PHYCOLOGICAL STUDIES X. Taxonomic Studies in the Oscillatoriaceae AILSIE F. BAKER AND HAROLD C. BOLD ' -< - / I Phycological Studies X. Taxonomic Studies in the Oscillatoriaceae 1 2 AILSIE F. BAKER AND HAROLD C. BOLD 1 Department of Biological Sciences, Chico State College, Chico, California 95926. 2 Department of Botany, University of Texas, Austin, Texas 78712. Acknow led gm ents The authors would like to thank Professors Donald Larson, Calvin McMillan, and Chase Van Baalen for their aid during this investigation and for reading the manuscript. They would also like to thank Dr. Francis Drouet of the Academy of Natural Sciences of Philadelphia for checking the identifications of many of the isolates and Dr. Hannah Croasdale of Dartmouth College for preparing the Latin diagnoses. They are grateful to Drs. R. D. Groover, P. T. Kantz, and Patricia Archibald, and to Miss Janet Winstead and Mr. Darryl Fritze for cultures and for their help­ful discussions of this work during the past three years. During this investigation the senior author was supported by National Science Foundation Graduate Traineeships. This research was also supported by N.S.F. grants 7987X and U.S. Dept. of the Interior, Federal Water Pollution Control Administration grants DBR 18050 and 16010 to the junior author. The University of Texas at Austin Table of Contents Instruction . 7 Materials and Methods 10 Taxonomic Criteria . 18 Descriptions of Certain Isolates of the Oscillatoriaceae in Culture . 20 Schizothrix calcicola (Agardh) Gomont. 21 v. vermiformis var. nov. 23 v. diffusa var. nov. 23 v. radiata var. nov. 23 v. glomerulata var. nov. 24 v. amorpha var. nov. 25 v. vaginata var. nov. 25 v. olivacea var. nov. 25 v. minuta . 26 v. actiniformis var. nov. 26 v. fuscoviridis var. nov. 26 v. densa var. nov. 27 v. scabella var. nov. 27 v. compacta var. nov. 28 v. nitida var. nov. 28 v. mucosa var. nov. 28 v. glabra var. nov. 29 v. fusca var. nov. 29 v. discreta var. nov. 30 Schizothrix arenaria (Berkeley) Gomont 30 v. vermiformis var. nov. 31 Key to Species of Schizothrix in Culture . 31 Microcoleus vaginatus (Vaucher) Gomont 33 v. glaber var. nov. 34 v. undulatus var. nov. 34 v. cyano-viridis var. nov. 34 v. funiformis var. nov. 35 v. conicus var. nov. 35 v. fuscus var. nov. . 35 v. fuscorubens var. nov. 36 v. radiatus var. nov. 36 v. araneaformis var. nov. 37 M icrocoleus lyngbyaceus (Kuetzing) Crouan 37 v. vermiformis var. nov. 38 M icrocoleus irriguus (Kuetzing) Drouet 38 Key to Species of Microcoleus in Culture. 39 Porphyrosiphon notarisii (Meneghini) Kuetzing 40 v. canus var. nov. 41 Oscillatoria lutea Agardh 41 v. auxotrophica var. nov. 42 v. contorta var. nov. 43 v. scabra var nov. 43 Physiological Studies . 44 Selection of Basal Medium . 44 Comparison of Unialgal and Axenic Cultures. 45 Comparisons of Cultures at Various Ages 46 Effects of Carbon Sources . 49 Effects of Various Nitrogen Sources 52 Hydrolysis of Gelatin 54 Hydrolysis of Starch 56 Sensitivity to Crystal Violet 57 Effect of Vitamins . 58 Effects of Varying pH . 59 Discussion 62 Summary 64 Literature Cited . 65 Illustrations . 71 Introduction The primary concern of this investigation was to examine in culture a number of isolates of blue-green algae belonging to the family Oscillatoriaceae to see which, if any, characteristics would prove consistent and, therefore, taxonomically useful. Members of the family Oscillatoriaceae (Suborder Oscillatorineae, Order Os­cillatoria!es, Division Cyanophyta) are characterized by the filamentous, uniseriate organization of the cells and by the occurrence of reproduction by fragmentation (formation of hormogonia) only. Specialized cells such as akinetes and hetero­cysts are absent. The species are found in a great variety of geographical areas. The problems encountered in trying to identify many blue-green algae, even in a general way, are numerous. Though the blue-green algae are generally conceded to have many affinities with the bacteria (Echlin and Morris, 1965; Pringsheim, 1968), they have only rarely been studied by or characterized on the basis of any microbiological techniques. Taxonomy depends upon the type specimen method. Taxa are described from natural collections, and keys for identification are fre­quently based on habitats, especially the type habitat, even though further collec­tions may have considerably expanded the original distribution of the species. The occurrence of environmental variation has long been recognized, but until recently ( Drouet and Daily, 1956; Drouet, 1962, 1963, 1964, 1968) it has not been seri­ously considered in the taxonomy of the group; also the consideration of variation was and is primarily field-based with few cultural studies supporting it. Reports of variation among the Oscillatoriaceae were published by Agardh, Kiitzing, Hansgirg, and other nineteenth-century workers, but some of the sup­posed variation appears to have been inferred from observations of mixed popula­tions. Gomont ( 1892) recognized that there could be some modification of the alga by the environment, but he thought reports of transformations of one species into a completely different one were the result of incorrect observations. In his monograph he retained a number of genera and species at least partly because he thought that uniting a large number of genera would result in one genus of such magnitude that it would be impossible to work with. In the twentieth century Crow ( l 923, 1924, 1925, 1928) grew numerous and varied members of the Cyanophyta in the laboratory and also examined them in the field. With different concentrations of organic materials he observed variation in the structure of the trichome and of the sheath. Despite the numerous variations which he found, he retained the idea that real groups (or species) do occur under field conditions, and he did not attempt to r~vise the classification of any group although in some genera he could quite possibly have done so. Canabaeus ( 1929), studying the effects of various salt concentrations on the akinetes of Anabaena, found that environmentally induced variation was indeed possible. She failed, how­ever, to offer an alternative, more stable character for taxonomic use. The variability in the false-branching genera has been investigated by several workers. Jaag ( 1943) collected Scytonema from very wet, moist, and very dry habitats; in the very wet places the sheath formation was that of the genus Peta­lonema and in the moist and dry habitats two different species of Scytonema oc­curred. He also found intermediates in the expected localities and accordingly grouped all three species as stata of Scytonema myochrous. The status designation (an infraspecific taxon applied to ecologically varying forms) has been used from time to time by other European workers (Golubic, 1965b; Komarek, 1958) in attempts either to describe more fully very large and diverse genera or possibly to avoid the problem of variation within the species. Variation in numbers and type of branching in Tolypothrix was investigated by Hollerbach ( 1928) and, more recently, by Stein ( 1963). Golubic and Kann ( 1967), on the basis of many measurements and statistical calculations, have concluded that cell measurements can be used to distinguish species in some cases. In Tolypothrix distorta v. penicillata they decided the sig­nificant cellular differences could not be attributed solely to ecological variation and advocated treating the two variants as separate species according to the orig­inal descriptions. Golubic ( 1965a) had earlier compared variability in marine Oscillatorias; based on variation in granulation, spiraling of trichomes, and term­inal cell shape, he divided them into two groups of species. Schwabe ( 1960, 1964) fully described the problems in blue-green algal system­atics and investigated relative stability of various characteristics in Plectonema spp. and in Oscillatoria acutissima in the field and, to a more limited extent, in the laboratory. He concluded that there were definite species in Plectonema, that ecological variation, while present, was not nearly as prevalent as in Lyngbya and in some other genera, and that the taxonomic value of false-branching was limited. In his paper on 0. acutissima ( 1964) he discussed the criteria given in various monographs and seen in his own investigations but refrained from a con­clusive description due to lack of knowledge of variation. Other experimental, morphologically oriented works with various blue-green algae have included those of McLachlan, Hammer, and Gorham ( 1963) and Gorham. et al. ( 1964) on Aphanizomenon and Anabaena, respectively. These in­vestigators have examined the effects of various nutrients and of pH upon several strains of these genera. Pearson and Kingsbury ( 1966) used four species ( Gloeo­capsa montana, Anabaena cylindrica, Calothrix membranacea, and Lyngbya sp.) of four different families in an investigation devised to test induction of morpho­logical variation. The characteristics of all varied, but in their isolates Anabaena and Gloeocapsa were changed less than Lyngbya and Calothrix. Demeter ( 1956) investigated 13 species (eight genera) with regard to variation in different media and in different concentrations of several components of the media. He noted the different plant-mass forms on agar and liquid and for the species he investigated, plant-mass form seemed to correspond with other generic attributes. He also found that increasing concentrations of salts generally produced changes in morphology. One ecologically and culturally coordinated investigation of the type called for by Pringsheim ( 1967) is that of Sorensen and Conover ( 1962) on the Lyngbya confervoides mats occurring in lagoons along the Texas coast. They identified five layers in the mats and cultured samples from them. Differences in appearance of the protoplasm and in sheath coloration and in thickness were observed that were constant for each zone in the field and under simulated culture conditions. N ostoc and Anabaena were extensively studied in culture by Kantz ( 1966). Kantz and Bold ( 1969) found that motility and configuration of the plant-mass correlated well with taxonomic position and that these attributes were much more constant in culture than some of the classical characteristics usually cited for these genera. Sharp ( 1969) observed three strains of Schizothrix calcicola from three ecologi­cally varying tropical marine habitats. He concluded that they were separate, in­fraspecific entities, but the exact meaning of his discussion is not clear. The correlation of environmental variation with ultrastructural variation has only begun. Peat and Whitton ( 1967) found great variation in the lamellar struc­ture of Chlorogloea fritschii at different ages and with or without a combined nitro­gen source. They also ( 1969) studied the variation in a species of Oscillatoria in culture and in the field; finding several cultural forms of the field species which they thought might correlate with seasonal variation in the field. It is no longer necessary to demonstrate that changes can occur in blue-green algae, for this is common knowledge. The need is for defining conditions which cause changes and for studying the range of possible variations-provided, of course, that someone can determine how much is "possible." The necessity of experimental work for future systematic evaluation of blue-green algae has been noted, among others, by Schwabe ( 1960, 1964), Koster ( 1966), Padmaja and Desikachary ( 1967), Allen and Stanier ( 1968) and Kantz and Bold ( 1969). Materials and Methods The cultures used in this investigation were obtained primarily by isolation from soil samples and from freshwater, air, and marine collections. Cultures available from the Culture Collection of Indiana University were also used as well as some isolated by Dr. Chase Van Baalen. Table 1 summarizes the sources and isolators of the cultures used. The culture medium most frequently used was Bald's Basal Medium-lN BBM (Bischoff and Bold, 1963) and modifications thereof, including 3N BBM (Brown and Bold, 1964), BBM-K (Kantz and Bold, 1969), and 3N BBMT (Groover and Bold 1969). Other media employed were biphasic soil water (Pringsheim, 1946), Shen-X (Shen, 1966), Kratz and Myers' Medium D-KMD (Kratz and Myers, 1955), Cg-l 0, a modification of Katz and Myers' Medium C (Van Baalen, 1967), and von Stosch's enriched sea water-VSE (von Stosch, 1964). The for­mulae for these media are given below. 1N BBM: 10 ml each of the following six major element stocks and 1 ml each of the four minor element solutions are added to 936 ml deionized water. The major element stock solutions are made by dissolving the indicated amount of salt in 400 ml deionized water. NaNOs _ _ _ 10.0 g K2HPO, 3.0 g CaCl2·2H20 __ __ _ _--···-----·· 1.0 g KH2PO, 7.0g MgS017H20 --··--· 3.0 g NaCl 1.0 g The minor element solutions are made as follows: l. 50 g NaEDTA and 31 g KOH are dissolved in 1 liter deionized water. 2. 4.98 g FeS0.-7H20 are dissolved in 1 liter acidified water. Acid water is made by adding 1 ml concentrated H 2SO, to 999 ml deionized water. 3. 11.42 g H 3B03 are dissolved in 1 liter deionized water. 4. The following are dissolved in 1 liter deionized water: ZnS0.-7H20 8.82 g CuS0.-5H20 _ -------· __ _ 1.57 g MnCl2·4H20 1.44 g Co(NOJ) 2·6H20 ___ _____ -··-·-· 0.49 g Mo03 0.71 g 3N BBM: This medium is made just as in lN BBM except that 30.0 g NaNOs instead of 10.0 g, are used in the stock solution of this salt. BBM-K: 5 ml each of the following major element stocks and 1 ml each of the BBM minor element solutions are added to 966 ml deionized water. The pH is adjusted to 7.5 with lN HCI. The major element stocks are made by dissolving the indicated amount of each salt in 400 ml deionized water. NaN03 ----···· ·--·-·--·-····-_10.0 g NaCl ····-·--············ --· 7.0 g CaCl2·2H20 _ _--·------·--·-··-··---· 1.0 g Tris (trihydroxy-methyl­MgS04·7H20 ----· -· . -· --·· ___ 3.0 g aminomethane ··-·--·------·---·-__ 20.0 g K2HP04 --··---·· -··· _ ·-·····---·-··· 3.0 g 31\' BBMT: 10 ml of Tris stock and of the 3N BBM macro-and microelement stocks are added to 926 ml deionized water. The pH is adjusted to the desired point with IN HCI. The Tris stock is made by dis.5olving 25 g Tris in I liter de­ionized water. Biphasic Soil water: A small pinch of CaCO:i and approximately one-fourth inch of garden soil are placed in the bottom of a test tube which is then filled three-fourths full with deionized water and autoclaved. S hen-X: 1 ml of each of the following major element stocks as well as I ml of each of the BBM minor element solutions are added to about 600 ml deionized water. The volume is then brought to 950 ml, and the pH is adjusted to 7.2 with IN HCI. Deionized water is then added to bring the total volume to I liter. The stocks are made by dis.5olving the indicated amount of salt in I00 ml deionized water. Co(l'\H2)2 ---·· 2.0g Na2Si03 ·----···· _. __ ·-·---·----1.0 g CaCl2 2H20 ----10.0 g KC! . -----· -··--· .. 0.055 g :M:gS01·7H20 _ 5.0 g K2HP04 ----·---· --·--· _ -··-__ 5.0 g Na2CO" ---2.0 g /\MD : 10 ml of each of the major element stocks cited below are added to 800 ml deionized water; I ml of H r. microelement solution ( = minor element solution No. 4 of BBM) is also added, and the total volume is brought to I liter with deion­ized water. The stocks are made by dissolving the indicated amount of each of the following salts in 500 ml deionized water: ?\1gSO,-7H20 7.5 g NaN03 . -· ·-·-· -----50.0 g K"HPO, _50.0g EDTA _ 2.5 g Ca ('.'\O,, 'i 2·-l-H20 0.05 g Fe2(S04)3·6HoO 0.2 g Cv-1 (): 10 ml of each of the stock solutions are added to 800 ml deionized water; I ml of the .-\, microelement solution is also added. The total volume is brought to I liter with deionized water, and the pH is adjusted to 8.0 with IN HCI. The stock solutions are made by dissolving the indicated amount of each of the following salt<; in 500 ml deionized water: :M:gSO4·7H20 12.5 g Na2EDTA 8.25g K2HPO, 2.5 g Fe2 (SO,) 3·6H20 0.2 g Cafl'\03) 2·-l-H20 ...... . ... 1.25 g glycylglycine ·-50.0 g Kl'\O" .. ... ... 50.0g TABLE 1. Sources of Cultures of Oscillatoriaceae No. Isolate Schizothrix calcicola v. vermiformis 2 Schizothrix calcicola \". compacta 3 Schizothrix calcicola v.densa 4 Microcoleus lyngbyaceus 5 Porphyrosiphon notarisii 6 Schizothrix calcicola v. discreta 7 Schizothrix calcicola v. glomerulata 8 Schizothrix calcicola v. diffusa 9 Schizothrix calcicola 10 Schizothrix calcicola v. radiata 11 S chizothrix calcicola v. vermiformis 12 Microcoleus lyngbyaceus v. vermiformis 16 Schizothrix ca!cicola 17 Microcoleus vaginatus v. fuscus 18 Schizothrix calcicola v. vaginata 19 Schizothrix calcicola v. vermiformis 20 Schizothrix calcicola 21* S chizoth rix calcicola 22* Oscillatoria lutea v. auxotrophica 23* Oscillatoria lutea Source Isolator soil, Univ. Texas Baker campus, Austin soil, Takamatsu Groover Plateau, Japan soil, Welsh Bog, Archibald Elkhart Co., Ind. pond, west of Austin, Baker Travis Co., Texas pool, Univ. Texas Baker greenhouse, Austin soil, Brackenridge Baker Field Tract, Austin soil, Brackenridge Baker Field Tract, Austin soil, Univ. Texas Baker campus, Austin Austin Sewage Baker Disposal Pond pond, Biol. Lab. Baker Bldg., UT, Austin Mobile, Alabama Baker soil, Pampa, Texas Baker temporary pond, Baker Brackenridge Field Tract, Austin soil, Univ. Texas Baker greenhouse, Austin sand, Marine Inst., Baker Port Aransas, Texas soil, Austin Texas Baker contaminant in culture Baker of Selaginella pond, west of Austin, Baker Travis Co., Texas Rock Garden Pool, Baker Zilker Park, Austin soil, Brackenridge Baker Field Tract, Austin No. Isolate Source Isolator 24* Oscillatoria lutea growing with Typha Baker v. auxotrophica spp., UT greenhouse, Austin 25* Oscillatoria lutea temporary pond, Baker v. contorta Brackenridge Field Tract, Austin 26* Microcoleus uaginatus contaminant of Van Baalen v. fuscus Marchantia culture 27* Schizothrix calcicola brackish water, east Van Baalen v. minuta coast, N.Y. 30 Schizothrix calcicola soil, roadside, Rt. 77, Baker v. oliuacea LaVaca Co., Texas 32 Schizothrix arenaria soil, roadside, Rt. 77, Baker v. uermif ormis LaVaca Co., Texas 33 Microcoleus uaginatus soil, roadside, Rt. 77, Baker v. araneaf ormis LaVaca Co., Texas 34 Microcoleus uaginatus stream, Landa Park, Baker v. fuscus New Braunfels, Texas 35 Microcoleus irriguus stream, Landa Park, Baker New Braunfels, Texas 38 Oscillatoria lutea soil, near Sacramento, Baker v. scabra California 39 Porphyrosiphon notarisii sand, beach, Monterey, Baker California 40 Porphyrosiphon notarisii roadside ditch, Rt. 16, Baker v. canus west of Sacramento, California 43 Schizothrix calcicola sulfur spring, Baker v. discreta Dripping Springs, Tex. 45 Schizothrix calcicola sulfur spring, Baker v. glabra Dripping Springs, Tex. 46 Schizothrix calcicola sulfur spring, Baker v. fusca Dripping Springs, Tex. K2 S chizothrix calcicola serpentine soil, Kantz v. circinalis near Austin, Texas K6 Schizothrix calcicola soil, between Kings-Kantz ville and Brownsville, Texas Kl7 Kl8* Schizothrix calcicola petri dii.h exposed to Kantz K44* air, Austin, Texas K63 K21 Schizothrix calcicola petri dish exposed to Kantz v. densa air, Austin, Texas No. Isolate Source Isolator K27 M icrocoleus vaginatus petri dish exposed to Kantz cyano-viridis air, Austin, Texas K35 Schizoihrix calcicola unknown Kantz K36 Schizothrix calcicola unknown Kantz v. actinof ormis K38 Schizothrix calcicola petri dish exposed to Kantz v. vaginata air, Austin, Texas K39 Schizothrix calcicola petri dish exposed to Kantz v. densa air, Austin, Texas K43 Schizothrix calcicola petri dish exposed to Kantz v~ nitida air, Austin, Texas K51 Schizothrix calcicola unknown Kantz K52 Schizothrix calcicola soil, between Kings-Kantz ville and Brownsville, Texas K55 A1icrocoleus vaginatus unknown R. Smith v. conzcus K56 Schizothrix calcicola petri dish exposed to Kantz v. vaginata air, Austin, Texas K57* Schizothrix calcicola unknown Kantz v. vermiformis K58 Schizothrix calcicola soil, between Kings-Kantz v. scabella ville and Brownsville, T exas K59 Schizothrix calcicola petri dish exposed to Kantz K60 v. vermiformis air, Austin, Texas K61 S chizothrix calcicola petri dish exposed to Kantz v. actinoformis air, Austin, Texas K62 S chizothrix calcicola petri dish exposed to Kantz v. f uscoviridis air, Austin, Texas K107* Schizothrix calcicola petri dish exposed to Kantz v. spiralis air, Austin, T exas K120 Schizothrix calcicola petri dish exposed to Kantz K123 v. mucosa air, Austin, Texas K182* Schizothrix calcicola soil, Hawaii Kantz v. mucosa I386 Oscillatoria lutea Indiana University Manten