99.6 119.6 Colorado R. (Total)106.1 102.4 96.0 GuadalupeR. Nueces R. 89.7 84.8 96.2 San Saba R. 115.6 106.4 Colorado R. (Time Lapse Fertilization)94.4 73.0 68.4 Guadalupe R. Nueces R. 60.0 67.2 135.7 least three taxa, one in the Nueces, one in the Colorado, and one or more in the Guadalupe. The Nueces River stock of E. lepidmn resembles the Brazos River stock E. spectahile in its inability to exchange genes with any sympatric species. The two stocks differ greatly in relative vitality of the sperm, the Nueces popula­tions having less vigorous sperm than comparable populations inhabitingother stream systems. The reduced vitality is in distinct contrast with the high sperm viability of Brazos River E. spectahile. Large eggs and reduced egg complements of Nueces system females (Hubbs and Delco, 1960) mayberesponsibleforreduced vitalityofsperminmalesfromthatstreamsystem. The survivaloffishesfrom the San SabaRiverisdistinctlylowerthanthat of comparable fishes from any other system. The summed survivals are 79 and 91 percent of the other Colorado system localities. Vitality is most re­duced during postlarval development, and survival of fertilized eggs throughthe larval stages is 40 to 50 percent of that of the other localities within the Colorado System. The low survival of the San Saba population may be due to genetic or environmental circumstances; more probably, environmental circumstances are involved because P. caprodes controls from the San Saba River alsoshowlow survival.Another set ofcircumstances seemstoindicate this. Extensive pecan groves are present at the headwaters and sprayed with insecticides in spring months. Two fish kills have occurred in the last decade. Survival of San Saba eggs is highest in the spring just before the recom­mended spraying interval and lowest in the fall after the spray interval. In­secticides are known to adversely affect bird reproduction (Bernard, 1963).These factors indicate but do not prove an external environmental effect causing the low survival of San Saba stocks. Whether the assumption is valid or not, the low survival necessitates treating the post-fertilization data sepa­rately. Theadverse effectseemstooccurinbothreciprocals ofhybridtests.That is, survival of intrastream system hybrids through larval stages using San Saba stocks is below that of the non-San Saba controls, regardless of which reciprocal is used. Similar within-Colorado system interpopulation hybrids are invariably heterotic. The prehatching figures are more or less equivalent,indicating that the mortality is posthatching. Despite reduced vitality of the San Saba hybrids, the survival data presented on Table 2 are above 100, be­cause comparisons were based on survival of both parental types. The ex­ceedingly low San Saba figures more than compensate for the somewhat reducedhybridsurvival.Despiteproblemsinevaluatingsurvival ofSan Saba hybrids, the relative survivals can be used. Hybrids with Nueces stocks in­variably have distinctly lower survival than do those with other Colorado stocks. The survival reductions approximate those of other (high survival)Colorado stocks crossed with Nueces River stocks. Etheostoma caeruleum. Relatively few tests were carried out on inter-population survival of rainbow darters. Survivals were either consistentlyhigh, witliin-White, or low, White River X Loose Creek (Table 3). The former are more likely to be valid because of the much larger number of experiments. Moreover, the single time lapse fertilization experiment, Loose CreekX WhiteRiver, wasmore successfulthanthe controls. Hadropterusscierus. Thenumberoftestsrun onrelative survival ofinter­population hybrids of blackside darters was rather limited. Three compari­sonswerebelow95,andsixabove 105,indicatinganoverallpatternofheter­osity (Table 4), The only apparent inhibition was within the Neches River System,andthe analyseswerebased ontoofeweggsfromtoomanydifferent creeks to base any definite conclusions on the results. The Guadalupe X Brazos experiments were contradictory by being low and high simultane­ously, depending on which stage was studied. The extensive Colorado River experiments were strongly heterotic. Table 3 Relative survival of interpapulation crosses of Etheostoma caeruleum. Other circumstances as described in Table 1. Loose Cr. White R. (Posthatch)White R. 91.4 164.5 (Total)White R. 91.6 154.5 (Time Lapse Fertilization)White R. 661.1 0.0 Table 4 Relative survival of interpopulation crosses of Hadropterus sciems. OthercircumstancesasdescribedinTable 1. Neches R. Brazos R. Colorado R. Guadalupe R. Neches R. 36.0 (Posthatch) 325.9 GuadalupeR. 114.4 105.3 98.3 Neches R. 84.5 (Total) 166.6 Guadalupe R. 80.0 108.4 122.1 Neches R. (Time Lapse Fertilization) 178.5 GuadalupeR. 296.1 34.9 Time lapse experiments were equally few but also indicate an overall heterosis. The low figure for Colorado X Guadalupe hybrids may be due to chance or may indicate that the morphologic distinction between the Guada­lupe and Colorado fish has an associated isolation mechanism. Percina caprodes. Intraspecific hybrid survival experiments on logperch were mostly more successful than the controls (Table 5). Only four tests were below 95, and 14 were above 105. Only one of the low figures was repeated on both analyses, and that (Brazos X Oklahoma) was based on 55 eggs. Two of the high figures that were repeated were based on 939 and 1152. It can therefore be concluded that interpopulation crosses of logperch tend to be heterotic. The survival rates for San Saba stocks are treated separatelyfrom the others in the Colorado system. The survival figures of San Saba eggs are approxi­mately one half those of the others. Most of the difficulty occurs after hatch­ing and apparently involves factors similar to those affecting E. lepidumsurvival. The time lapse fertilization tests again tend to be less successful than the controls. The Brazos X Oklahoma (high) and within-Guadalupe (low)samples are based on less than 500 eggs; those using Colorado stocks (all low) had many more than 1,000 eggs. Hubbs (1957) reported that Percina caprodes sperm was more vigorousthan E. spectabilesperm.The differenceinspermvitalityisveryapparentin Table 5 Relative survival of interpopulation crosses of Percina caprodes. Othercircumstances asdescribedinTable1. LooseCr. IllinoisR. Oklahoma BrazosR.ColoradoR.GuadalupeR. (Posthatch) Brazos R. 65.5 206.8 341.2 Colorado R. 188.1 197.6 Guadalupe R. 111.6 457.4 104.3 San Saba R. 434.5 0.0 214.0 (Total)Brazos R. 65.7 153.8 218.9 Colorado R. 124.6 106.2 Guadalupe R. 84.7 171.2 100.0 San Saba R. 109.6 103.2 101.7 (Time Lapse Fertilization)Brazos R. 133.7 0.0 Colorado R. 56.4 77.2 Guadalupe R. 61.0 the experiments reported here because the sperm vitality of Central Texas logperch is at least one and one half times that of any population of Etheo­stoma analyzed in this study. The difference is probably because the log-perch female produces ten to 20 eggs per spawning act, and the orangethroatproduces two to seven eggs per spawning act (Winn, 1958a). Moreover, the logperch female begins spawning on the stream bottom and the orangethroatfemale buries herself in the gravel before spawning (Winn, 1958b). Both of these factors would result in more precise control of an individual egg’sdeposition site, and consequently less need for extra sperm to fertilize the scattered eggs of orangethroat darters than of logperch. The need to fertilize the “wandering” eggs would necessitate a longer sperm vitality as it is found in logperch. The reproductive pattern of the greenthroat darter resembles that of the orangethroat except that eggs are carefully applied to aquaticvegetation (Strawn, 1955). Despite the “high vigor” of logperch sperm, the duration of its vitality is relatively short with less than 1 percent fertilization after a delay of25 seconds at 15° C. Overall Intraspecific Survival. The interspecific hybridization experiments were mostly more successful than the controls. Only ten of 40 posthatching tests had indices below 100, and the median figure was between 116.2 and 116.6. The total hybrid survival was somewhat similar with 12 of 40 tests beingbelow 100,andamedianfigurebetween106.4and107.6.Timelapsefertilization tests were very different. Fifteen of 24 had indices below 100,and the median figure was between 73.0 and 77.2. It is readily apparent that incipient hybridization in darters is much more easily recognized with a fertilization test than by hybrid survival. The patterns of intraspecific hybridization resemble those for interspecifichybridization listed below bybeingtypicallyheterotic insurvivalandhaving a reduced fertilization rate. The heterotic responses of intraspecific hybridshad no consistent pattern with regard to distance of the separation. That is,those hybrids from the localities within the same system usually were as likely to be heterotic as those between distinct systems. The exception involves the Nueces populations ofE. lepidum which seemtobeinthe process ofseparat­ing from those inhabiting the Guadalupe and Colorado Systems. Except for E.lepidumthe timelapsehybridizationdataalso donot showgeographicallyassociated variations in fertilization potential. Sometimes the within-systemhybrids are very much less successful than those between systems (Table 3),and at other times they may be relatively successful (Table 1). If this isola­tion mechanism were to be established in allopatry, the intensity of the mechanism should typically increase with the degree of allopatry. There were occasional instances in which the within-system hybrids were rather unsuccessful. These may have been by chance; however, this did not occurinColoradoSystem tests(seebelow). Intrageneric Etheostoma Hybrids Comparative data on the relative survival of interspecific hybrids can pro­videinformationabouttheselectiveprocesses.Differencesinrelative survival involving direct environmental factors should have been apparent in intra­specificexperiments;therefore,therepeatablepatternsprobablyarebased on genetic incompatibility. Etheostoma lepidum X Etheostoma spectahile. Both reciprocals of the experiments involving these two related species were done many times. The survival of the hybrids seems to demonstrate an overall heterosis. Only two comparisons based on E. spectahile eggs are below 95, and excluding tests based on parents from the San Saba River, 25 are above 105 (Table 6). The Table 6 Relative survival of Etheostoma spectabile $ X E. lepidum $ hybrids. A figure of one hundred(100)wotddbeequaltothatofthecontrolexperiments. Crosses within a stream system are divided into those with both parents from within five stream miles (superscript “s”) and those with parentsfrom localitiesseparatedby morethanfive stream miles (superscript “d”). $ | $ Colorado R. Guadalupe R. Nueces R. San Saba R. (Posthatch)Loose Cr. 140.3 137.3 White R. 179.9 147.2 Illinois R. 62.1 150.0 Rrazos R. 133.8 105.3 133.6 sdColorado R. 114.6 120.6 125.6 109.6 134.2 sdGuadalupe R. 125.8 119.8 112.1 139.1 145.2 (Total)Loose Cr. 107.5 104.5 White R. 134.1 122.5 IllinoisR. 59.0 102.5 Rrazos R. 120.1 104.4 119.4 Colorado R. 102.5 100.2 105.3 100.6 102.5 Guadalupe R. 112.5 105.6 104.0 115.7 123.9 (Time Lapse Fertilization)Loose Cr. 137.1 White R. 68.5 43.0 Rrazos R. 106.2 108.7 40.2 sdColorado R. 65.0 40.5 87.4 75.1 sd Guadalupe R. 67.2 88.5 67.2 150.2 two low tests are both Illinois X Colorado. The low survivals are difficult to explain, but they are not repeated on the reciprocal or on other tests involv­ing either parental population. The anomalous figures are caused by a hatch­ingratetwothirdsthatofthe controls. Nootherlowsurvival testshavealow hatching figure and some adverse circumstances may have affected the eggs, more than 99 percent of which were collected from one locality on one day.Tenofthereciprocalfiguresarebelow 95,and21areabove 105(Table7). The somewhat reduced effect of heterosis is undoubtedly based on biologicphenomena because the low survival examples are often based on largesample sizes collected at several intervals. Two contrasting survival patterns are apparent. That using Colorado River females is invariably heterotic ex­cept when two different stocks from the same system are the parents, A simi­larreductioncanbenotedinthe SanSabaRivertests.Comparingthe success within Central Texas, the lowest survival is in experiments involving crosses between the San Saba and other Colorado stocks, or in effect, the lowest survival occurs in the within Colorado tests. Therefore, the reduced hybridvitality occurs in experiments in which both controls had high survival rates and in those in which the controls’ survival differed widely. If some extrinsic factor caused the reduced vitality of the within-system hybrids it should also Table7 RelativesurvivalofEtheostomalepidum $ XE.spectabile$hybrids.Other circumstances asdescribedinTable 6. 2 | $ LooseCr. WhiteR. IllinoisR. BrazosR.ColoradoR.GuadalupeR. (Posthatch) Colorado R. 162.4 135.1 120.7 s137.7 d74.1 140.0 GuadalupeR. Nueces R. 75.5 105.9 65.2 172.2 84.7 128.3 124.4 96.7 92.4 108.0 132.2 San Saba R. 133.6 120.3 172.6 127.6 163.7 Colorado R. 110.6 (Total) 123.2 109.2 s117.9 d85.4 122.4 GuadalupeR. Nueces R. San Saba R. 54.1 87.6 116.4 96.6 109.0 112.9 89.4 111.8 125.1 111.1 84.2 106.7 102.9 111.9 111.4 117.1 (Time Lapse Fertilization) Colorado R. 61.8 154.0 61.4 s128.6 d105.8 371.0 Guadalupe R. Nueces R. 118.1 81.3 67.7 63.3 86.6 142.7 51.1 231.1 be apparent in the comparable intraspecific experiments which in contrast had high survival rates. In experiments using Guadalupe system eggs there is relatively low sur­vival, except if Colorado or other Guadalupe system males are used. These data are somewhat less reliable because the sample sizes range around 1,000,whereas the Colorado samples range about 4,000 eggs. Both the Colorado and Guadalupe comparisons show similarities between the distant and iden­tical locality experiments, and the nearby locality tests are very different. The series are somewhat contradictory in that with Colorado system eggs the nearby survival is low and with Guadalupe eggs the nearby survival is high. The survivals using Nueces River stocks tend to be high. Of the five com­binations using E. spectabile males, only one has both post-hatch and total survival figures below 100 (Nueces X Colorado). This is in striking contrast to the universally low survivals when greenthroats from different stream systems are tested. The time lapse fertilization figures tend to be much less heterotic with only 11 figures above 105, and 17 below 95, These figures are often suspectbecause of small sample sizes. The crosses using E. lepidum eggs have seven high and seven low figures and do not show a pattern. In contrast, the re­ciprocals have a repeatable pattern. The one series that was studied inten­sively was that using Colorado River E. lepidum sperm. In this series onlythe Brazos River sample has an index above 70. Because there is no sym­patric congener in the Brazos system, the eggs would not be expected to be isolated against foreign sperm. Otherwise the different Colorado sample is distinctly less successful than the others. The series based on Guadalupelepidum males is nearly identical except for the successful cross with Loose Creek eggs based on a single hybrid experiment. That is, the Brazos system eggsaremosteasilyfertilizedand thosefromanotherlocalitywithinthe same system the least easily fertilized. If one assumes that sympatric E. spectabile eggs reject foreign sperm, it should not be surprising that they also rejectallopatric E. lepidum sperm from the Nueces River. The sample sizes for the Nueces River experiments are very low, and individual variations in the fertilization success may be due to chance. Repeatability of the survival pattern of Colorado system hybrids in fertili­zation tests and the contradictions of the pattern of Guadalupe system hy­brids further indicates that the former reflects a selective phenomenon. Etheostoma caeruleum X Etheostoma lepidum. Relatively few tests were made on these exclusively allopatric species. Excluding San Saba tests, six of the survival comparisons had indices over 105, and five were below 95 (Tables 8 and 9)—figures not discordant with a heterotic pattern. Moreover, the only two adequate samples present (both reciprocals of the Colorado X White)wereabove 105twiceandneverbelow95.Thetestswithtimelapse Table 8 SurviavlofEtheostomalepidium $XE.caeruleum$hybrids.Other circumstances as described in Table 6. $ $ Loose Cr. White R. (Posthatch) Colorado R. 36.9 99.2 Guadalupe R. 102.1 San Saba R. 30.2 (Total) Colorado R. 66.8 112.8 Guadalupe R. 94.2 San Saba R. 69.0 (Time Lapse Fertilization) Colorado R. 14.7 Guadalupe R. 30.3 Table 9 Survival of Etheostoma caemleum $ X E. lepidum $ hybrids. Other circumstances as described in Table 6. $ 1 s Colorado R. GuadalupeR. Nueces R. San Saba R. Loose Cr. 102.3 (Posthatch)110.3 White R. 107.9 70.4 168.4 114.3 Loose Cr. 84.0 (Total)107.2 White R. 96.0 97.4 161.2 99.8 (Time Lapse Fertilization)WhiteR. 41.5 fertilizations are also based on small samples but all three are distinctly less successful than the controls. Etheostoma caeruleum X Etheostoma spectahile. The survival of hybridsbetween these closely related species occupying similar ecologic nitches is distinctly heterotic. Only four of the comparisons were below 95, and 29 wereabove 105.Survivalofhybrids(Tables 10and 11)whoseparentswere from two localities within the same system tends to be low. All of the indices below 100 are in this group, and none of the figures are as high as those for the same hybrid combinations taken from the same locality. Therefore, the nearbylocalities dofarworsethaneithertheidenticalorthedistantparentallocality hybrids. Table 10 Survival of Etheostoma caeruleum 2 X E, spectabile 3 hybrids.Other circumstances as described in Table 6. Colorado+ $ $ Loose Cr. White R. Illinois R. Brazos R. GuadalupeR. (Posthatch) sd Loose Cr. 119.8 103.1 176.4 109.0 sd White R. 128.1 159.6 96.9 141.1 125.0 145.0 (Total) sd Loose Cr. 100.9 89.4 146.8 107.9 sd White R. 110.2 122.5 78.5 115.2 112.1 98.9 (Time Lapse Fertilization)Loose Cr. 285.1 sd WhiteR. 144.9 435.8 347.4 70.4 91.4 The time lapse fertilization experiments are somewhat contradictory. The sperm of E. caeruleum seems to be inhibited by E. spectabile eggs, but the E. spectabile sperm seems to be invigorated by E. caeruleum eggs. The in­hibition is slightly greater than the apparent invigoration because the figures range between one half and one twelfth of the controls, a distinctly greaterdeviation than the up-to-fivefold increase, indicating an overall inhibition. There is no indication of a pattern of geographic variationexcept that the allopatric populations often have the least fertilization potential. Miscellaneous Intrageneric Crosses. A variety of experiments have been carried out using two species of Etheostoma as parents. Most of the speciescombinations are based on too few populations to be useful for racial com­parisons of success. The only one showing a pattern that may be meaningful is E. punctulatum X E. spectabile (Table 12). Texas E. spectabile seem to produce heterotic hybrids with E. punctulatum females; when Arkansas and Missouri orangethroat males are used there seems to be a reduced overall vitality. The overall success of intrageneric Etheostoma hybrids is better than the controls.ExcludingtheSanSabatests,only21ofthe 118posthatchingcom­parisonsarebelow100,andthemedianfigureisbetween 134.4and135.1;and 30 of the 118 total survival indices are below 100, and the median figure is 112.2. The time lapse fertilization experiments are distinctly different with 42of67fallingbelow 100,andthemedianfigureis67.2. Other circumstances as described in Table 6. 9 | $ Loose Cr. White R. (Posthatch) Table 11 Survival of Etheostoma spectabile $ X E. caeruleum $ hybrids. Loose Cr. s198.1 d85.7 White R. 134.0 s178.8 d94.6 Illinois R. 170.0 Brazos R. 139.2 Colorado R. 132.4 GuadalupeR. 130.3 Loose Cr. (Total) s d154.6 132.4 WhiteR. 118.4 s117.1 d104.0 Illinois R. 122.8 Brazos R. 117.3 Colorado R. 116.0 Guadalupe R. 115.3 Loose Cr. (Time Lapse Fertilization) 40.5 White R. s34.0 d53.5 Illinois R. 24.4 Mountain Cr. 50.9 Brazos R. 8.5 Colorado R. 15.2 Interspecific Hybrids of Primitive Darters Only three species of primitive darters were studied extensively, Percina caprodes, Hadropterus scierus, and 11. shumardi. Bailey, et al. (1954) have suggested that the species comprising the two genera are closely related and haveplacedall oftheminPercina.Regardless ofwhetherthe speciesshould be congeneric or not, there is no question that they are much more closelyrelated among themselves than they are to any species of Etheostoma. Allofthe speciesstudiedaredifficulttorearinthelaboratory.Theyhave narrow ranges of thermal tolerance, tend to be bruised by swimming into the edges of their rearing containers, and seem to need large quantities of small food items. Consequently, survival rates vary widely, and the validity of comparisons maysufferfromextrinsicfactors. Hadropterus scierus X Percina caprodes. The survival of Fi hybrids is Table 12 Survival of various intrageneric Etheostoma hybrids. Othercircumstances asdescribedinTable 6. Posthatch Total Time Lapse E. punctulatum 9 X E. spectabile $ Illinois R. X Loose Cr. Illinois R. X Same Illinois R. Illinois R. X Different Illinois R. Illinois R. X Colorado R. IllinoisR. X Guadalupe R. 68.1 70.9 192.2 146.0 oo 87.0 81.8 83.3 115.4 125.2 E. spectabile 9 X E. punctulatum $ Loose Cr. X Illinois R. WhiteR. X Illinois R. IllinoisR. X Same Illinois R. Illinois R. X Different Illinois R. Brazos R. X Illinois R. Colorado R. X Illinois R. Guadalupe R. X Illinois R. 218.8 215.0 239.4 160.0 206.2 194.8 192.7 120.7 154.3 129.2 124.2 127.0 132.4 165.2 46.0 139.6 40.5 283.9 41.5 E. punctulatum 9 X E. lepidum $ Illinois R. X Nueces R. 158.7 117.1 E. lepidum 9 X E. punctulatum $ ColoradoR. X Illinois R. GuadalupeR. X Illinois R. Nueces R. X Illinois R. 183.6 134.4 136.8 129.2 128.4 111.0 E. tetrazona 9 X E. punctulatum $ Loose Cr. X Illinois R. 331.8 160.3 184.9 E. punctulatum 9 X E. tetrazona $ Illinois R. X Loose Cr. 235.6 67.2 E. tetrazona 9 X E. caerulum $ Loose Cr. X White R. 199.1 97.9 56.6 E. caerulum 9 X E. tetrazona S Loose Cr. X Same Loose Cr. White R. X Loose Cr. 379.8 485.1 148.8 164.2 E. tetrazona 9 X E. spectabile S Loose Cr. X Different Loose Cr. Loose Cr. X Same Loose Cr. Loose Cr. X Illinois R. 306.2 398.6 215.8 118.8 129.2 82.4 Table 12 (Continued) Posthatch Total Time Lapse E. spectabile 9 X E. tetrazona $ Loose Cr. X SameLoose Cr. Illinois R. X Loose Cr. 247.8 498.6 112.2 161.3 E. tetrazona 9 X E. lepidum S Loose Cr. X Colorado R. 187.1 79.6 E. lepidwn 9 X E. tetrazona $ Colorado R. X Loose Cr. 252.8 112.2 E. tetrazona 9 X E. juliae $ Loose Cr. X White R. 12.0 59.8 E. juliae 9 X E. tetrazona $ White R. X Loose Cr. 367.2 136.5 E. juliae 9 X E. caeruleuin S White R. X Same White R. 105.0 87.2 E. caeruleum 9 X E. juliae S White R. X Same White R. White R. X Different White R. 306.8 319.9 146.8 123.7 E. juliae 9 X E. spectabile $ White R. X Loose Cr. White R. X Illinois R. 77.8 228.4 97.7 153.8 E. spectabile 9 X E. juliae $ Loose Cr. X White R. 500.1 145.2 E. juliae 9 X E. lepidum $ White R. X Colorado R. White R. X GuadalupeR. E. lepidum 9 X E. juliae $ GuadalupeR. X White R. E. juliae 9 X E. punctulatum $ White R. X Illinois R. 340.1 338.2 398.2 