v. contorta Culture Collection (IUCC) No. B386 as 0. chalybea Maertens 1390 Oscillatoria lutea IUCC No. LB390 as Pringsheim v. contorta 0. formosa Bory 1426* Schizothrix calcicola IUCC No 426 as Boresch v. glomerulata Phormidium luridum v. olivacea Boresch No. Isolate Source Isolator Ailsie F. Baker and Harold C. Bold 1427* Schizothrix calcicola IUCC No. B427 as De Marz v. glomerulata Phormidium f aveolarum Gomont H28 Microcoleus vaginatus IUCC No. B-1-28 as Manten v. radiatus Oscillatoria tenuis Agardh 1482* Schizothrix calcicola lUCC No. B482 as Dyar v. glomerulata Platonema notatum Schmidle 1485* Schizothrix calcicola IUCC No. 485 as Allen v. amorpha Phormidium sp. 1487* Schizothrix calcicola lUCC Nos. 487, 488 Dyar 1488 v. globerulata as L)'ngbya sp. 1596 Schizothrix calcicola IUCC Nos. B596, 597 Dyar 1597* v. glomerulata Plectonema boryanum Gomont 1598* Schizothrix calcicola lUCC No. 598 as Allen v. glomernlata Plectonema calothri­ coides Gomont 1617 Microcoleus vaginatus lUCC No. B 617 Hughes v. fuscorubens as S~mzploca muscorum 1621* M icrocoleus vaginatus IUCC No. 621 as Lewin Lyngbya sp. 1622 M icrocoleus vaginatus IUCC No. 622 as Lewin v. undulatus Lyngbya sp. 11270 lvficrocoleus vaginatus IUCC No. 1270 Lewin v. fuscus as Orcillatoria brolif era 11306 .tlicrocoleus vaginatus lUCC No. LB1306 as unknown v. funi/or mis 0fcillatoria amoena (Kiitz). Gomont (Gottingen LB 1459-7) 11309 M icrocoleus vaginatus IUCC No. LB1309 as unknown v. funiformis Oscillatoria animalis (Got tin gen LB1459-6 ) 11546 Microcoleus vaginatus IUCC No. 1546 as M. M. Allen v. glaber Lynghya sp. 11547 Microcoleus vaginatus IUCC No. 1547 as Pringsheim v. glaber LJ·ngbva kuetzingii Schmidle * Indicates axenic culture. The Ar. microelement solution is made by dissolving the following amounts of salts in 1 liter deionized water: HaBOa ------·-·· ------············ .2.86 g Mo03 (85%) ·-·-······-·------·-0.Ql 77 g MnC12·7H20 ··-····--···-···------1.81 g CuS04·5H20 --------·-·····--·····0.079 g ZnS0.-7H20 __ __ __ _ __ _______ 0.222 g VSE: 1 ml of each of the salt and vitamin stock solutions is added to 1 liter of natural seawater, which has been filtered through No. 1 filter paper. The final solu­tion is autoclaved at 15 psi for 25 min. The stock solutions are made by dissolving the indicated amount of each salt listed below 200 ml deionized water: NaN03 -----··-····------·······--·--·-8.5 g MnC12·4H20 ---··-··-··--··--------3.9 mg N3.:?HP0.-12H20 __ ___·-·-····--2.15 g Na2EDTA-2H20 ----····----·· . 0.74 g FeS04·7H2 0 .... ···········--·--···55.6 mg The vitamins listed below are dissolved in a single aliquot of 200 ml deionized water: biotin ·--·-· ········-·····--·······---· 0.2 mg B12 soln (1,000 y/ml) -······· 0.2 ml thiamine-RC! -···· ··-······ ·········40.0 ml At the beginning of the investigation two drops (0.1 ml) vitamin B12 solution ( 1000 y/ml solution ) and 10 ml lOOX Eagle's vitamin solution were added rou­tinely to the media. In an attempt to define more precisely the nutrition of the isolates, vitamins were omitted from the media for 3 months. The deterioration of some of the isolates during this time led to including vitamins once again in the media. Cultures were maintained on agar-solidified 3N BBM medium ( 1.5% Difeo Bacto-Agar) under standard conditions of light and temperature ( 150-300 ft c, 12-12 hr light-dark cycle, 22°C). Unialgal cultures from soil samples were generally obtained by plating out ali­quots of suspensions of the soil in sterile water on 3N BBM or BBM-K plates. Parts of the developing plant-masses were transferred to fresh media. Single hormogonia or minute portions of the plant-mass were used to establish stock cultures. For some samples, a slurry of deionized water and the sample was prepared; this was sprayed directly onto a 3N BBM or BBM-K plate from which unialgal plant-masses were subsequently transferred to new media. Freshwater and some marine collections were placed in liquid 3N BBM or BBM-K or on agar plates. Single filaments were isolated as they moved out from the center of the inoculum or were taken directly from the original collection, washed in 5-10 drops of sterile, deionized water, and placed in 3N BBM or BBM-K media. Growth of solitary filaments frequently was increased when washed agar was used; possibly the increased growth was due to removal of peroxides or phenolic compounds (Van Baalen, 1965; Gorham, in Kantz and Bold, 1969). Washed agar is prepared by solidifying 1 liter deionized water with 32 g agar. This is cut into small pieces, about 1-2 cm square, and immersed in deionized water. The water is changed about 10 times over a period of 72 hr. The water is then poured off, and the agar is mixed with 1 liter of double-strength medium and prepared for either plates or tubes (Kantz and Bold, 1969). The advantages of washing agar were equaled by the use of Cg-10 medium, which, as Van Baalen ( 1967) discussses, was designed especially to facilitate growth of single cells of blue-green algae. Perox­ide formation is eliminated and there is no precipitation at pH 8.0. Growth of very short single filaments or trichomes was excellent in the isolates tested. The axenic cultures used were all clonal; not all of the unialgal cultures were, however. Several isolates were received in an axenic state. Other cultures were puri­fied by several methods. The spraying technique of Wiedeman, Walne, and Trainor ( 1964), with some slight modifications, was first used. With this method the ma­terial in an actively growing culture is borken up using a diSONtegrator unit (if agar slants are used, the slant is flooded with sterile water, the plant material scraped off, and then broken up). The material is then placed in a sterile centrifuge tube and centrifuged for a short time at a low speed. The supernatant is poured off, and 5-8 ml sterile water and a drop of Tween 80 are added. The tube is then placed in the diSONtegrator unit or on a test-tube stirrer for 1-2 min. The material is left in the Tween 80 solution for about 20 min; it is stirred or sonicated several times dur­ing this period. The tube is then centrifuged again, the supernatant poured off, and fresh sterile water is added. Sonication or stirring, centrifugation, and addition of fresh water follow. This step is repeated 7-10 times. After the successive washings, the remaining material is aspirated onto an agar plate. The plate is incubated under standard conditions for 3 days to 1 week; at the end of this period any filaments appearing uncontaminated are transferred to new media. The difficulty in break­ing up the plant-mass with the diSONtegrator unit is the main drawback to this method. Some axenic cultures were obtained by isolating single hormogonia that moved rapidly away from the plant-mass. Repeated isolations were made. This method was also used after apparently clean cultures had been obtained by spraying to insure clonal isolates. The most successful method for obtaining axenic cultures was one developed by Dr. Chase Van Baalen1 and used in his laboratory. This combines sonication and ultra-violet irradiation. Filaments from an actively growing culture are placed in distilled water and broken up with a sonicator (Branson 100 unit) to the point where most fragments are 1, 2, or 3 cells in length. The cell-liquid suspension is then placed on a magnetic stirrer in an open petri dish under an ultra-violet light source for 5 min ( 19 in from a GE 15T8 rod) . At the end of this time small por­tions of the suspension are drawn into capillary tubes and pipetted into melted 1 D<'pt. of Botany, University of Texas at Austin and Marine Science Institute, Port Aransas, Texas. agar, and pour-plates are made. The plates are immediately returned to light (non­fluorescent) and allowed to incubate for 48-72 hr. At that time axenic filaments can be selected with a dissecting microscope and transferred to new media. Cultures were checked for bacterial and fungal contaminants over a period of 2-3 weeks, on a number of different media, including: Nutrient broth (Difeo); Nutrient agar (Difeo); Protease peptone agar ( 1 g/ liter 3N BBM or BBM-K and 15 g agar) ; Yeast extract agar ( 5 g/ liter pecimens, Drouet retained six genera. These genera were characterized by cytological differences­primarily arrangement of the granules in the cells and the morphology of the terminal cell. The morphology of sheath and trichome were considered to be too variable to be reliable taxonomically. Each of the 23 species recognized by Drouet has very broad morphological limits. During the course of the present investigation, numerous specimens have been collected, many of which were established in unialgal and/or axenic cultures (Table l). Observations over a 2-year period have repeatedly shown that while the Gomontian system of classification may have included unreliable character­istics, that of Drouet seemingly does also. Perhaps the most variable characteristic in culture was that of granulation. The larger (in trichome width) isolates all contained distinct granules in the cells, but the number and arrangement varied considerably in the cells of individual tri­chomes. Since the presence or absence of dark granules along the crosswalls is a prime generic distinction in Drouet's system, difficulties in identification were greatly compounded by these variations. Drouet, recognizing that some variation in granulation does occur, suggests staining with I2KI followed by ZnCl2 as a method of clearly demonstrating granules that were not already visible. Identifications were made on the assumption that granules were characteristic if they had ever been observed in an isolate. The morphology of the terminal cell is more constant in culture than the granu­lations along crosswalls (the granules represent stored metabolites and thus varia­tion in them is not surprising). Fluctuations in terminal cells did occur, but usually several observations of a culture at different ages revealed the pattern of develop­ment from rounded (newly formed) to the capitate, conical, etc. type characteristic of a given taxon. There are several isolates in which the thickened outer wall of the terminal cell was never observed or in which it was only very rarely observed; in spite of this, these isolates were assigned to the genus Microcoleus upon the recom­mendation of Dr. Drouet. In culture, the