453.0 118.8 125.0 125.8 135.5 E. blennioides 9 X E. spectabile $ Loose Cr. X Different Loose Cr. Loose Cr. X Illinois R. Loose Cr. X Brazos R. 99.1 90.1 90.5 118.2 105.8 92.4 0 0 0 E. spectabile 9 X E. blennioides $ Loose Cr. X Same Loose Cr. 164.1 189.2 E. blennioides 9 X E. punctulatum $ Loose Cr. X Illinois R. White R. X Illinois R. Illinois R. X Same Illinois R. Illinois R. X Different Illinois R. 117.6 79.0 23.9 28.2 32.2 0.0 E. blennioides 9 X E. caeruleum $ Loose Cr. X White R. 107.9 80.7 174.9 E. gracile 9 X E. lepidum $ Neches R. X Colorado R. 11.1 E. chlorosomum 9 X E. lepidum $ Neches R. X Colorado R. 35.8 E. chlorosomum 9 X E. spectabile $ Neches R. X Colorado R. 28.0 E. caeruleum 9 X E. punctulatum S Loose Cr. X Illinois R. WhiteR. X Illinois R. 121.2 177.3 123.0 127.7 122.3 102.7 E. zonale 9 X E. spectabile $ Loose Cr. X Illinois R. White R. X Illinois R. White R. X Same White R. 70.5 149.3 360.0 definitelybetterthanthatofthecontrols(Tables 13and14).Onlyfivefigures arebelow95,and22areabove 105.ApatternseemsapparentintheColorado experiments using P. caprodes sperm. The indices based on samples from two differentlocalities within the system are distinctly (and significantly) lower than the others. Similar to the survival results based on E. lepidum and E. spectabile hybrids, this pattern is not apparent in Guadalupe River stocks. Unfortunately, the reciprocal experiments do not include any tests between two localities within the same system; there is, however, a distinctly lower survival of sympatric hybrids than allopatric hybrids. Thetimelapse experimentsaremuchlowerthanthoseofthe controlswith 13below95,andtwoabove 105.Itisobviousthatgameticinhibitionissig­nificant in reducing natural hybridization between these two species. The patterns are less obvious, however; the lowest figures for logperch sperm Table 13 Survival of Hadropterus scierus $ X Percina caprodes $ hybrids. Other circumstances as described in Table 6. 9 | $ Illinois R. Brazos R. Colorado R. Guadalupe R. (Posthatch)Neches R. 167.8 314.2 Brazos R. 124.0 sd Colorado R. 243.2 149.5 0.0 42.8 sd Guadalupe R. oo 172.7 167.4 385.4 (Total)Neches R. 103.8 132.1 Brazos R. 94.0 sdColorado R. 147.2 113.2 64.4 66.6 sd Guadalupe R. 391.4 120.9 117.1 136.6 (Time Lapse Fertilization)Neches R. 80.8 Brazos R. 29.1 sd Colorado R. 43.8 68.3 39.8 16.9 sd GuadalupeR. 124.5 47.9 58.4 10.1 5.1 Table 14 Survival of Percina caprodes 2 X Hadropterus scierus $ hybrids. Other circumstances as described in Table 6. $ 1 $ Brazos R. Colorado R. Guadalupe R. (Posthatch) Colorado R. oo 106.6 243.9 (Total)Colorado R. 141.6 97.6 130.9 (Time Lapse Fertilization)Rrazos R. 9.2 Colorado R. 19.0 127.2 GuadalupeR. 0.0 survival for any stream system was for those tested against female H. scierus from adifferentspotinthe same stream system.Hybridization Experiments Involving H. shumardi. The stocks used for H. shumardi experimentscamefromasinglelocality sothenumberofracial comparisons is limited. The H. shumardi X H. scierus survival experimentsare contradictory, showing low survival with H. shumardi eggs and highsurvival with H. shumardi sperm (Table 15). Perhaps as a consequence no pattern can be noted. The H. shumardi X P. caprodes experiments are based on many more ex­perimentsandconformbettertothegeneralpatternofhigh survivalofinter­specific hybrids. Two of the comparisons are low in both indices. The low survivalofGuadalupeH.shumardiXSanSaba P.caprodesmayinvolvethe adverse environmental circumstances associated with San Saba stocks as well as the small sample available. Low survival in P. caprodes X H. shumardi tests from the same site in the Guadalupe System seems tobe real because of the large sample size. These hybrids are distinctly less viable than compar­ablehybrids inwhich thematernalparent isfromadifferentlocality. Results of time lapse experiments based on males of either species of Hadropterus were high, but the number of tests was minimal because Had­ropterus males seldom yield adequate quantities of sperm more than thirty-six hours after capture. The time lapse tests with P. caprodes sperm are probably more meaningful, and seem to parallel the hybrid survival tests Table 15 SurvivalofhybridsusingH. shumardiasaparentalspecies.Other circumstances as described in Table 6. Posthatch Total Time Lapse H. scierus X H. shumardi Same GuadalupeDifferent Guadalupe 505.2 378.0 178.5 176.0 194.3 H. shumardi X H. scierus GuadalupeX Neches Same GuadalupeDifferent Guadalupe 11.0 75.2 57.6 62.8 76.4 56.0 259.3 H. shumardi X P. caprodes GuadalupeX Colorado Guadalupe X San Saba Same GuadalupeDifferent Guadalupe 240.7 69.6 134.1 204.0 111.0 70.4 105.4 93.6 181.8 28.3 9.3 P. caprodes X H. shumardi Colorado X GuadalupeSame GuadalupeDifferent Guadalupe 351.6 25.4 200.4 128.6 94.6 142.3 with the best results occurring when allopatric parents are used. The absence of H. shumardi in all collections from the Colorado System makes the allo­patric aspect of Colorado P. caprodes X Guadalupe H. shumardi more sig­nificant. The overall successofhybrids amongprimitive darters isbetterthanthat of the controls. Only seven of 21 posthatching comparisons are below 100, and the median figures are between 167.8 and 172.7. Ten of 26 total survival indicesarebelow 100,andthemedianfigureisbetween 111.0and113.2.The timelapseexperimentsaremuchlesssuccessfulwith 15of20below 100,and the median figure between 29.8 and 32.8. Compared to the Etheostoma hy­brids the effect of heterosis on Fi survival seems to be extreme and fertiliza­tion tests show considerable inhibition. The total survival figure is intermedi­ate, perhaps because it incorporates both Fi survival and fertilization per­ centages. The apparent increase in hybrid vitality may be because the rela­tively low control survival rates permit greater improvement. Many Etheo­stoma controls had survivals over 70 percent so that survival of all hybridswould only result in an index of 143, a figure less than the median for primi­tive darters. The exceedingly low fertilization figures may well be based on biologiccriteria. The primitive darters have less control over individual egg release (Hubbs, 1955; Winn, 1958a; Hubbs and Strawn, 1957a). Therefore, the eggsmight not be fertilized by sperm from the mated male and be available for fertilization by “stray” sperm. Such eggs would be expected to have a mech­anism to reduce fertilization potential of foreign sperm. Hybrids Between Primitive and Advanced Darters Many of the experiments were carried out with hybrids between the ad­vanced (Etheostoma)andprimitive(HadropterusandPercina)darters. Most species of Etheostoma studied (and all of these studied extensively) are relatively easily reared, and most species of primitive darters studied are difficult to rear. Therefore, the hybrid survivals are contrasted separatelywith the survival rates of each parent. Percina caprodes X Etheostoma spectabile. The posthatching survival of the orangethroat female X logperch male hybrids is usually much greaterthan that of the controls (Table 16). It is not surprising that the hybridsalways do better than the logperch controls; however, only two comparisonswith orangethroats are below 95, one based on San Saba parents, and 19 are above 105. This is a very convincing example of heterosis. Both of the low comparisons with paternal orangethroats must be balanced against the com­parisons with the maternal logperch survival in which the hybrid survival is more than eight and sixteen times as successful as the comparable logperch Table 16 Relative survival of Etheostoma spectabile 9 X Percina caprodes $ hybrids. A figure of 100outsidetheparentheses would meansurvivalequaltothatofthematernalspecies,and inside the parentheses would mean survival equal to that of the paternalspecies. Crosses within a stream system are divided into those with both parentsfromwithinfivestreammiles(superscript “s”),and thosewithboth parentsfrom localitiesseparatedby morethanfivestreammiles(superscript“d”). Loose White Illinois Brazos Colorado GuadalupeSan Saba $|9 Cr.R. R.R. R. R. R. (Posthatch)Loose Cr. 128.0 199.3 35.1 (3622)(1139) (854)White R. 209.8 234.8 (8033) (1021) sdIllinoisR. 142.8 101.8 121.8 127.8 (307) (1060) (952) (2529)Brazos R. 112.7 125.3 122.8 (589) (537) (4708) sdColorado R. 122.9 141.8 107.8 116.4 133.6 69.9 No control (1001) (414) (663) (440) (1687) sd GuadalupeR. 127.6 143.2 136.0 129.4 (555) (594) (561) (548) (Total)Loose Cr. 94.6 120.0 70.3 (167.6) (267.5) (154.1)White R. 126.9 92.9 (275.6) (210.2) Illinois R. 116.0 102.3 102.0 113.4 95.2 (161.7) (115.8)(304.8)(288.3) (127.1)Brazos R. 112.2 122.8 113.8 102.3 (231.1) (177.4) (177.4) (188.6) Colorado R. 116.9 120.0 97.6 82.4 109.4 90.3 No control (254.4)(137.1)(105.6)(168.4) (190.0) GuadalupeR. 125.0 136.3 123.5 109.1 (241.1) (175.7)(168.1)(151.8) (Time Lapse Fertilization) WhiteR. 70.3 sd Illinois R. 33.7 84.1 297.3 Brazos R. 159.8 156.2 140.0 Colorado R. 29.5 225.0 103.4 3.9 64.8 cl GuadalupeR. 113.9 180.6 49.6 survival. Moreover, the Loose Creek X Guadalupe River comparison is based on only three experiments and may not represent the actual hybrid survival. The Colorado X San Saba comparison may reflect reduced survival of hy­brids in which the parents are from two localities within the same stream system. The comparisons also may have been affected by environmental factors associated with the San Saba locality; however, the contrast with logperch is much lower than that of other hybrids whose logperch fathers camefromthe San Saba.Posthatchingcomparisons oftheotherinterpopula­tion hybrids whose parents both came from within the Colorado System have a relatively high index. In contrast, overall survival figures show a low survival index for the hybrid crosses involving two Colorado System locali­ties. The comparison with logperch is by far the lowest figure, and that with orangethroats is second to the statistically questionable Loose Creek X Guadalupe River survival. There is also some indication of reduced vitalityofGuadalupe Systemhybrids withparentsfromtwolocalitiesinthesystem. Theoverall survivalis somewhatlessthan theposthatchingsurvival,butstill compares favorably with that of the controls. All comparisons with logperch exceed 105; 14 of the comparisons with orangethroats are above 105 and five below 95. The posthatching survivals of reciprocal experiments are also typically better than those of the controls (Table 17). All 17 comparisons with log-perch are above 105 as are five of the contrasts with orangethroats. Seven of the orangethroat comparisons are below 95. The overall 23 to eight survival comparison is in accord with a heterotic pattern. On the other hand, the figures are considerably lower than those of the reciprocal. Whereas all 21 hybrid combinations with orangethroat mothers have more than four times the logperch posthatching survival rate, only six of 17 reciprocals do. Like­wise, survival values of 18 of 22 comparisons of hybrids with orangethroatmothersand onlyfiveof 18reciprocalsexceedvaluesof 105.Therefore,there is no question that a difference exists between the posthatching survival potential of the two reciprocals and that this difference parallels survival potential of the maternalparent. The pattern of hybrid survival resembles that of Etheostoma lepidumfemales and E. spectahile males. The Colorado River samples from Central Texas are heterotic except for those involving two different Colorado Systemlocalities. The low figure for Colorado X Illinois hybrids is very suspect be­cause all of the fertilized eggs in experiments were run at 29° C., a tempera­ture at which survivals are typically low. As in the intrageneric comparisons,the results of Guadalupe River experiments diverge with the best results obtainedincrossesfromnearbylocalitiesandwiththe worstresults obtained in experiments involving sympatric or distinctly allopatric parents. The simi­larity in the comparison of Colorado system hybrids is striking in that both Table 17 Relative survivalof Percina caprodes $ X Etheostoma spectabile $ hybrids. Other circumstances as described in Table 16. Loose White Illinois Brazos Colorado Guadalupe5|$ Cr.R.R. R. R. R. (Posthatch)Loose Cr. 333 (160.6)White R. 261 (86.8)Illinois R. 548 (68.0)Brazos R. 1347 1472 (141.4) (143.2) sd Colorado R. 1547 467 477 262 545 (67.6) (105.9) (104.6) (69.9) (119.4) sd Guadalupe R. 257 156 317 295 318 250 317 (95.5) (67.6) (95.7) (104.1) (101.6) (88.9) (89.2) San Saba R. 1299 1560 (60.4) (92.0) (Total)Loose Cr. 153.8 (120.3)White R. 121.4 (93.3) Illinois R. 174.7 (84.2) 344.5 Brazos R. 286.3 (108.0) (125.4) sd Colorado R. 59.7 163.1 150.4 105.3 141.3 (48.2) (99.7) (104.0) (71.1) (114.1) sd Guadalupe R. 135.9 59.6 124.4 141.8 128.2 126.6 140.3 (101.8) (43.3) (92.2) (103.6) (92.0) (88.1) (92.1) San Saba R. 165.4 242.8 (70.4) (104.0) (Time Lapse Fertilization)Loose Cr. CO 0 Illinois R. 359.9 sd Colorado R. 44.1 55.0 63.0 0 Guadalupe R. 0 28.5 are based on male orangethroats and the pattern is much less apparent when female orangethroats were used. The time lapse fertilization experiments are not heterotic, with nine com­parisonsabove 105,and 14below95.Thetworeciprocalsarequitedifferent. The tests with P. caprodes sperm provide results approximately the same as the controls, especially the Texas tests in which the sample sizes are ade­quate. There is no question that one of the Texas comparisons is exceedinglylow (different Colorado) and that it is based on a large sample. The within- Guadalupe and Illinois samples are contradictory and may not representdifferential inhibition. Comparisons using E. spectabile sperm clearly show an overall inhibition. The two high figures are based on exceedingly small samples at time lapses in which the controls were unusually unsuccessful,resulting in an artificially high index. The adequate Texas samples are all exceedingly low,indicating that P. caprodes eggshavelarge quantities ofthe factor that inhibits heterospecific sperm. Percina caprodes X Etheostoma lepidum. The survival of logperch X greenthroat darter hybrids is distinctly lower than comparable logperch X orangethroathybrids(Tables 18and19).Theposthatchingcomparisonswith greenthroatsresultintenbeingabove 105,and24below95.Thecomparisonswith logperch survivals are much higher, with all above 105. Total survival figures are comparable with the posthatching indices; eight indices above 105, and 25 below 95 when contrasted with E. lepidum, and 29 above 105, and two below 95 when contrasted with F. caprodes controls. The two re­ciprocals differed by having a greater survival rate when greenthroats were used as maternal parents than when logperch females were used. This is essentially the same as occurred with logperch X orangethroat hybrids. The comparable low survival indices of this hybrid combination as con­trasted with similar indices for orangethroat X logperch hybrids is in large-part based on a low survival of sympatric crosses. The comparisons based on survival of hybrids whose logperch parent is from outside the greenthroat range indicates a reasonable degree of heterosis. Ten of the posthatchingcomparisons with greenthroats exceeded 105, seven of which involved allo­patric logperch and inone other allopatric greenthroats were used. The other two are of dubious significance because the greenthroats used for comparison were from the San Saba River, a locality at which the control survivals were unusually (and probably artificially) low, perhaps causing a high index. The patterncanperhapsbestbeshownbycomparingtheindexrankings (exclud­ing those involving the San Saba System). The highest relative success was clearly in those crosses in which both parents were from allopatric popula­tions (Table 20). There was no significant difference among the various com­parisons within the sympatric range. Thetimelapsefertilizationsrepeatthepatterns ofcomparabletestsinvolv­ Table 18 Relative survival of Etheostoma lepidum $ X Percina caprodes S hybrids. Other circumstances as in Table 16 9 | $ WhiteR BrazosR ColoradoR. GuadalupeR. SanSabaR. (Posthatch) Colorado R. 112.9 90.2 88.4 90.8 63.4 (628) (492) (382) (505) (1882) sd Guadalupe R. 11.6 77.1 78.5 85.9 61.8 51.4 (6240) (808) (356) (383) (321) (1350)Nueces R. 158.6 139.6 87.8 88.4 61.3 (209) (435) (389) (379) (1204) San Saba R. 147.0 135.4 87.3 90.5 (1440) (388) (243) (1589) (Total) sd Colorado R. 102.3 95.0 90.8 90.3 86.1 (241.4) (152.3) (151.7) (161.2) (151.5) sd GuadalupeR. 52.3 85.3 93.1 89.0 81.4 81.8 (100.6) (266.0) (144.6) (151.3) (137.3) (212.2)Nueces R. 122.0 115.7 92.5 91.8 75.7 (122.3) (216.7) (138.4) (142.2) (152.8)San Saba R. 120.0 105.0 73.8 98.1 (347.9) (142.5) (110.8) (179.9) (Time Lapse Fertilization) j Colorado R. 91.8 156.1 134.9 162.7 Guadalupe R. 188.5 110.5 122.6 Nueces R. 121.6 119.6 129.3 ing orangethroat X logperch hybrids. That is, logperch sperm is not inhibited by greenthroat eggs but the reciprocal experiments show a high degree of inhibition. Miscellaneous Intergroup Hybrids. A large number of inter-group hybridshave been reared that do not provide reliable patterns of sympatric versus allopatric survival rates (Table 21). For example, the H. scierus Brazos X E. lepidum Colorado hybrids are relatively heterotic (both reciprocals); how­ever, both reciprocals of the equally allopotric H. scierus Colorado X E. lepidum Nueces hybrids, have low vitality and the comparable within-Colorado hybrids tend to be intermediate. Because of the small sample sizes Table 19 RelativesurvivalofPercinacaprodes $XEtheostomalepidumShybrids. Other circumstances as in Table 16. 5 | $ Colorado R. GuadalupeR. Nueces R. San Saba R. Loose Cr. (Posthatch)345 White R. (322.2) 135 Brazos R. 366 1440 (826.0) Colorado R. (100.4) s d260 318 291 (139.6) 444 283 GuadalupeR. San Saba R. (49.8) (70.7) 252 (65.1)1667 (54.1) s d276 377 (84.2) (66.8) (141.3) 271 (90.3) (122.5) 1495 (49.2) (71.9) Loose Cr. (Total)175.3 White R. (205.1) 91.3 Brazos R. 261.2 320.2 (137.8) Colorado R. (107.0) s d96.0 120.2 94.9 (112.5) 130.3 101.6 Guadalupe R. San Saba R. (64.4) (74.4) 114.2 (66.5)159.2 (59.9) s d132.0 99.7 (80.3) (77.0) (91.2) 116.4 (85.2) (73.3) 200.3 (60.4) (75.4) Brazos R. 45.1 (Time Lapse Fertilization) Colorado R. s37.2 d21.1 5.3 Guadalupe R. 25.5 s19.9 d42.4 14.0 available, those variations make no sense and may be influenced by extrinsic factors. Several items are available that substantiate the circumstances present in other intergroup hybrids. The two reciprocals differ in survival potential in Table 20 Average rankings of survival indices of greenthroat X logperch hybrids P. caprodes eggs E. lepidum eggs Total Both parents allopatric 1.8 5.0 6.8 P. caprodes allopatric 2.9 6.3 9.2 E. lepidum allopatric 4.8 7.9 12.7 Different sympatric systems 9.5 6.9 16.4 Different sympatric localities 6.8 9.5 16.3 Same localities 8.1 6.6 14.7 Table 21 Survivalofvariousintergroup darterhybrids. Other circumstances as in Table 16. Posthatch Total Time Lapse Fertilization (H. scierus 2 X E. lepidum $ ) Neches R. X Guadalupe R. 0.0 Neches R. X Nueces R. 0.0 Brazos R. X Colorado R. 165.4 128.0 12.8 (112.4) (102.8)Colorado R. X Same 196.9 143.3 31.2 Colorado R. (58.0) (76.4)Colorado R. X San Saba R. 617.9 300.8 57.2 (115.0) (107.1)Colorado R. X Nueces R. 76.6 62.0 10.7 (70.6) (57.9)Guadalupe R. X Colorado R. 425.9 177.0 4.9 (87.2) (92.1)Guadalupe R. X Different 272.4 101.8 GuadalupeR. (25.6) (44.6)Guadalupe R. X Nueces R. 678.9 152.5 (67.6) (68.1) (E. lepidum 2 X H. scierus $ ) Colorado R. X Brazos R. 134.2 120.6 692.4 ( oo ) (302.4)Colorado R. X Same 79.2 89.5 94.9 Colorado R. (245.4) (159.9)Colorado R. X Guadalupe R. 101.4 103.6 (303.9) (158.2)Guadalupe R. X Neches R. 0 3.6 970.6 (0) (7.4)Guadalupe R. X Brazos R. 96.9 106.7 (181.7) (143.5) Table21 (Continued) Posthatch Total Time Lapse Fertilization GuadalupeR.XColoradoR. 126.3 117.9 2428.6 (378.9) (240.8)GuadalupeR. X Different 145.4 117.6 GuadalupeR. (400.6) (176.1)Nueces R. X Neelies R. 54.2 70.0 481.2 (284.1) (128.6)Nueces R. X Colorado R. 86.8 88.7 22.3 (278.6) (137.0)Nueces R. X GuadalupeR. 116.0 95.2 (294.1) (136.0) San Saba R. X Brazos R. 137.2 120.6 (121.1) (117.9)San Saba R. X Colorado R. 121.3 106.8 (149.9) (133.7)San Saba R. X GuadalupeR. 160.3 118.4 (376.1) (166.6) (H. scierus $ X E. caeruleum $ ) Colorado R. X WhiteR. 87.3 87.3 (52.8) (34.0)GuadalupeR. X White R. 208.8 61.0 (58.2) (50.9) (H. scierus 9 X E. spectahile $ ) Neches R. X Colorado R. 187.0 102.8 18.7 (78.4) (69.6)Colorado R. X White R. 109.5 96.2 2.7 (67.4) (65.0)Colorado R. X Brazos R. 197.6 126.1 53.4 (90.0) (83.5)Colorado R. X Same 313.6 170.1 81.8 Colorado R. (113.3) (102.4)Colorado R. X Different 661.1 219.9 31.7 Colorado R. (69.0) (109.5)GuadalupeR. X White R. 242.2 121.7 53.7 (75.4) (68.4)GuadalupeR.XIllinoisR. 51.0 94.1 8.3 (9.5) (54.8)GuadalupeR. X Brazos R. 347.9 167.2 22.9 (72.8) (89.2)GuadalupeR. X Colorado R. 557.1 213.1 47.5 (131.8) (110.0) GuadalupeR. X Same 473.2 167.2 24.5 Guadalupe R. (149.2) (117.0) Guadalupe R. X Different 311.9 153.8 Guadalupe R. (87.8) (92.9) ( E. spectabile 2 X H. scierus S ) White R. X Colorado R. 125.0 107.3 (92.1) (103.2)Brazos R. X Colorado R. 98.9 106.3 264.1 (269.2) (176.0)Brazos R. X GuadalupeR. 103.2 103.9 240.0 (343.2) (159.8)Colorado R. X Neches R. 162.0 134.8 151.1 (307.4) (165.3)Colorado R. X Same 125.4 104.1 68.9 Colorado R. (271.4) (151.3)Colorado R. X Guadalupe R. 85.6 82.6 25.6 (304.0) (146.9)Guadalupe R. X Colorado R. 265.6 189.0 (302.1) (176.9) GuadalupeR. X Same 130.4 139.4 236.2 Guadalupe R. (446.9) (190.9) ( H. shumardi 2 X E. spectabile $ ) GuadalupeR. X Brazos R. 338.0 156.8 (67.0) (79.1) Guadalupe R. X Different 449.2 187.4 44.6 Guadalupe R. (118.2) (111.7) (E. .spectabile 2 X H. shumardi $ ) White R. X GuadalupeR. 0 Brazos R. X Guadalupe R. 160.8 145.4 (454.7) (258.0)Colorado R. X Guadalupe R. 129.0 119.8 0 (744.3) (249.6) Guadalupe R. X Different 108.2 106.2 0 GuadalupeR. (827.8) (229.3) (H. shumardi 2 X E. lepidum $ ) Guadalupe R. X Colorado R. 281.9 164.1 (48.8) (67.4) GuadalupeR. X Different 70.2 108.0 GuadalupeR. (9.8) (44.1) (£. lepidum 2 X H. shumardi $ ) Colorado R. X Guadalupe R. 95.4 96.0 (376) (185.0) Nueces R. X Guadalupe R. 133.7 118.7 (1135) (268.9) Table21 (Continued) Posthatch Total Time Lapse Fertilization (E. caeruleum $ X Percina caprodes S ) 00 Loose Cr. X Loose Cr. 133.4 116.9 (818) (211.4)Loose Cr. X White R. 133.3 125.2 (195) (122.3)Loose Cr. X Illinois R. 132.9 125.8 No Control No Control Loose Cr. X GuadalupeR. 