sheath was in our experience as reliable and constant as the morphology of the terminal cell and considerably more reliable than the presence and disposition of granules. The isolates herein described included three types, with respect to sheaths: ( 1) those in which some sheaths were always seen, no matter how young the culture; these algae would thus be considered as Lyngbya sensu Gomont; (2) those in which sheaths were never seen, no matter how old the culture ( Oscillatoria sensu Gomont) ; and (3) those in which sheaths appeared with age. The consistency of the sheath was also constant in most isolates. The form of the plant-mass on agar also proved to be quite constant and ac­cordingly taxonomically significant. Several different types of plant-mass were consistently observed in some instances for a single morphological type of trichome. That these plant-mass types are truly indicative of other differences seems to be borne out by the rudimentary physiological analysis herein conducted. A given plant-mass configuration could not, however, be correlated with a given species or genus as these are currently delimited by Drouet ( 1968) or as they were by Gomont ( 1892). Plant-mass type is related to motility and to other, in part undetermined, attributes. It is affected by light, pH, and mineral nutrition, but it is always, apparently, constant under a constant set of conditions. Plant-mass form is used in the descriptions of the isolates given in this dissertation as an infraspecific at­ tribute. Since the nomenclature follows that of Drouet, the sheath type is also herein considered an infraspecific characteristic. Descriptions of Certain Isolates of the Oscillatoriaceae in Culture The descriptions of the isolates used in this investigation are based on observa­tions of 2-to 4-week-old cultures grown on agar-solidified 3N BBM medium. As is discussed in the section on comparative studies with several media, there are constant differences in plant-mas.5 form, cell structure, and trichome structure in different liquid or solid media. The need for standard growth conditions as a basis for descriptions is thus obvious. The attempt was made to identify the isolates with previously described species a5 summarized by Drouet ( 1968). For each isolate or group of isolates the generic and specific names listed are taken from Drouet's publication. The varietal names were appended during the course of this investigation to recognize certain isolates with constantly occurring attributes. The brief discus.5ion of the genera that follows is based on Drouet's descriptions; his complete description of each species represented in these isolates is also given. The additional descriptions of the isolates of each species are based on the differ­ences observed in culture during this investigation. Keys to the varieties of each species are given following the descriptions. The genera of Oscillatoriaceae recognized in Drouet's revision of the family are Spirulina, Schizothrix, Oscillatoria, Porphyrosiphon, Microcoleus, and Arthro­spira. Spirulina is unicellular, or at least crosswalls can not be distinguished. The other five genera have been separated by Drouet on the basis of terminal cell form and type of granulation in the cells. Schizothrix is not granulated along the cross­walls, the outer wall of the terminal cell is never thickened, and only the terminal cell, if any, is attenuated. If the ends of the trichomes do taper, and the other char­acteristics remain as in Srhizothrix, the genus is Porphyrosiphon. Oscillatoria in­cludes those organisms without granules along the crosswalls that have terminal cells with thickened outer walls. Two additional genera are distinguished by the presence of many granules along each side of the crosswall; of these, Arthrospira includes those organisms in which the terminal cell has a thin outer wall and 1!Jicro­coleus those in which the outer wall is thickened. Species of Schizothrix, Microcoleus, Porphyrosiphon, and Oscillatoria were studied during this im·estigation. Schi::.othrix calciwla (Agardh) Gomont The most widely encountered taxon in this work was the unwieldy complex designated by Drouet as Schizothrix calcicola. Isolates with the cell structure de­scribed for this species were obtained from soils, freshwater ponds, marine and brackish en\'ironments, and from the air. The species is distinguished from others of the genus by Drouet by its small cellular diameter and its rounded terminal cell. The 52 isolates studied in culture during this in\'estigation, which, according to Drouet's system are Schizothrix calcicola, are described as 21 different taxa in this publication on the basis of morphological and physiological studies. The type of the plant-mas.s is the ~ingle most obvious attribute which varies consistently among the isolates, but there is also variation in cell shape, presence or absence of a sheath, and type of sheath, if present. For purposes of this investigation the most commonly encountered of the various isolates of S. calcicola is considered to represent the species; others are listed as varieties. The distinctness of the varieties (both in S. calcicola and other species) is often much more apparent from macro-or micro­scopic observation, or from photographs, than from the written descriptions. Whether or not more widespread collection and subsequent culturing would always yield this form as the most common remains to be seen. Drouet ( 1968) described Schizothrix calcicola as follows: Trichomes blue-green, yellow-green, yellow, olive, brown, red, violet, or gray-green, cylindrical or torulose or somewhat constricted at the cross walls, 0.2-3.5 µ. (4.5 µ.) 1 in diameter, here and there and in part increasing or decreasing in diameter, straight or curving or spiraled, capable of determinate or indeterminate growth in length, breaking by means of the destruction of an intercalary cell or by constriction at a cross wall. Cells quadrate or shorter, sometimes longer, than broad, 0.2-6 µ. long, the pro­toplasm homogeneous or granulose, often pseudovacuolate, often with one or two granules at either side of a cross wall. Terminal cell at first cylindrical, becoming bulbose, often enlarged, the outer membrane not thickened, at first quasitruncate, then becoming rotund or rarely exccntrically swollen. Sheath material hyaline, sometimes developing yellow, brovro, blue, violet, or red pigments, often turning blue in chlor­zinc-iodide. Plant consisting of long or short naked trichomes, or of trichomes in a homogeneous or laminose mucus, or of solitary or few or many trichomes within a more or less discrete cylindrical, often branched, sheath. Descriptions of the isolates representing this species are given below. Schizothrix calcicola (Agardh) Gomont (Figs. 2-4) . Isolates 9, 16, 21, K6, Kl 7, Kl8, K35, K44, K51, K52, K63 The plant-mass is rough-vermiform macroscopically (Fig. 2) . At 14X, curved, smooth bundles (Fig. 3) of filaments are easily distinguished at the edges, extend­ing outward from the central, densely vermiform mat. The color of the plant-mass is deep yellowish-green ( 132) at 2 weeks, becoming some shade of olive-green (125, 126) at4weeks. The cells (Fig. 4) are 1.5-3.0 µ.wide and 1.5-3.0 µ.long. The terminal cell is rounded. The trichomes are constricted at the crosswalls, but the constrictions are often not apparent in the filamentous condition. The cell contents may be homoge­neous, or the central portion may appear as a bright translucent spot, or (as cul­tures age) several distinct granules may appear in the protoplast. The sheaths are colorless and narrow (to 0.5 µ.wide). Isolates 1, 8, 10, 11, 19, K2, K57, K59, and K60 are indistinguishable from the above-described isolates when observed microscopically, but they are charac­terized by different plant-mass types. There are slight, but ever-present, physio­ 1 Parts of the description enclosed in parentheses indicate additions to it. Isolate 30 was identified by Dr. Drouet as S. calcicola; the cells, however, are wider than 3.5 µ.. Several other isolates also at times were wider than 3.5 µ.. logical differences in K57 as compared to K44 and 21, which are the only isolates of these groups that have been thus investigated. These isolates and their variations from S. calcicola are summarized in the several varieties proposed below. Schizothrix calcicola (Agardh) Gomont v. vermiformis var. nov. Fig. 5. Isolates 1, 11, 19, K57, K59, K 60 Varietas a specie typica differens eo qttod magnificatione 14X pars centralis massae plantarum aspera apparel. The plant-mass is very similar to that of isolates listed as belonging to S. calcicola, but a separation seems to be tenable as there are certain small, but consistent, dif­ferences. The plant-mass is macroscopically a dense vermiform mat with rough, cur\'ing bundles of filaments visible at the edges (Fig. 5). At 14X, the central part of the plant-mass appears rough. The color of the plant-mass is deep yellow-green ( 132) at 2 weeks and dark olive-green ( 126) at 4 weeks. The change in color from yellowish-green to reddish-orange ( 51 ) , which is the ultimate color of both groups, is much slower than in the first group of isolates. Schizothrix calcicola (Agardh) Gomont v. diffusa var. nov. Figs. 6-7. Isolate 8 Varietas a specie typica differens eo quod massa plantarum sine microscopo observata diffusa et sine proprietalP. The plant-mass is macroscopically nondescript. At 14X, it appears rough and dense in the center (Fig. 7) with very thin bundles of filaments at the edge (Fig. 6) . Growth on agar is usually more diffuse than in the two previously discussed taxa. The color of the plant-mass is deep yellow-green ( 118) at 2 weeks and deep yellowish-green ( 138) at 4 weeks. Schi:.othrix calcicola (Agardh) Gomont v. circinalis var. nov. Figs. 8-9. Isolate K 2 V arietas a specie typica diff erens eo quod massa plantarum sine microscopo observata tegetem asper am format; singula trichomata anfractus arctos e lficientia. The plant-mass is macroscopically a rough mat. At 14X, it appears as a mounded sheet of filaments except at the extreme edge where rough ropes of filaments may be seen (Fig. 8). The color of the plant-mass is deep yellow-green ( 118) at 2 weeks, and medium olive-green ( 125) at 4 weeks. Single trichomes from time to time form tight coils on the