115.0 108.8 113.6 (343) (188.6)WhiteR. X Loose Cr. 133.0 108.8 100.0 (2689) (212.1)White R. X White R. 387.1 155.7 (39043) (234.5)White R. X Illinois R. 130.4 117.9 77.9 No Control No Control White R. X Colorado R. + 148.2 117.2 110.4 GuadalupeR. (433) (162.4) White R. X San Saba R. 124.8 101.9 (1864) (239.2) {Perdmcaprodes $ X Etheostoma caeruleum $ ) White R. X White R. 190.0 137.2 29.4 (115.0) (124.8)GuadalupeR. X White R. 200.5 91.3 40.2 (49.8) (60.4) (E. tetrazona $ X Percina caprodes $) Loose Cr. X Loose Cr. 55.3 117.0 (232.1) (167.8)Loose Cr. X White R. 175.8 174.8 (316.4) (162.5) {Percim caprodes, $ X Etheostoma punctulatum $ ) Colorado R. X Illinois R. 345.0 150.3 15.3 (180.6) (139.3) GuadalupeR. X Illinois R. 303.4 106.9 63.8 (180.0) (91.0) that the hybrids based on Etheostoma eggs are more viable than those based on Percina or Hadropterus eggs. Similarly, both reciprocals of hybrids with E. spectabile tend to be more successful than those based on E. lepidum.Finally, Percina and Hadropterus eggs tend to inhibit Etheostoma sperm,whereas the reverse is less apparent. Overall Intergroup Hybridization. The relative survival of intergroup hybrids follows the same pattern regardless of species involved (Table 22).The reciprocal based on Etheostoma eggs does better than that based on primitive darter eggs. The median survival figure is invariably higher, when either E. lepidum or E. spectabile are crossed with primitive darters. Com­parisons using only P. caprodes eggs follow the same pattern but include a slightly higher figure in one comparison with E. lepidum. Using all primitive darters, three of the differences between reciprocals are significantly more successful with Etheostoma eggs than with primitive darter eggs at the 0.05,0.01, and 0.0001 levels. Superimposed on this pattern is a difference in the success of E. lepidumand E. spectabile when they are crossed with primitive darters. All 16 pos­sible ways of comparing the survival medians result in higher survival when E. spectabile is used as the specialized darter for hybridizations. The 16-0 comparison is slightly exaggerated because many of the tests are in effect using the same data. One of the comparisons is significant in itself at a level between 0.01 and 0.001. It is quite apparent that the E. spectabile X primi­tive darter are much more likely to survive than are E. lepidum X primitivedarter hybrids. The results with time lapse fertilization tests are somewhat similar in that primitive darter eggs significantly inhibit Etheostoma sperm, and primitivedarter sperm does not seem to be adversely affected by Etheostoma eggs. Table 22 Median index of success of various intergroup hybrids. The indices are obtained fromTables16-21.ThoselimitedtoP.caprodes astheprimitiveparent are shown in parentheses. If even numbers of indices are available, thetwomedianswereaveraged. spectabile 9 Primitive 9 lepidum 9 Primitive 9 Posthatch Etheostoma comparisonPosthatch 127.6 (127.7) 90.0 (98.6) 90.5 (88.1) 71.3 (90.3) Primitive comparisonTotal Ethostoma 561 (575) 333 (400) 382 (464) 287 (318) comparisonTotal Primative 109.4 (110.8) 92.6 (93.3) 95.0 (91.3) 75.9 (77.0) comparison 176.4 (177.4) 146.1 (141.3) 151.7 (151.6) 129.2 (120.2) Time lapsereduction in sperm activity 93.9 (103.4) 38.2 (44.1) 132.1 (126.0) 19.9 (23.3) This circumstance cannot account for the differential results with hybridsurvival because that pattern is very apparent in posthatching comparisons. The differences between the results using the two specialized darters is not retained in the fertilization tests. One comparison favors E. spectahile and the other (incidentally that one that is statistically significant), favors E. lepidum. Obviouslv, the factors that caused the survival of intergroup E. lepidum hybrids to be lower than that of comparable E. spectahile hybridsdonotaffectfertilizationpotential, and itseemslikelythat the circumstances are reversed. The overall hybrid survivals are less easily determined because the two controls are so different that comparisons with both parents together is im­possible. The values listed below are those in which all figures are used and then each parent is contrasted separately. Seventy of 280 posthatching com­parisons are below 100 (65 of 143 Etheostoma and 5 of 137 primitive), and the median figure is 161 (104 in Etheostoma, and 379 for primitive con­trols). Eighty-two of the 280 total comparisons are below 100 (70 of 143 Etheostoma,and12of 137primitivedartercontrols),andthemedianfigure is118(102Etheostoma,and 154forprimitivecontrols).Thefiguresfortime lapse fertilization are much lower with 52 of 83 below 100 (36 of 38 for Etheostoma, and 16 of 44 for primitive darters); the median figure is 57 (28 forEtheostomaand 120forprimitivedarters). Discussion A variety of conclusions are available concerning geographic variation of adaptive characters, and those emphasized in this discussion relate to the action of one species on another. The best approach to this problem is to use a wide-ranging species. Those populations occupying areas outside the rangeof most other species would be subject to different competitive factors than would those populations sympatric with a number of near relatives. Those species with narrow geographic limits could of course be modified by selec­tion influenced by the presence ofrelatives; but the effects would be difficult toascertainbecause oftheabsence ofanunaffected homospecificpopulation.Egg Size. The egg size of a darter is roughly inversely proportional to the egg number. The abdominal cavity of a female can contain a given volume ofeggs. Any increaseineggnumber wouldconsequentlyresultinan equiv­alent decreaseineggsize(See HubbsandJohnson, 1961,formeasurements of darter eggs associated with egg complements). Longer females have con­siderablylargerabdominalcavitiesandconsequentlyahigherfecundity. The increase in egg number with size should optimally approximate the cube of thelengthincrease.Whenfoodisinshortsupplythe increaseislessbecause food intakewouldlimit thenutrientsavailable for eggproduction.Thelargest femalesoftenhaveveryfeweggs,probablybecausefood intakemustincrease more or less in a linear fashion, and metabolic needs increase logarithmically(See Hubbs, 1964b, for a discussion of some aspects of this problem). Be­cause of these non-genetic influences, egg size is a more meaningful index of fecundity than egg numbers obtained at a single observation interval. No seasonal or significant growth changes in egg size have been noted in the darters studied. The space available for mature eggs also would be affected by require­ments for other abdominal structures. Overlooking this factor may have led Williams (1959), who used ovary weight as an index of fecundity, to draw an erroneous conclusion. Darters with extensive parental care had propor­tionally heavier ovaries than did those with little parental care. Superficiallythis indicates a greater fecundity with increased parental care. On the other hand, if parental care is extensive, much postspawning effort would be de­voted to the care of the eggs, delaying the next spawning interval and re­ ducing the need to maintain a series of nearly ripe eggs in the ovary. Over­crowding the ovary might crush the developing eggs in ‘non-care” darter females. As aresult therewould be selective pressure tomaintain arelativelysmall ovary. Therefore, Williams’ (1959) data can be interpreted to show that parental care is associated with low fecundity. Actually, fecundity in­volves the number of mature eggs produced optimally in a reproductive season. Those darters with many spawning intervals (no parental care)wouldbemuch morelikelytoproducemanymore eggsthanthosewithfew spawning periods (much parental care). Egg sizes have been ascertained for a number of populations of darters. The two most extensively and intensively studies were E. spectabile and P. caprodes. Both have small eggs in Missouri, intermediately sized eggs in Arkansas, and big eggs in Texas. Similarly, within Texas the western popula­tions have bigger eggs than eastern populations at the same latitude. The intraspecific association of small northern or eastern eggs is widespread; E. zonale,E. hlennioides,and E. caeruleumfromMissouriallhaveeggsatleast one-tenth of a millimeter less in diameter than those from Arkansas. The smallereasternandnorthernH. scierusandE.lepidumeggscanreflecteither of the two dines. These data indicate increased fecundity in eastern and northern darters. The latter is in distinct contrast with the generalization byHesse et al. (1937, pp. 159 and 160), and Moore (1942) that large eggs are associated with low temperatures. The pattern of increased fecundity with northern samples is similar to that obtained by Tinkle (1961) for a turtle. It is possible that the apparent discordance does not exist but merely reflects the interpretation of the phenomena. Most of the literature cited by Hesse et al. (1937) concerns cold temperature animals. Both Tinkle’s and the pres­ent studies include a large warm temperate element. A similar parallel can be drawn with Moore’s Rana work. His early work (1942) showed large eggsassociated with cold (northern) waters; however, later (1949) he obtained largeeggs inMexico .Additionofwarm temperate samplesagainresultedin large southern eggs. The available comparisons indicate that the north-south cline in darter fecundity is not as striking north of Missouri, ComparisonswithWinn’s(1958 a)eggsizedatafromMichiganshowthatwhileP.caprodesand E. spectabile have slightly smaller eggs than those recorded here from Missouri, the Michigan measurements for E. hlennioides, E. caeruleum, and E. flabellare show that these species have bigger eggs than in Missouri. Like­wise, the last two also have bigger eggs in New York, a west-east increase inegg size.Bothdines seemtobereversed.Itispossiblethatreversalofthe dines is associated with a single selective factor. Missouri is near the center of distribution of darters. Far more species are available within one general area than would occur in Michigan, New York, or Texas. Similarly, more species are found in East Texas than West Texas, and in southern Michiganthan in New York. Therefore, large eggs and low fecundity are associated with few species of darters, and small eggs and high fecundity occur to­gether with many species. It is obvious that intraspecific competition is usu­ally more severe than interspecific competition. This seems likely in darters, especially in the postlarval stages when some occupy midwater and the others are on the bottom. All adult darters are bottom fishes. Therefore, more habitats are available to larval darters than to their parents so that two sym­patric species are more likely to compete as adults than as larvae. An area with few species would have more larval competition than a similararea with many species. Large eggs would permit greater individual survival throughintensively competitive larval stages because of ample yolk supplies. Small eggswouldpermit morelarvaeandiflarvalcompetitionwerereduced,more subadults when that species entered the competitive environment. This is essentially the conclusion made by Hubbs and Delco (1960) for fecundityvariations in Etheostoma lepidum, and is extended here to the majority of the darter egg size geographic variations. The weakest aspect in the differ­ence in fecundity of darters is north and east of Missouri. This part of the U-shaped pattern of darter egg sizes happens to resemble that reported byHesse et al. (1937). Not only are the darter estimates based on small sized samples, they are also subject to potential differences in measuring tech­niques.Forinstance,Fahy (1954)measuredE. blennioideseggsthataveragedlarger than any others. His measurements were obtained from eggs extracted fromfemalespreservedinalcohol. Theseeggs,iflaid,wouldhavebeenmuch larger than Fahy’s measurements indicated, because they had not had an opportunitytoswellinnaturalwatersandinfact hadbeensubjecttoalcohol shrinkage. The competition-based cause for egg size variation may also be applied to Moore’s (1949) Rana data. The smallest eggs come from Florida where many anuran species are found; the largest occur in Quebec where there are few anurans, and in Mexico where there are only two species of ranids. The differences in egg size between the Illinois and White systems may be somewhat discordanttothe generalpatternlisted above. Threeoffour specieswith different sized eggs, P. caprodes, E. stigmaeum and E. spectabile, have largereggsintheWhite,andonlyE. blennioideshaslargeeggsintheIllinois. The White collections are on approximately the same latitude as those from the Illinois, but the White River drains toward