agar (Fig. 9). Granules are rarely observed in the cells. Schizothrix calcicola (Agardh) Gomont v. radiata var. nov. Figs. 10-11. Isolate 10 V arietas a specie typica differens eo quod massa plantarum sine microscopo observata radiata, fasciculis filamentorum et ad centrum et ad marginem arcte tzppressis. The plant-mass is macroscopically radially arranged, with the bundles of fila­ments tightly appressed both at the center and at the edge (Fig. 10). At 14X, the ropes of filaments are rough, frequently confluent with one another, and cen­tripetally oriented instead of curving in all directions (Fig. 11). The color of the plant-mass is very dark yellowish-green ( 138) at 2 weeks and dark olive-green ( 126) at 4 weeks. The remaining isolates falling into the Schizothrix calcicola group vary from the "type" of the species not only in plant-mass but also have at least one other consistent difference. A large group of isolates obtained from the Indiana Uni­versity Culture Collection belong in this caterogry. These isolates ( 1426, 1427, 1482, 1485, 1487, !488, I596, I597, I598) were identified originally (Starr, 1964) as members of three different genera (Plectonema, Lyngbya, and Phormidium). It would be interesting to have descriptions of these isolates when they were first taken into culture to see if there were differences then which in a common environ­ment are no longer expressed. The possibility of a "laboratory species" was seriously considered until isolate 7 was studied; it has the same plant-mass characteristics and cell structure and has exhibited them in culture continuously from the time of its isolation. Two plant-mass types occur among these algae (I485 is one type, the rest are the other) from the Culture Collection of Algae at Indiana University. The cell morphology is the same in all except for minor and somewhat variable differences. The maximum width of the trichomes in I598 is always narrower than that in the other isolates, but the width falls into the lower limits of the range of the rest. Sheaths are not of general occurrence; they are present to a limited extent in 1485, I597, and !426. The general descriptions of this group of isolates are given below under S. calcicola v. glomerulata and v. amorpha. Schizothrix calcicola (Agardh) Gomont v. glomerulata var. nov. Figs. 12-15. Isolates 7, 1426, 1427, 1482, !487, 1488, I596, I597, I598 V arietas a specie typica differens eo quod mass plantarum sine microscopo observata in area centrali densa, fasciculis trichomatum arcuatis ad marginem; magnificatione 14 X observata, pars central is minute glomerulata apparel; cellulae interdum dolioformes; culturae post 2-3 hebdomades omnino e hormogoniis plerumque compositae. The plant-mass macroscopically has a dense central area with curving bundles of trichomes at the edges (Fig. 12). At 14X, the center appears minutely glomeru­late (Fig. 13) ; the bundles of trichomes appear zigzagged and fade into single trichomes, which may be very intricately curved, at the margins (Fig. 14). The color of the plant-mass is deep, to very dark, yellowish-green ( 132/138) at 2 weeks and dark olive-green ( 126) at 4 weeks. The cells (Fig. 15) are 1.5-4.0 µ. ( 1.5-2.5 µ.in I598) wide and 1.0-4.0 µ.long. There are very great variations in width of cells in the same trichome and of tri­chomes in the same culture. The terminal cell is rounded. The trichomes are con­stricted at the crosswalls; constrictions may be slight or pronounced. At times the cells are slightly barrel-shaped. The cell contents may have one central translucent zone or several translucent granules ( 3-4-week-old cultures and older.) The cul­tures are generally in a hormogonial state after 2-3 weeks (except for 7, 1427 and I598). Sheaths occur in older cultures of 1426 and I597 and are colorless, narrow, and soft. Schizothrix calcicola (Agardh) Gomont v. amorpha var. nov. Figs. 16-17. Isolate 1485 V arietas a specie typica difjerens eo quod massa plantarum sine microscopo observata sine proprietate; vaginae rare in culturis vel in periodo immobili, sporadice, autem, apparentes. The plant-mass is macroscopically nondescript. At 14X, it appears rough and dense in the center and has rough bundles of filaments at the edges which fade into single filaments (Figs. 16-17 ). The color of the plant-mass is deep yellowish­green ( 132) at 2 weeks and medium olive-green ( 125) at 4 weeks. Sheaths occur only on some individuals in cultures 3 to 4 weeks old and older. The following isolates are distinctive either in plant-mass type or cell morphol­ogy or both. Schizothrix calcicola (Agardh) Comont v. vaginata var. nov. Figs. 18-19. Isolates 18, K38, K56 Varietas a specie typica dilferens eo quod massa plantarum sine microscopo observata sine proprietate; filament a vaginas solidissimas persistent es ( usque ad 0.8 µ.lat.) habentia. The plant-mass is macroscopically nondescript. At l 4X, it is rough; thin bundles of filaments are seen at the edges (Fig. 18). The color of the plant-mass is deep yellowish-green ( 132) at 2 weeks and very dark yellowish-green ( 138) at 4 weeks. The cells (Fig. 19) are 2.0-3.5 µ.wide and 1.2-2.5 µ.long. The terminal cell is rounded. The trichomes are constricted at the crosswalls. The cells have one to several dark or translucent granules. Gas vacuoles have been observed in isolate K56 in very small numbers. The sheaths are very firm, up to 0.8 µ. wide, and colorless. Schizothrix calcicola (Agardh) Gomont v. olivacea var. nov. Figs. 20-21. Isolate 30 Varietas a specie typica difjerens eo quod massa plantarum sine microscopo observata sine proprietate; cellulae alquantulo minores ( 1.2-3.0 µ.lat., 1.0-2.2 µ. long.) quam in planta typica; trichomata saepe in spiram contorta aut undulata. The plant-mass is macroscopically nondescript and has feathery, thin bundles of filaments radiating from the central mat (Fig. 20). At 14X, the individual filaments may be seen growing among the bundles of filaments. The color of the plant-mass is dark olive-green ( 126) at 2 and 4 weeks. The cells (Fig. 21) are 3.7-4.5 µ.wide and 1.5-3.0 µ.long. The terminal cell is rounded. The trichomes are constricted at the crosswalls, but the constrictions are not apparent in the filamentous condition. The cells may contain several dark and translucent granules. The trichomes are frequently undulate. The sheaths are firm, 0.5-1 .0 µ. wide, and colorless to pale yellow-brown. Schizothrix calcicola (Agardh) Gomont v. minuta var. nov. Figs. 22-24. Isolate 27 V arietas a specie typica differens eo quod massa plantarum sine microscopo observata sine proprietate; magnificatione 14 X observata ut massa fasciculorum tenuium asperorumque filamentorum visa. Massa plantarum profunde flavovirens 117 ( 118) pos post duas quattuorque hebdomades. The plant-mass is macroscopically nondescript. At 14X, it appears as a mass of thin, rough bundles of filaments (Figs. 22-23). The color of the plant-mass is deep yellow-green ( 117I 118 ) at 2 and 4 weeks. The cells (Fig. 24) are 1.2-2.0 µ.wide and 1.0-2.2 µ.long. The terminal cell is rounded. The trichomes are slightly constricted at the crosswalls. The cells fre­quently contain several translucent and dark granules. Some of the trichomes may be spiraled or undulate. The sheaths are narrow and colorless. Schizothrix calcicola (Agardh) Gomont v. actiniformis var. nov. Figs. 25-26. Isolate K36, K61 Varietas a specie typica differens eo quod massa plantarum sine microscopo observata radiata; cellulae 2.5-4.5 µ.lat., 1.0-3.0 µ.long.; trichomata per 3.6 cellu­las ultra vaginam typice extensa; vagina solida ( usque ad 0.5 µ.lat.), sine colore ad dilute fiavam. The plant-mass is macroscopically radial with ropes of filaments swirling close to the center of the plant-mass (Fig. 25 ). The color of the plant-mass is deep yel­lowish-green ( 132 ) at 2 weeks and dark olive-green ( 126) at 4 weeks. The cells (Fig. 26) are 2.5-4.5 µ.wide and 1.0-3.0 µ.long. The terminal cell is rounded. The trichomes are constricted at the crosswalls; constrictions are easily seen in the trichomes which characteristically extend for 3-6 cells beyond the sheath. The cell contents are granular. False branches may occur sparsely as the culture ages. The sheaths are firm, distinct (up to 0.5 µ. wide ) , and colorless to pale yellow. Schizothrix calcicola (Agardh) Gomont v. fuscoviridis var. nov. Figs. 27-28. Isolate K62 Varietas a specie typica differens eo quod massa plantarum sine microscopo observata radiata, usque ad duas menses semper atro-virides; cellulae 2.5-4.0 µ. lat., 1.5-2.5 µ.long., vaginae nullae. The plant-mass is macroscopically radial. At 14X, straight bundles of trichomes are seen to radiate from a dense, rough central mass (Fig. 27). The color of the plant-mass is dark olive-green ( 126) at 2 and 4 weeks. The cells (Fig. 28) are 2.5-4.0 µwide and 1.5-2.5 µlong. The terminal cell is rounded. The trichomes are constricted at the crosswalls. The cell contents may be granular or the central portion of the cell may appear as a translucent area. Sheaths have not been observed. Schizothrix calcicola (Agardh) Gomont v. spiralis var. nov. Figs. 29-30. Isolate K 107 V arietar a specie typica differens eo quad massa plantarum sine microscopo observata radiata; trichomata vaginis distinctis ( usque ad 0.8 µlat. ) praedita; trichomata cum aut sine vagina, per tantam longitudinem in spiram perspicue contorta. The plant-mass is macroscopically radially arranged with very short bundles of filaments extending out from the central mat (Fig. 29). The color of the plant­mass is very dark yellowish-green ( 138 ) at 2 and 4 weeks. The cells (Fig. 30) are 2.0-3.0 µwide and 1.0-2.5 µlong. The terminal cell is rounded. The trichomes may be very slightly constricted at the crosswalls. The cell content<; may include a translucent zone or several translucent granules. The sheaths are 0.2-0.8 µwide, firm, and colorles.c;. The filaments are distinctly spiraled along their entire length. Schizothrix calcicola (Agardh) Gomont v. densa var. nov. Figs. 31-32. Isolates 3, K2 l , K39 Varietar a specie typica di!Jerens eo quad massa plantarum sine microscopo observata compacta, non patens; magnificatione 14X observata aspectu aspera; cellulae saepe piluliformes; vagina solida (usque ad 1.0 µlat. ), sine colore. The plant-mass is macroscopically compact. At 14X, it is rough (Fig. 31 ) . The color of the plant-mass is very dark yellowish-green ( 138) at 2 and 4 weeks. The cells are 2.0-4.0 µwide and 1.0-2.5 µlong (Fig. 32). The terminal cell is rounded. The trichomes are constricted at the cros.c;walls and may be bead-like in appearance. In very old cultures some false branches occur. The sheaths are up to 1.0 µwide, colorless, and firm. Schizothrix calcicola (Agardh) Gomont v. scabella var. nov. Figs. 33-34. Isolate K58 Varietas a specie typica di!Jerens eo quad massa plantarum sine microscopo observata compacta, non patens; magnificatione 14X observ'lta, penicilli erecti e superficie emergentes. The plant-mass is macroscopically compact. At l4X, it appears as a dense, rough mat with tufts of filaments on the surf ace (Fig. 33). The color of the plant-mass is very dark olive-green ( 128) at 2 and 4 weeks. The cells (Fig. 34) are 2.5-3.0 µ.wide and 1.5-2.5 p. long. The terminal cell is rounded. The trichomes are frequently slightly constricted; this is usually not ap­parent in the filamentous condition. The cells contain several granules; occasionally one or two dark granules are seen on either side of the crosswalls. The sheaths are firm, colorless and 0.2-0.5 µ.wide. Schizothrix calcicola (Agardh) Gomont v. compacta var. nov. Figs. 35-36. Isolate 2 V arietas a specie typica differens eo qttod massa plantarum sine microscopo observata compacta, non patens; magnification 14X observata minute glomulata; cellulae 2.5-3.5 µ.lat., 1.5-4.0 µ.long.; culturae vetustiones (4-8 hebdomadum) omnino e hornzongoniis brevibus saepe consistantes; vaginae nullae. The plant-mass is macroscopically compact. At 14X, it appears minutely glom­erulate except at the extreme edges where some intricately curved and coiled bundles of trichomes are seen (Fig. 35). The color of the plant-mass is very dark yellowish-green ( 138) at 2 and 4 weeks. The cells (Fig. 36) are 2.5-3.5 µ.wide and 1.5-4.0 µ.long. The terminal cell is rounded. The trichomes are slightly constricted; the cells may be barrel-shaped. Older cultures ( 4-8 weeks) frequently consist entirely of short hormogonia. The central area of the cell frequently appears as a translucent zone. Sheaths have not been observed. Schizothrix calcicola (Agardh) Gomont v. nitida var. nov. Figs. 37-38. Isolate K43 V arietas a specie typica differens eo quod massa plantarum sine microscopo observata compacta, non patens, atque butyracea; magnificatione 14X observata .• minute glomerulata. The plant-mass is macroscopically compact. At 14X, it is a dense, undulate to minutely glomerulate mass (Fig. 3 7). The color of the plant-mass is dark olive­green ( 126) at 2 weeks, very dark olive-green ( 128) at 4 weeks. The cells (Fig. 38) are 1.5-2.0 µ. wide and 1.5-2.5 µ. long. The terminal cell is rounded. The trichomes are constricted at the crosswalls. The cell contents may be granular. Sheaths have not been observed, but the butyrous consistency of the plant-mass would seem to indicate the presence of a colloidal material. Schizothrix calcicola (Agardh) Gomont v. mucosa var. nov. Figs. 39-40. Isolate K120, K123, Kl82 Varietas a specie typica differens eo qttod trichomata in materia communi col­loidali dispersa, vaginis singulis distinctis raris. The plant-mass is vermiform macroscopically with many curved bundles of tri­chomes (Fig. 39). At 14X, among the bundles, growing both on and into the agar, solitary trichomes occur. The color of the plant-mas.5 is very dark yellowish-green ( 138) at 2 weeks and deep yellowish-green ( 118) at 4 weeks. The trichomes are positively phototactic. The cells (Fig. 40) are 2.0-4.0 µ. wide and 1.5-4.0 µ. long. The terminal cell is rounded. The trichomes are constricted at the crosswalls; the constrictions may be so great as to give a bead-like appearance to the cells. The cells may have several translucent granules. Distinct sheaths are of very rare occurrence, but diffuse col­loidal material may usually be demonstrated with methylene blue stain. In isolate K 182 sheath material is better defined; in cultures 3-4 weeks old staining with methylene blue or India ink frequently revealed wide sheaths with several tri­chomes scattered through them. Schizothrix calcicola (Agardh) Gomont v. glabra var. nov. Figs. 41--42. Isolate 45 Varietas a specie typica differens eo quod massa plantarum sine microscopo observata tegetem dens am levemque format; cellulae 2 .0-2.5 µ. lat., 2 .5-4 .5 µ. long.; unica granula translucens in utraque cellulae extremitate saepe repert a; vaginae distinctae non apparent, multa materia colloidalis dilfusa, autem, adest. The plant-mas.5 is macroscopically a smooth, dence mat, which shows bundles of trichomes in several layers in the center and single trichomes at the edges (Fig. 41 ) . At l 4X, the trichomes and bundles at the margin appear rough. The color of the plant-mass is deep yellowish-green ( 132) at 2 weeks and deep yellow-green ( 118) at 4 weeks. The cells (Fig. 42) are 2.0-2.5 µ. wide and 2.5--4.5 /" long (usually 3.5-4.0 µ. long). The terminal cell is rounded. The cells frequently contain one translucent granule at either end near the crosswalls; there may also be several granules scat­tered throughout the cell. The crosswalls are often hard to distinguish. The tri­chomes are not constricted at the crosswalls. Distinct sheaths have not been ob­served, but diffuse colloidal material may usually be demonstrated with methylene blue stain. The trichomes adhere very firmly to one another, and a large sheet-like portion of the plant-mas.sis easily removed from an agar surface. Schizothrix calcicola (Agardh) Gomont v. fusca var. nov. Figs. 43-45. Isolate 46 Varietas a specie typica dilferens eo quod massa plantarum sine microscopo observata in centro confertissima brevisque; ad marginem fasciculos filamentorum arcuatas praebens; brunnea. The plant-mass is macroscopically very dense and smooth in the center with many curving bundles of filaments at the edge (Fig. 43). At 14X, the bundles of trichomes are smooth (Fig. 44). The color of the plant-mas.5 is dark olive-brown (96) at 2 and 4 weeks. The cells (Fig. 45) are 2.0-2.5 µ. wide and 1.5-2.5 .u long. The terminal cell is rounded to slightly conical. The cells contain no distinct granules. The trichomes are slightly constricted at the crosswalls; the crosswalls appear very thick. Sheaths are not ubiquitous, but they are narrow ( 0.3 µ. wide) and colorless when they are seen. Schizothrix calcicola (Agardh) Gomont v. discreta var. nov. Figs. 46-48. Isolates 6, 43 Varietas a specie typica dif!erens eo quod massa plantarum sine microscopo observata massas rotundatas discretas ad margtnem massae centralis confiuentis praebet; trichomata ad dissepimenta valde constrict a; vaginae nullae. The plant-mass macroscopically shows discrete, round masses at the margin; the central area is a confluent mass of indeterminate shape (Figs. ·1-6-4 7). At 14X, a few trichomes are seen growing between the central and exterior portions. The color of the plant-mass is deep yellowish-green ( 132) at 2 weeks and medium olive-green ( 125) at 4 weeks. The isolate exhibits negative photoaxis. The cells (Fig. 48) are 1.8-2.5 µ. wide and 1.5-3.5 µ. long. The terminal cell is slightly rounded; several dark granules usually occur in an otherwise clear area in the outer end of the terminal cell. The trichomes are distinctly constricted at the crosswalls. There is a dark granule at each end of the cell usually; chromatoplasm appears to be located mainly at the periphery of the cell. Sheaths have not been observed. Schizot hrix arenaria (Berkeley) Gomont Schizothrix arenaria is distingui5hed from S. calcicola by the elongate, conical, terminal cell in contrast to a rounded one no longer than the others of the trichome. The species was described by Drouet ( 1968) as follows: Trichomes blue-green, yellow-green, olive, brown, red, violet, or gray-green, pre­dominantly constricted at the crosswalls, often torulose-cylindrical, 1-6 µ. in diameter, here and there and in part increasing or decreasing in diameter, straight or curving or spiraled, capable of indeterminate growth in length, breaking by means of the de­struction of an intercalary cell or by separ:"ltion of adjacent cells, becoming abruptly conical at the tips. Cells quadrate or longer than broad, 2-10 /L long, the protoplasm homogeneous or granulose, rarely pseudovacuolate, with rarely a single granule de­veloping on either side of a cross wall. Terminal cells becoming acutely or obtusely conical, rarely more or less cylindric-conical, the outer wall not becoming thickened. Sheath material hyaline or in part becoming yellow or brown, rarely red, violet, or blue, not at all or only here and there turning blue in chlor-zinc-iodide. Plant consist­ing of long or short naked trichomes, or of trichomes in a homogeneous or laminose mucus, or of one, few, or many trichomes in a more or less discrete cylindrical, often sheath. Descriptions of the two isolates of this species studied in culture are given below. Schizothrix arenaria (Berkeley) Gomont (Figs. 49-51.) Isolate 20 The plant-mas.5 is macroscopically distinguishable by the presence of small dark dots scattered over the surface (Fig. 49). At 14X, these dots are seen to be tightly coiled ropes of trichomes. The ropes of trichomes are elsewhere arranged in an undulate manner (Fig. 50). The color of the plant-mass is medium olive-green (125) at 2 weeks and dark olive-green ( 126) at 4 weeks. The cells (Fig. 51) are 1.5-2.0 IL wide and 2.5-6.5 IL long. The terminal cell is long, tapered, and may bend back and forth. The trichomes are constricted at the crosswalls. The cell contents include one dark granule (very rarely two) at each end of the cell next to the crosswall. Sheaths are of very infrequent occurrence and are narrow, soft, and colorless. Schizothrix arenaria (Berkeley) Gomont v. vermifonnis var. nov. Figs. 52-53. Isolate 32 V arietas a specie typica diff erens eo quod massa plantarum sine microscopo observata vermiformis. The plant-mass is microscopically vermiform with curved and coiled bundles of filaments. At 14X, the bundles are seen to be smooth to confluent (Fig. 52). The color of the plant-mass is deep yellow-green ( 118) at 2 and 4 weeks. The cells (Fig. 53) are 1.5-2.5 IL wide and 3.5-6.0 IL long. TI1e terminal cell is long and slightly conical. The trichomes are slightly constricted at the crosswalls. One large dark granule occurs at either end of the cell; in very long cells one or two granulec; may be seen in the center of the cell-perhaps an indication of imminent cell division. Sheaths are narrow and colorless. A key to above-characterized isolates of Schizothrix follows. A KEY TO SPECIES OF SCHIZOTHRIX IN CULTURE1.2 1. Tenninal cell hemispherical to almost spherical _ __ __ _______ __ __ S. calcicola. 