the north and has a more northern fauna. On the other hand, both have a variety of species that is ap­proximately equal to the other. The details of the species distributions mayshowthatthe competitive conditions are severeintheWhite. It is apparent that interspecific fecundity variations do not follow a con­sistent north-south pattern. There is some evidence, however, that the inter-specific variations may show fewer larger eggs in the north. Interspecificvariationsare ofcoursesubjecttomanyselectivefactors. Forinstance,Winn (1958a) statedthatE.fabellarehadthemostelaborateparentalcareofanydarter.Thisspeciesalsohasthelargest eggsofany dartermeasuredbyWinn, Lake (1936), and me. Therefore, large eggs and low fecundity are associated with parental care. Because parental care tends to be equivalent within taxa, thepatternwithinspecies groupsmaybe thebestindexofgeographiceffects on species. Wherever they are sympatric, E. caeruleum has larger eggs than E. spectabile, and the former is of more northern distribution than the latter. Similarly, the northern E. nigrum has bigger eggs (1.5 millimeters, Winn, 1958a; and Speare, 1965) than the southern E. stigmaeum (1.1-1.4 milli­meters ), and E. chlorosomum (1.1 millimeters). Hybrid Fertility. Although any darter hybrid combination can be reared tomaturesizeaseasilyasthecontrols,this doesnotdemonstratethatgeneticmaterial can be exchanged. The minimal data available indicate that hybridfertilityisassociated withmorphologicsimilarity,andineffectreflects phylo­genetic divergence. Etheostoma grahami and E. lepidum are closely related allopatricspeciesthathavefertilehybrids.Bothspecies arereasonably closelyrelated to E. spectabile and the hybrid females are fertile, the males sterile. Similarly,E.radiosumisreasonablycloselyrelatedtoE.spectabileand these hybridsare alsofertileiffemaleand sterileifmale.Afewhybrids havebeen reared between one or more of the above species and several less closely re­lated species of Etheostoma; and no viable gametes in the sexual dimorphicadult-sized hybrids were noted. Similarly, in the putative hybrids between Percina caprodes and Hadropterus scierus both sexes seem to be sterile (HubbsandLaritz, 1961b).Allhybridsrearedfromparentsbelongingtothe two groups of darters show no signs of sexual dimorphism. Examination of the gonads shows that they are reduced or absent. Hybrid fertility varies ex­ tensively andthe degreeoffertilityisassociated withthephylogeneticrela­tionship of the parents.PatternsofHybridSurvival—PhylogeneticDifferentiation. Thepotentialof ,„ producing viable hybrid adults seems to be absolute. It is likely that anydarter hybrid combination can be raised to adult size (Hubbs and Strawn,1957a; and Hubbs, 1959) and that darters crossed with members of other taxa cannot be reared past prolarval stages (Hubbs, 1967). The only po­tential intermediate stage might occur in etheostomatine X percine hybrids, acombination not yettested. Thedefinitivehybridsuccess seemstobetypi­calofmanyteleosts(Hubbs, 1955)anddistinctlydifferentfromthecircum­stances in other vertebrates such as Bufo (Blair, 1961) or many insects such as Drosophila (Patterson and Stone, 1952). Cyprinodont fishes are the onlyNorth American ones clearly shown to have intermediate levels of hybridiza­tionpotential (Hubbs and Drewry, 1960); however,reduced survival of dis­tant centrarchid hybrids (West and Hester, 1966) and deformation of sal­monid hybrids (Crossman and Buss, 1966) indicates that intermediate sur­vival rates may be widespread. The cobitid fishes of Asia also seem to have reduced survival of some hybrid combinations (Suzuki, 1957, etc.) Comparison of survival invariably results in relatively greater success in hybrids (Table 23). This heterotic survival parallels typically increased growth rate of hybrids (Ricker, 1948; Smirnov, 1953; and Guidice, 1966).Thesurvivalofindividual crosses varieswidelyduetochancecircumstances;however, the crosses done extensively are typically heterotic as are the ma­jority of those seldom done. The percentage above the control survival rate and the median indices would show the general effect of phylogenetic dif­ferentation. It is apparent that the hybrid survivals are better than those of thecontrols.Morecritically,theintergenerichybridsareatleast as successful as the controls. For the most part they are intermediate between the survival of the interspecific hybrids within either group. The high incidence of het­erosis is especially significant when one considers that hybrids of species not yet reared were excluded from the various hybrid survival rates discussed. Obviously, inclusionofthosesuccessful combinations,regardless ofthelevel of success, would raise the various indices. Therefore, phylogenetically the hybrids are either very successful or, if between families, unable to be reared. The high survival rates for intergenetic hybrids clearly shows that the ca­pacitytoproduce hybridshasnodirectapplicationtoconspecificrelationshipof the parental taxa. It merely signifies that the parental taxa are relativelyclosely related. Patterns of Hybrid Survival—Differences between reciprocals. Many hy­brids have been shown to have differential survival of the reciprocals (Pat­tersonandStone,1952;Thornton, 1955;HubbsandDrewry,1960;andmanyothers).Onereciprocal isviable(andoftenheterotic) andtheotherinviable. None of the darter hybridization tests provide such absolute differentiation of hybrid survival. Hybrids between primitive and advanced darters do varyquantitatively in survival, depending upon which reciprocal is used. Such survival variations are less easily recognized than the absolute ones typicallyrecorded in the literature. The intergroup hybrids based on primitive darter eggs are less viable than are the intergroup hybrids based on specializeddarter eggs. The difference between reciprocals parallels difference between rates of the maternal parents. These reciprocal differences indicate considerable maternal influence on the hybrid. It is likely that maternal influence also may apply to morphologi­cal characters because both reciprocals of atherinid hybrids are more like their maternal than their paternal parents (Rubinoff and Shaw, 1960). Differences between reciprocals is somewhat discordant with Nikoljukin’s(1952) hypothesis of hybridization indicating the primitive parent. He theo­rized that Fi hybrids should more closely resemble primitive parents than specialized parents. Reciprocal differences clearly show that both reciprocalsshould be reared in order to determine primitive parents, because one re­ciprocal might resemble its mother. Similarly, many Fi hybrids show fan­tastic morphological variation (Hubbs, 1956; Hubbs and Strawn, 1957c; Suzuki, 1957; etc.) so that small samples might not be at all meaningful. All of these problems could distort data so that Nikoljukin’s hypothesis should beapplied onlywithextremecaution. The overall survival data for intergroup experiments are more discordant withNikoljukin’shvpothesis.Eachhybridreciprocal oftheE. spectabile XP. caprodes hybrids does as well as or better than both controls. Because E. spectabilealways hasahighersurvivalpotentialthan P. caprodes,thehybridsurvival is invariably more like E. spectabile than like that of P. caprodes.Following Nikoljukin,thespecializeddarterswouldthenbeprimitiveandthe primitive darters specialized, a conclusion discordant with all morphologicevidence. Patterns ofHybridSurvival—Ecologic vs. Geographic Factors.Therelative significance of various factors in speciation has been discussed extensively by many authors. A number of workers, notably Mayr (1963), have held that species separation nearly always involves geographic isolation, and others such as C. L. Hubbs (1961) have held that ecologic selection can be suffi­ciently strong to permit species to separate in sympatry. Analysis of the rela­tivesurvivalofhybridsmayapplytothisdiscussion. Thetwomostintensivelystudied species of Etheostoma lepidum and spectabile have distinctly dif­ferent ecologic niches as larvae.-E. spectabile lives on the bottom and larvae ofE.lepidumarefreeswimming. Thelarvae ofall species ofprimitivedarters are also free swimming. Therefore, during the larval stages studied, E. lepi­dum was competing with primitive darters and E. spectabile was not. In contrast, the geographic relationship is reversed. That is, E. lepidum fre­quently is allopatric to primitive darters and E. spectabile seldom, if ever, is allopatric. Contrasted with then-controls hybrids between primitive darters andE.lepidumaredistinctlylessviablethanthosebetweenprimitive darters andE.spectabile(Table22).Thereduction inhybridvitalityofecologicallyoverlapping forms compared with geographically overlapping taxa indicates ecologic factors have a greater effectiveness than geographic factors. The time lapse data appears to contradict the relative effectiveness of geo­graphic and ecologic selection because E. lepidum eggs inhibit primitive sperm significantly less than do E. spectabile eggs. The contradiction is ap­parentlynotreal becausethe ecologiccircumstances arereversedduringeggdeposition. That is, the primitive darters studied here and E. spectabile lay eggs in or on gravel and E. lepidum attaches its eggs to aquatic vegetation.Therefore, this comparison also substantiates the significance of ecologicfactors in developing isolation mechanisms. The above evidence supports ecologic isolation as being highly significant in speciation. Livingstone (1965) also has emphasized the role of ecologicfactors as isolating mechanisms by showing that geographic isolation in African lakes could not have occurred as hypothesized by Brooks (1950).Patterns ofHybrid Survival—Gametic Inhibition. The indicesfortime lapsefertilization (Table 23) are distinctly lower than those of the controls. This reduction in fertilization potential clearly involves the time lapse phenome­non, because a similar analysis of fertilization indices obtained from “damppan”experimentsresultin48percentoftheindicesbelow 100,andamedian of 97. The results do not deviate significantly from 50 percent and a median index of 100, but do deviate significantly from all of the time lapse sum- Table 23 Comparison of hybridization success with level of phylogenetic differentiation. InterpopulationInterspecific InterspecificSpecialized Primitive Between Groups % combinations reared % posthatchingindices above 100 56 (all controls) 75 82 69 67 62 75 Median index 116 135 170 161 % total indices above 100 70 75 62 71 Median index 107 112 112 118 % time lapseindices above 100 38 37 25 37 Median index 75 67 37 57 Maries presented on Table 23. The relative significance of sperm delay in the gametic inhibition of darters is also shown by tire equivalent results of experiments with up to ten seconds delay and the strikingly divergent results of experiments with greater delay. The slightly (and insignificantly) reduced fertilization percentages of “damp pan” experiments may reflect a time lapserelated circumstance. Some of the eggs would have been located distant from anyofthe spermandthetwogametesmighthavecomeincontactafter considerabledelay.Thefertilizationofthoseeggswouldthereforereflect in­hibitionof“tired” spermbyheterospecificeggs. The great difference in the relative fertilization potential between fresh and exposed sperm shows that this isolating mechanism would have little ef­fect on behavioral accidents. That is, those heterospecific pairs that mismated would not have a significantly reduced rate of fertilization (if the sum­marized dataare correctapproximately97percentofthehomospecificrate). In contrast, those sperm that are washed away from the vicinity of the mated pair might be swept by the current to the vicinity of a second mated pair. If this pair were of the same species the chances of the “tired” sperm fertilizing an egg would be considerably less than the fresh sperm, and the relative suc­cess would depend upon the time of the exposure and temperature of the water.If,onthe otherhand,thesecondmatedpairwereofadifferentspecies,the phenomena responsible for the time lapse results would also apply to the fertilization potential. At best, the probability of heterospecific fertilization would be 67 percent of that of comparable homospecific pairs from the same locality. Therefore, this isolation mechanism is useful in preventing the chance meeting of gametes, and shows the strong selection in darters againsthybridization. This factor is further shown by the exceedingly short duration of sperm activity—up to 25 seconds at 15° C. This figure is notably shorter than all listed by Lindroth (1947) for Swedish fresh-water fishes, by Fink and Hayden (1960) for marine cottoids, by Miller (1952) for a marine clupeid, and by Hubbs and Drewry (1958) for euryhaline cyprinodonts.Darters breed in rapids, a factor that otherwise would increase potentialchance hybridization. The very low duration of sperm vitality would be re­quired to prevent massive hybridization in oviparous animals breeding in rapid water. The short duration of brown trout sperm fertilization potential(abouttwominutesat 9°C.)