1. Terminal cell blunt-to acute-conical S . arenaria. KEY TO THE VARIETIES 01' SCHIZOTHRIX ARENARIA 1. Plant-mass showing macroscopically small dark dots (tightly coiled tri­chomes) scattered over the surface S. arenaria. 1. Plant-mass macroscopically without any small dark dots scattered over the surface. _ _____ S. arenaria v. vermiformis. KEY TO THE VARIETIES OF SCHIZOTHRJX CALCICOLA l. Plant-mass vermiform 2 1. Plant-mass otherwise __ _ ___ _ 4 2. Plant-mass appearing rough or minutely glomerulate in the center ex­cept when young (vermiform then), becoming distinctly vermiform at the margin ___ _ S . calcicola v. vermiformis. 2. Plant-mass vermiform over the entire surface at all times _ 3 1 The keys to the species are taken from Drouet ( 1968). 2 The keys to the varieties are based on 2 to 4-week-old cultures grown on 3N BBM agar under standard conditions. 3. Trichomes with narrow, but firm, sheaths___ _ ____ ______ __ ---------------------S . calcicola. 3. Trichomes occurring in a mass of colloidal material, sometimes several tri­chomes loosely arranged in a sheath ___ __ S. calcicola v. mucosa. 4. Plant-mass minutely glomerulate --------------------· 5 4. Plant-mass otherwise ___ __ _____ _____ 6 5. Plant-mass distinctly minutely glomerulate in the center; minutely glom­erulate and "zigzagging" bundles of trichomes at the margin _ _ S. calcicola v. glomerulata. 5. Plant-mass minutely glomerulate and compact ___ _____ __S. calcicola v. compacta. 6. Plant-mass compact; rough or smooth__ ______________ ---------------------7 6. Plant-mass otherwise ____ __ _ __ ----------------__________________ ____ _________ 10 7. Plant-mass brownish-black---------·-_______________ ________S. calcicola v. scabella. 7. Plant-mass green ______ _____ _____ ___ ________ _ 8 8. Plant-mass smooth ____ ___ __ _______ __ ___________--------------------------------S. calcicola v. nitida. 8. Plant-mass rough _____________ ______ __ ___ ____ __________ _ . __ ___ _____________ 9 9. Plant-mass very compact and rough; filaments straight or (if older) irregu­larly curved with very firm sheaths _____ ____ ______ ___ ___ ____________ S. calcicola v. densa. 9. Plant-mass an extended, rough, dense mat; filaments straight and at trmes tightly coiled with thin sheaths ____ ____ ____ _________ ___ ____ _______________ S. calcicola v. circinalis. 10. Plant-mass distinctly radially arranged--------------------__________ --------------------11 10. Plant-mass otherwise ____ _____ _ 14 11. Trichomes noticeably spiraled along their entire length _ __ S. calcicola v. spiralis. 11. Trichomes not spiraled, but straight __ ___ ________ ____ __ ________ _ ____ _____ ______ 12 12. Plant-mass with long, tightly appressed bundles of trichomes radiating from the inoculum _ _____ _____________ ___________________ S . calcicola v. radiata. 12. Plant-mass with central compact mass and short bundles of trichomes radiating from it. _ _____________ ________ __ ___ __ __ __ __ ___________ ___ ________ ____ ____ ___ 13 13. Trichomes with firm, distinct sheaths ______ _____ __ _________ S . calcicola v. actinoformis. 13. Trichomes without sheaths_ _______ _________ S. calcicola v. fuscoviridis. 14. Plant-mass dense and smooth in center with bundles of filaments visi­ble at the margin ____ __________ -----------------_ ________________ 15 14. Plant-mass otherwise ___ ___ ·----·-___ --------------------------------16 15. Plant-mass green ---·-----·-----_ ____ S. calcicola v. glabra. 15. Plant-mass brown ________ ____ _______ __ ____ __ __ -----------------__ _____ S. calcicola v. fusca. 16. Plant-mass with distinct globular masses at the margin -----------------------­ S. calcicola v. discreta. 16. Plant-mass otherwise-nondescript-not included in any of the above descriptions __ ___ _ __ __ ____ __ _____ _____ __ 17 17. Sheaths occurring only rarely and then in older cultures __ S. calcicola v. amorpha. 17. Sheaths occurring regularly and abundantly _____________ ___ ____ ___ _____ ____________ 18 18. Trichomes (filaments) spiraled or undulate in part ___ __ S. calcicola v. minuta. 18. Trichomes (filaments) straight _____ ___ _______________ __ _____ __ _____ __ __ __ 19 19. Sheaths thin, frequently seen only where empty, to 0.5 p. wide ______ ___ _ ___ ____ _ S . calcicola v. diffusa. 19. Sheaths very firm, easily seen, to 1.0 p. wide _--------·-----------------------------------------20 20. Trichomes generally no wider than 3.5 µ. (most about 3.0 µ.);sheaths colorless ...... .... .. .. ...... .. .. . ..... ... .. .... ........ S . calcicola v. vaginata. 20. Trichomes generally 4.0-4.5 ,u wide, sheaths colorless and becoming pale yellow-brown with age . S. calcicola v. olivacea. M icrocoleus uaginatus (Vaucher) Gomont Most of the fast-growing, extremely motile isolates studied in this investigation were identified as M icrocoleus uaginatus. The species is distinguished by the con­spicuously attenuated ends of the trichomes, according to Drouet ( 1962, 1968). Isolates were both terrestrial and aquatic in origin. Drouet ( 1968) described the species as follows: Trichomes blue-green, yellow-green, olive, brown, red, violet, or gray-green cylindri­cal, rarely constricted at the cross walls, 2.5-9 Jl in diameter, straight or curving or spiraled, capable of growth to an indeterminate length, breaking by means of the de­struction of an intercalary cell or rarely by separation of two cells at a cross wall, at­tenuated through usually several cells at the tips, the tips not rarely capitate. Cells quadrate or shorter or longer than the diameter, 1-10 µ.long, the protoplasm homoge­neous or granulose, often pseudovacuolate, the cross walls lined on either side with a layer of granules. Terminal cell conical, hemispherical, truncate-cylindrical, the outer membrane becoming thickened into a rigid cone, cup, or convex disc. Sheath material hyaline, not at all or only in part and here and there turning blue in chlor-zinc-iodide. Plant consisting of long or short naked trichomes, or of trichomes in a homogeneous or laminose mucus, or of single, few, or many trichomes within a more or less discrete cylindrical, often branched, sheath. The plant-mass types and the morphology of the cells and trichomes encountered in these isolates divided them into ten groups, which are described below. M icrocoleus vaginatus (Vaucher) Gomont (Figs. 54-5 7.) Isolate 1621 The plant-mass is vermiform macroscopically with curved bundles of filaments (Fig. 54) . At 14X, among the bundles, growing both on and into the agar, solitary filaments are seen. The bundles may be rugose (Fig. 55), thus resulting in a striped appearance when the plant-mass is examined at low magnification, or they may be rough (Fig. 56) . The color of the plant-mass is deep yellowish-green ( 13 2) at 2 weeks and yellowish-brown ( 74) at 4 weeks. The cells (Fig. 57) are 5.0-7.0 µ.wide and 2.0-5.0 µ.long. The terminal cell is rounded, pointed, or rarely capitate; the terminal one to five cells may taper. The trichomes are usually not constricted at the crosswalls, but slight constrictions occur sometimes. The cell contents are granular; dark granules may occur at the cross­walls. The sheaths are narrow (0.8 µ.), firm, and colorless. Microcoleus uaginatus (Vaucher) Gomont v. glaber var. nov. Figs. 58-59. Isolates I1546, !1547 V arietas a specie typica differens eo quad massa plantarum sine microscopo obseruata uermiformis, multos fasciculos trichomatum conuolutos praebens; mag­nificatione 14X fasciculi leves apparentes. The plant-mass is macroscopically vermiform with curved and coiled thin bundles of trichomes. At 14X, the bundles are seen to be smooth, and solitary tri­chomes among them are not numerous (Fig. 58). The color of the plant-mass is deep yellow-green ( 118) aat 2 weeks and medium olive-green ( 125) at 4 weeks. The cells (Fig. 59) are 3.5-4.5 µ.wide and 2.0-4.0 µ.long. The terminal cell is rounded or rostrate and may bend and have a thickened outer wall; the end sev­eral cells may taper (to 2 .0 µ. wide) . The trichomes are not constricted at the cross­walls. The cell contents are granular and translucent granules occur throughout, including either side of the crosswalls. Sheaths have not been observed. Microcoleus uaginatus (Vaucher) Gomont v. undulatus var. nov. Figs. 60-62. Isolate 1622 Varietas a specie typica differens eo quod massa plantarum sine microscopo obseruata uermiformis, f asciculos trichomatum crassos habens; trichomata post circa duas hebdomades hie illic abrupte inftectunt, ut fasciculi torulosi uideantur. The plant-mass is macroscopically vermiform with thick bundles of trichomes, which from about 2 weeks on, are abruptly curved at intervals, giving a knobby appearance to the bundles (Fig. 60) . At 14X, the bundles are smooth and solitary trichomes are present among them (Fig. 61). The color of the plant-mass is dark olive-green ( 126) at 2 and 4 weeks. The cells (Fig. 62) are 5.0-7.0 µ.wide and 2.5-5.0 µ.long. The terminal cell is rounded, capitate, or pointed; the outer wall may be thickened. The end few cells may taper (to 4.0 µ.wide). The trichomes are not constricted at the crosswalls. The cell contents are granular; dark granules may occur along either side of the cross­walls as well as in the central portion of the cell. The crosswalls are often indis­tinct. Sheaths have not been observed. Microcoleus uaginatus (Va~cher) Gomont v. cyano-viridis var. nov. Figs. 63­ 64. Isolate K27 Varietas a specie typica differens eo quod massa plantarum sine microscopo obseruata uermiformis, colore semper atrouirens usque ad duas hebdomades. The plant-mass is vermiform macroscopically with curved bundles of filaments. At 14X, solitary filaments may be seen among the bundles, growing both on and into the agar (Fig. 63). The color of the plant-mass is medium olive-green ( 125) at 2 weeks and deep yellow-green ( 118) at 4 weeks. The cells (fig. 64) are 4.5-6.0 µ. wide and 1.8-5.0 