(BussandCarl,1966)probablyreflectstheir rapid water spawning site. The exceedingly low incidence of natural darter hybrids indicates the effectiveness of this isolation mechanism. Only the in­tergrouphybridreportedbyHubbsandLaritz(1961 a)clearlyresultedfrom chance gametic contact. There is considerable difference in the effects of primitive darter eggs and thoseofspecialized darters onspermvitality.Thedifferenceisapparentfrom the data presented in Table 23 for within-group hybrids (median indices of 37 and 67 respectively) and equally divergent in the between-group hybridslistedonp.57(medianindicesof28and 120respectively).Therefore,primi­tivedartereggshave amuch greaterinhibition offoreignsperm than dothose ofspecializeddarters.Primitivedarterfemalesproducemanyeggsat spawn­ing time and typically lay their eggs on the surface of the substrate. Spe­cialized darters produce fewer eggs and have greater control over deposition of eggs which are typically placed under gravel, on the underside of rocks, or on aquatic vegetation. Scattered eggs would be most exposed to chance fertilization andthesehavethegreatestinhibitionagainstspermwhichmight becarriedtothem by currents. Gametic inhibition is somewhat intermediate between premating and post-mating isolating mechanisms. It does inhibit fertilizaton and therefore could be considered to be premating. The egg that was exposed to foreign spermwould be likely to have been washed away from the mated pair before a homospecific sperm joined it. Therefore, this type of egg would be unlikely to produce a homospecific embryo and would be already wasted. If so, the population model for selection discussed below would apply. In contrast, any eggthatwascontactedbytheforeignspermwhilestillinthe swarmofhomo­specificsperm and beforeithadbeenfertilizedbyhomospecificspermcould, of course, produce a homospecific embryo from a subsequent sperm contact and be subject to individual selection if a means of avoiding heterospecificfertilization were present.Patterns of Hybrid Survival—Reinforcement. Only one series of experi­ments (with logperch and greenthroat darters) provided distinct evidence forreinforcement. Bothreciprocals ofthishybridcombinationhaddistinctly higher survival potential if both parents were from allopatric stocks; inter­mediatesurvivalpotentialifonlyoneparentwasallopatric;and lowsurvival potential if both parents were from sympatric stocks (Tables 18-20). The relative difference is indicated by the fact that hybrids with both parents from allopatric stocks never had indices below 100 (median 150), those with oneallopatricparentwerebelow onehundred,29percentofthe time(median138), and those with both parents sympatric had indices below one hundred 49 percent of the time (median 101). Except for the most extreme figures,thepercentageaboveagivenfigure isalwayshighestinallopatriccrossesand always lowest in sympatric crosses, and in most instances with a probabilityof less than 0.01 of being by chance. The exceptional circumstances never approach statistical significance;therefore,theprobabilityofallopatricpopu­lations producingviablehybridsisapproximately Vk timesthatofcomparable sympatric populations assuming the geographic isolation is eliminated. A similar reinforcement of isolation mechanisms is indicated by the H. shumardi X P. caprodes hybrids and for E. punctulatum female X E. spec­ tabile male crosses. The only possible reduced hybrid vitality in allopatry is in the E. tetrazona X E. spectahile hybrids; however, too few eggs from too few localities were used to draw even tentative conclusions. Low survival of greenthroat X rainbow darter hybrids may indicate low hybrid potential in allopatric crosses but these data are contradicted by the highly successful hybridsofE.lepidumandE. juliaewhichareequallyallopatric. Reinforcement of isolation mechanisms also is supported by the exceed­inglyhigh vigorofE. spectahilesperm incontrol experimentsfrom the Brazos system, an areainwhichnootherspeciesofEtheostoma have beencollected. This seems alsotobereflectedinthenil effectofBrazosE. spectahileeggs on E. lepidum sperm from sympatric areas. The low vigor of equally allopatric E. lepidum sperm may be rationally explained by the small quantities neces­sary for fertilizing the reduced egg complements there. The Brazos E. spec­tahile egg complements are nearly equivalent to those of the Colorado and Guadalupe systems. The logperch X greenthroat darter combination clearly involves greaterhybridizationpotentialinallopatry thaninsympatry.Itfollowsthetheoreti­cal model set up by many authors for reinforcing isolation mechanisms. It is,however,thefirstexample ofreinforcementapplicable topostmating isolatingmechanisms. Thestandardexplanationofreinforcement basedonprematingmechanisms—those individuals that do not waste gametes in producing hy­brids have more gametes available for homospecific zygotes—cannot applyhere. Any zygote whose survival potential is reduced must already have re­sulted in wasted gametes. Because individual selection is not readily applied, population level selec­tion may be utilized. Most darters occupy similar habitats and will occupyadjacent habitats if the usual occupant is absent. For example, E. spectahileisfoundingravelrifflesandE.lepiduminriffleswithabundantaquatic vege­tation when sympatric. In contrast, each will occupy both habitats when the otherisabsent.It islikelythathybriddartersoccupyhabitatssimilartothose of their parents. If these darters are heterotic they might easily displace one or both parental species in their preferred habitat. For example, large num­ bers of putative hybrids between E. lepidum and E. spectahile have been collected in the Guadalupe River near Kerrville State Park. These hybrids aretypically obtained togetherwithlargenumbers ofparental types. Inhabi­tats where the pure stocks are rare, no hybrids are obtained. The presence of hybrids only in the highly preferred habitats of the parental types can onlybe interpreted as a similar habitat preference and the displacement of the parental types by the hybrids. If one assumes an equal probability of pro­ducinghybrids, areduction inhybrid vigorcouldbeselectivelyadvantageous to the population. That is, each vigorous hybrid would displace a parentaltype whereas the feeble hybrids would be displaced by members of their parental taxa. As a consequence, the number of breeding adults in the two areas would differ and the difference would be associated with the relative vigor of the Fi hybrids. During subsequent reproductive intervals, the pro­duction ofyounginariffle wouldbeinversely associated withthe vigorofthe hybrids.Therealsowouldbeastrongtendencyforthe morenumerousyoung to invade the riffle inhabited by the populations producing vigorous hybrids.Therefore, thepopulationswithreduced hybridvitality dueto geneticfactors would have aselectiveadvantage overthosethatproducedvigoroushybrids. Two other ecologic circumstances may be involved in the requirements for selection of postmating reinforcement. The first would be stable adult popu­lations andmigratoryyoung,asituationclearlyapplyingtomostdarterpopu­lations. A riffle extensively collected for breeding adults soon becomes de­populatedalthoughyoungaboundinthesamehabitats. Theseyoungcould, of course, have been produced locally, but the numerous young occupying new stream channels after floods, etc., must have come in from elsewhere. Perhaps the best example may be provided by a series of collections from the North Llano River at Junction. Adults were obtained for laboratory experi­ments in November, 1963, at the start of the breeding season. Other collec­tions indicated that the eggs obtained were those first to ripen that season. Although adults abounded in all riffles, the one that was collected most in­tensively yielded more than two hundred adults. On all subsequent collec­tions this riffle was re-examined and a total of fewer than ten adults was ob­tained during the remainder of that season. All samples contained numerous young of all sizes, indicating continuous immigration from adjacent riffles. Many adults were collected in each sample from each adjacent riffle, even those separated by less than twenty meters of quiet water. The next yearearly collections were distributed more evenly and all riffles contained breed­ing adults throughout the season. Adult darters therefore tend to remain within limited areas and dispersal occurs duringlarval oryoungstages.This is not surprising because it is typical of many vertebrates. The second ecologic circumstance that might relate to the selection model is genetic uniformity within individual populations. If each riffle were to maintaina high population density, considerable genetic diversity should oc­cur on each; this is not likely to occur, however, in semiarid regions such as CentralTexaswherenumerousnaturalcatastrophesoccur.Some localities are subject to repeated floods and after each flood most fish populations are re­ducedinnumber.Similarly, droughtsmaybesevereandprolonged,radicallyreducing surface flow and as a consequence the numbers of fish. The popula­tions could resume their former abundance upon re-establishment of suitable conditions and by chance would be expected to be relatively uniform ge­netically.Thefirst catastrophemightsetupthedifferenceinhybridpotential,and those populations producing less vigorous hybrids would spread into other areas. Soon the populations might be mixed with regard to selection for reduced hybrid vigor; but since natural catastrophes are seldom widelyspaced in Central Texas the populations would be repeatedly reduced to minimal levels. Each reduction would produce many homogeneous popula­tionsandthosewithfactorsreducinghybridvigorwouldbe selectedforfol­lowing each catastrophe. Obviously, strong selection would be closely linked with frequent natural catastrophes. It is not surprising, then, that the best example ofreinforcement can be found in fishes inhabiting catastrophe-prone semi-arid streams. The only distinct support for posthatching reinforcement lies in the log-perch X greenthroat darter hybrids. Perhaps by coincidence these two spe­cies occupy similar ecologic niches (upper waters near the surface film)during the larval stages that were used for analysis of hybrid survival. Both E. spectabile and E. punctulatum occupy bottom habitats, and H. shumardi and F. caprodes occupy upperwaters during the same periods; their hybridsurvival also indicates posthatching reinforcement. Patterns of Hybrid Survival—lntrogression., If introgression were to have o a major role in selection of darters, one would expect to find an increase in hybridization potential of sympatric populations. None of the comparisons are distinctlymore viableif sympatric. Likewise, introgression would require maintenance of some degree of hy­ bridfertility. Mostdarterhybrid combinationsaresterileand thosethathave some fertility typically have much less than do comparable controls. As a consequence, transfer of genetic material between distinct biological species would be difficult. The only example of reasonably high hybrid fertility is between allopatric species. If these taxa were to exchange genes it would not result from the breakdown of an intrinsic isolation mechanism, and would merely reflect sec­ ondarycontact betweenmorphologicallydistinctraces. Insummary,introgression doesnotseemtoapplytodarters. Patterns of Hybrid Survival—Mutual Coexistence. There is little question that most speciation does involve geographic separations and if so, there should be a variety of levels of divergence. Therefore, it is unlikely that one series of tests would necessarily show strong evidence for isolation mecha­ nismsin geographicraces,whichshouldbemostapparentinpremating situa­ tions. The intraspecific patterns of hybridization potential show that E. lepidwn is speciating in geographic isolation. Other intraspecific hybrids may have reduced fertilization potential; however, it is no more noticeable in within­systemthaninbetween-systemhybrids.Ifgeographicisolation weretobe of extreme significance the intraspecific results should show considerably re­duced fertilizationpercentagewith increasedallopatry.Althoughthe above seems easilyapplicabletoE.lepidwnracial variation,thismaynotbe agood example of mutual coexistence. If the races are in the process of dividing into distinct species, their interactions should parallel inter-species interac­tions; however, the hybrid viability is distinctly not heterotic as is typical of interspecific hybrids. Obviously those incipient species have more gameticisolation than do “standard sympatric” pure breeding units; perhaps the hybrids would be heterotic when the species are sympatric and behavioral isolation mechanisms are well developed. Patterns of Hybrid Survival—Proximate Inhibition. A large number of hy­brid combinations exhibit an unexpected pattern of survival indices. This is the heterotic survival of hybrids between parents from distant or identical localities and the corresponding inhibition of hybrids between parents from two localities within the same stream system. There is some evidence that the converse may occasionally occur; however, the probabilities are relativelylow and the phenomenon is less frequently observed. The best example of proximate inhibition is Colorado River E. lepidum females X E. spectabilemales. All hybrids but those from two different localities in the Colorado System are heterotic and deviation from the control has a probability of less than0.001ofbeingbychance inallbutthesmallsampleusingmalesfrom the Illinois River. Moreover, the reduced vitality of the hybrids from two Colo­rado localities also has a probability of less than 0.001 of being by chance. Therefore,it isexceedingly unlikelythatthewholepatternhasresultedfrom chance occurrence. The low fertilization (time lapse) potential of Colorado River E. lepidum sperm exposed to other Colorado System E. spectabile eggsis distinctive, significant, and follows the low survival potential of the re­ciprocal.AllofthesegreenthroatX orangethroatColoradoRiverhybridsare based on large samples from several localities and each shows the same phenomenon, indicating that it is not an isolated example. Perhaps of more significance is the presence of the same pattern in all combinations of the E. caeruleum X E. spectabile hybrids from two different localities in the Mississippi-Missouri system (both Loose Creek and White River); Colorado System H. scierus X P. caprodes hybrids; and Colorado System P. caprodesfemale X E. spectabile male hybrids. This pattern is obviously widespread,both phylogenetically and geographically. Its frequent occurrence in the Colorado System experiments is undoubtedly caused by the more extensive use of Colorado stocks for tests so that individual experimental error is unlikely to mask selective results. Moreover, the species in which the patternof proximate reduction of hybrid vitality is most apparent are also those most intensively studied, all of which indicates its widespread occurrence. This totally unexpected occurrence has misled me previously. In 1961, I pooled all within-Colonrad oSystem stocks in my report of E. lepidum sperm vitality. The drastically reduced fertilization potential of proximatepopulations combined with the reasonably high potential of sympatric hy­brids caused an overall reduction of fertilization potential within the system so that reinforcement was reported. Separation of the Colorado stocks into proximate and identical localities shows that reinforcement per se does not explain theresults. The frequency of proximate inhibition indicates that it must be significantin interspecies interactions; however, it cannot easily be adapted to any of the threepreviouslydiscussedpatternsofinterspeciesinteractions: introgression,reinforcement, or mutual coexistence. Clearly mutual coexistence cannot ap­ply because if it did, no pattern should appear. Superficially a mixture of in­ trogression and reinforcement seems to occur. That is, introgression within the stream system and reinforcement between systems. It is obvious that simultaneous action of introgression and reinforcement would tend toward equal results regardless of the level of sympatry. This does not occur here so one must examine the data to determine if another explanation is available. The within-system introgression is perhaps more apparent than real. Intro­gression would also require a reasonably high hybrid fertility. The E. spec­tabile X E. lepidum hybrids are partly fertile and by extrapolation it seems likely that the E. spectabile X E. caeruleum hybrids also are partly fertile. TheavailableH.scierusXP.caprodeshybridsshownosignofmature gam­etes and those with “male phenotypes” do not father young when stripped.Finally and most conclusively, the E. spectabile X P. caprodes hybrids are not sexually dimorphic and the gonads are minute. It is inconceivable that these hybrids would be other than a dead end eliminating any selective value resulting from introduced foreign chromatin. There is no reason to suspect that between-system reinforcement does not apply. If so, the apparently contradictory increase in hybridization potentialof sympatric stocks must be examined carefully. If some other series of isola­tion mechanisms were to prevent hybridization of sympatric populations, and these mechanisms were relatively ineffective in isolating allopatric popula­tions, the apparently contradictory reversal of hybridization potential would make sense. Almost every author who has discussed isolation mechanisms (C. L. Hubbs, 1961; Mayr, 1963; Mecham, 1961; Stebbins, 1950; etc.) considers that premating mechanisms are more effective than postmating mechanisms. Similarly, among the premating mechanisms those relating to courtship be­havior usually are considered the most effective. Therefore, if a behavioral mechanism were to prevent sympatric mating and its effect were negligiblein allopatric matings the theoretical requirements would be attained. Pre­liminary tests indicate that behavioral mechanisms operate much more effec­tivelyonsympatricthan onallopatricpopulations.If so,the onlynaturalwayby which behavioral isolation mechanisms might break down would be if a darter was displaced to another locality within its stream system. Such a circumstance is precisely that to which proximate inhibition would apply. A variety of data supports the above hypothesis. Because darters are known to break up into a variety of morphologic (Strawn, 1961; Hubbs, 1958b;Hubbs and Delco, 1960; Hubbs and Johnson, 1961) and physiologic races (Hubbs and Armstrong, 1962; Hubbs and Strawn, 1963) and these races can be separated by exceedingly short distances (Hubbs, 1964a), racial variation exists that might provide the differential behavioral responses. Moreover,hybridization in fishes is most likely if one species is abundant and the other rare (C.L. Hubbs, 1955,1961).Theapplicationoflightstimuli(abehavioral response) to the differential population density hypothesis for hybrid pro­duction was discussed by Hubbs and Martin (1965). The available evidence indicated that hybridization was most likely to occur when an upstreamspecies was displaced downstream by a flood into the range of the second species. Not only would floods establish differential population densities;they would also reduce the effectiveness of light stimuli as isolation mecha­nisms. One of the classic hybrid swarms between Gila and Siphateles (Hubbsand Miller, 1943) clearly resulted from one species being introduced into the range of another. A darter hybrid swarm in the Guadalupe River clearlyresulted from an upstream species ( E. lepidum) being washed downstream into the area of a related species (E. spectahile) Therefore, proximate in­ . hibition seems to supplement a behavioral mechanism and occurs when the two populations might get together in a situation where the finely tuned be­havioral mechanism would not be effective. Obviously the populations in­habiting different stream systems would be unlikely to be mixed naturallyand wouldnotbe involvedinthis problem. Acknowledgments The experiments reported here were supported by National Science Foun­dation Grants G328, G2214, G4776, G11272, G8707, and G83206. Permis­sionto obtainstocksfromprivatepropertywas givenbyMrs.Ford Boulware, Mr. Frank Cullum, Mr. C. L. Secor, and Mr. C. L. Boren, among others. Mr. Marion Toole approved the work at the State Fish Hatchery in Mountain Home. 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Observationsofthereproductivehabitsofthedarters(Pisces-Percidae).Amer. Midi. Nat., 59: 190-212. Bulletins of The Texas Memorial Museum Price Tax No. 1. Mylohyus nasutus, Long-nosed PeccaryoftheTexas Pleistocene, by E. L. Lundelius, Jr., 1960 $l.OO .02 No, 2. TheFreisenhahnCave(PartI)byGlenL. Evans; The Saber-toothed Cat, Dinobastis serus (Part II)by Grayson E. Meade, 1961 1.00 .02 No. 3. A Bibliography of RecentTexas Mammals by Gerald G. Raun, 1962 1.00 .02 No. 4. Handbookof Texas Archeology: TypeDescriptions. Editedby DeeAnn Suhm & Edward B. Jelks, 1962 6.00 .12 No. 5. SalvageArcheologyofCanyonReservoir: TheWunderlich, Footbridge, & Oblate Sites, by Johnson, Suhm, & Tunnell, 1962 2.00 .04 No. 6. The Ethnography & Ethnology of Franz Boas, by Leslie A. White, 1963 2.00 .04 No. 7. Fossil Vertebrates from Miller’s Cave, Llano County, Texas, by Thomas Patton, 1963 2.00 .04 No. 8. Interactions Between a Bisexual Fish Species & its Gynogenetic Sexual Parasite, by Clark Hubbs, 1964 2.00 .04 No. 9. Oedaleops campi (Reptilia: Pelycosauria)Anew genusandspeciesfromtheLower Permian ofNew Mexico, and thefamilyEothyrididae, by W. Langston, Jr., 1965 1.00 .02 No. 10. Blancan MammalianFauna and Pleistocene Formations, Hudspeth County, Texas, by William S. Strain, 1966 2.00 .04 No. 11. APopulationofWoodrats (Neotomamicropus) in Southern Texas, by G. G. Raun, 1966 2.00 .04 No. 12. Toward a Statistical Overview of the Archaic Cultures ofCentraland SouthwesternTexas by Leßoy Johnson, Jr., 1967 2.00 .04