µ. long. The terminal cell is rounded and very slightly tapered or (rarely) rostrate or capitate; the outer wall is occasionally slightly thickened. The terminal 1-5 cells may taper (to 2.0-3.5 µ. wide). The trichomes are not constricted at the crosswalls. The cell contents are granular; dark granules occur at the crosswalls frequently and dark or translucent ones in the center of the cell. The sheaths are firm, distinctly visible (to 1.0 µ. wide), and colorless. Microcoleus vaginatus (Vaucher) Gomont c. funiformis var. nov. Figs. 65-66. Isolates I 1306, I 1309 Varietas a specie typica differens eo quod massa plantarum sine microscopo observata ad centrum dense vermiformis, f asciculos filamentorum longos crassos radiantes habens. The plant-mass macroscopically has a densely vermiform center from which radiate long, frequently thick, curving bundles of filaments. At 14X, many soli­tary filaments are seen among the smooth bundles (Fig. 65). The color of the plant-mass is deep yellow-green ( 118) at 2 weeks and dark olive-green ( 126) at 4 weeks. The cells (Fig. 66) are 4.5-5.0 µ.wide and 2.0-5.0 µ.long. The terminal cell is conical or rounded; the end few cells may taper (to 3.0 µ.wide). Thickened ter­minal cells are of very rare occurrence. The trichomes are not constricted at the crosswalls. The cell contents are granular; dark granules occur throughout the cell and may be arranged in lines along either side of the crosswalls. Sheaths are pres­ent in cultures more than 2 weeks old; they are firm, colorless, and narrow. Microcoleus vaginatus (Vaucher) Gomont v. conicus var. nov. Figs. 67-68. Isolate K55 Varietas a specie typica diff erens eo quod massa plantarum sine microscopo observata vermiformis, fasciculis filamentorum levibus; cellula terminalis conica. The plant-mass is vermiform macroscopically with curved bundles of filaments. At 14X, the bundles of filaments appear smooth, and numerous solitary filaments are also seen (Fig. 67). The color of the plant-mass is dark olive-green ( 126) at 2 and 4 weeks. The cells (Fig. 68) are 6.0-8.5 µ.wide and 2.0-5.0 µ.long. The terminal cell is rounded or conical; the end several cells may taper (to 4 .0 µ. wide). The outer wall of the terminal cell may infrequently be slightly thickened. The trichomes are frequently slightly constricted at the crosswalls. The cell contents are granular; at times dark granules are seen on either side of each crosswall. The sheaths are 0.5­ 1.5 µ. wide, firm and colorless. Microcoleus vaginatus (Vaucher) Gomont v. fuscus var. nov. Figs. 69-72. Isolates 17, 26, 34, I1270 Varietas a specie typica di/Jerens eo quod massa plantarum colore fusco­brunnea. The plant-mas.5 is macroscopically vermiform with curved bundles of filaments (Fig. 69). At 14X, many of the bundles appear angularly curved, and numerous solitary filaments are seen among them (Fig. 70). The color of the plant-mass is dark olive-brown ( 96) at 2 and 4 weeks. The cells are 5.5-8.5 µ.wide and 2.0-5.5 µ long. The terminal cells are widely rounded, capitate (Fig. 71), or conical; the outer wall is frequently thickened. The end few cells often taper (to 4.0 µwide). The cells contain granules scattered throughout the protoplasm; sometimes there may be dark granules along either side of the crosswalls (Fig. 72). The sheaths are 0.5-1.5 µ. wide, firm, colorless, and sometimes lamellated. Microcoleus vaginatus (Vaucher) Gomont v. fuscorubens var. nov. Figs. 73-75. Isolate I617 Varietas a specie typica di!Jerens eo quod massa plantarum sine microscopo observata e fasciculis trichomatum curvatis convolutisque, quae post ca. duas heb­domades fimbriata videntur, composita. The plant-mas.5 is macroscopically composed of curving and coiling bundles of filaments (Fig. 73). At 14X, these bundles appear smooth to somewhat rough, and solitary filaments, growing both on and into the agar, are seen (Fig. 74). These solitary filaments are so numerous as to give a pale color to the agar. The color of the plant-mass is deep yellow-brown ( 7 8) at 2 and 4 weeks. The cells (Fig. 75) are 6.0-8.5 µwide and 2.5-4.5 µ long. The terminal cell is rounded and is infrequently thickened; the end few cells may taper (to 4.0 µ. wide). The trichomes are not constricted at the crosswalls. The cell contents are granular; translucent and a few dark granules may be seen throughout the cell. In 3N BBM, the granules were not observed in rows along either side of the cross­walls, but they were thus seen in 3N BBM at a lowered initial pH ( 5.0 and 6.0). The sheaths are narrow ( 0.3 µ. wide) , firm, and colorless to pale yellow. Microcoleus vaginatus (Vaucher) Gomont v. radiatus var. nov. Figs. 76-78. Isolate 1428 V arietas a specie typical differens eo quod massa plantarum sine microscopo observata radiata, fasciculos trichomatum distinctos diziergentes qui post duas heb­domades fimbriati videntur, habens. The plant-mass is macroscopically radial with distinct, divergent bundles of tri­chomes that appear fimbriate from about 2 weeks on (Fig. 76). At 14X, the fringed look is seen to be the result of intricately curved trichomes (Fig. 77). The color of the plant-mas.5 is very dark yellowish-green ( 138) at 2 weeks and deep yellow-green ( 118) at 4 weeks. The cells (Fig. 78) are 4.0-5.5 µ wide and 3.5-5.0 µ long. The terminal cell is rounded or conical; the end few cells may taper. Thickened outer walls of the terminal cell have not been seen. The trichomes are not constricted at the cross­walls. The cell contents are granular; granules of ten occur along each side of the crosswalls. Sheaths have not been observed. Microcoleus vaginatus (Vaucher) Gomont v. araneaformis var. nov. Figs.79­ 80. Isolate 33 Varietas a specie typica difjerens eo quod massa plantarum sine microscopo observata arachnoidea; cellulae 7.0-10.0 p. [at., 2.5-4.0 p. long. The plant-mass is macroscopically arachnoid. At 14X, the individual filaments and bundles of filaments are seen (Fig. 79). The color of the plant-mass is very dark yellowish-green ( 138) at 2 and 4 weeks. The cells (Fig. 80) are 7.0-10.0 p. wide and 2.5-4.0 p. long. The terminal cell is rounded. The trichomes are not constricted at the crosswalls. The cells contain numerous granules in a random arrangement and along either side of the cross­walls. Some sheaths are always seen though they never seem to be ubiquitous; they are to 1.0 p. wide and colorless. Microcoleus lyngbyaceus (Klitzing) Crouan This species is distinguished by Drouet ( 1968) from the other species by the densely granular protoplasm. This characteristic is best demonstrated by isolate 4 when growing under apparently less than optimal conditions. The dense granules are not, however, present when growth is good, except in scattered cells. Drouet ( 1968) described the species as follows: Trichomes blue-green, yellow-green, yellow, olive, brown, red, violet, or gray-green, cylindrical, constricted (sometimes only here and there or in parts) at the cross walls, 3.5-8 p. in diameter, here and there or in part decreasing or increasing in diameter, straight or curving or spiraled, capable of growth to an indeterminate length, breaking by means of the destruction of intercalary cells or by the separation of cells at a cross­wall, cylindrical or long-or short-attenuate at the tips. Cells commonly shorter than broad, up to 15 times as short as broad, rarely quadratic, 1.5-8 p. long, the protoplasm homogeneous or granulose, often pseudovacuolate, the cross walls and side walls lined with a layer of granules. Terminal cell rotund, the outer membrane at first thin, be­coming thickened in a depressed-rotund, depressed-hemispherical, or depressed-conical shape. Sheath material hyaline, often developing yellow or brown pigments, not at all or only in part turning blue in chlor-zinc-iodide. Plant consisting of long or short naked trichomes, or of trichomes in a homogeneous or laminose mucus, or of one or few or many trichomes in a more or less discrete, often branched, cylindrical sheath. The two isolates belonging to this species are described as two varieties based on plant-mas.s form, size, and nature of the sheath in the descriptions given below. Microcoleus lyngbyaceus (Klitzing) Crouan Figs. 81-83, 127. Isolate 4 The plant-mass is macroscopically arachnoid (Fig. 81). At 14X, the individual filaments may be seen (Fig. 82). The color of the plant-mass is very dark yellow­ish-green ( 138) at 2 and 4 weeks. The cells (Fig. 83) are 16.0-24.0µ.wide and 2.5-7.0µ. (mostly about 4.0µ.) long. The terminal cell is broadly rounded; thickened outer walls have not been observed. The trichomes are not constricted at the crosswalls. The cells contain many dark and translucent granules, which may appear at times to form almost a solid mass in the cells (Fig. 127). The sheaths are 1.0-4.0 µ. wide, colorless to slightly yellow, lamellated ( 4-8 layers), and firm. Microcoleus lyngbyaceus (Klitzing) Crouan v. vermiformis var. nov. Figs. 84­ 86. Isolate 12 Varietas a specie typica differens eo quod massa plantarum sine microscopo observata vermiformis; cellulae 5.0-6.0 µ.lat., 1.0-2.5 µlong. The plant-mass is vermiform macroscopically (Fig. 84) with many fine curved bundles of filaments (Fig. 85). At 14X, many solitary filaments can also be seen growing both on and into the agar. The color of the plant-mass is deep yellowish­green ( 118) at 2 and 4 weeks. The cells (Fig. 86) are 5.0-6.0 µ.wide and 1.0-2.5 µ.long. The terminal cell is rounded and may contain many dark granules; thickened outer walls have not been observed. The trichomes are not constricted at the crosswalls. The cell con­tents are granular; granules are usually distributed throughout the cell, but dark granules may also occur at the crosswalls and along the sides of the cells. The sheaths are 0.5-1.5 µ. wide, firm, and colorless. M icrocoleus irriguus (Klitzing) Drouet This species is distinguished from the two other species by Drouet ( 1968) by the very slight attenuation of the tips of the trichomes. Drouet described the species as follows: Trichomes blue-green, yellow-green, olive, brown, red, violet, or gray-green, cylindri­cal, constricted here and there only lightly at the crosswalls, 6-18 µ. in diameter, here and there and in part increasing or decreasing in diameter, straight or curving or spiraled, capable of growth to an indeterminate length, breaking by means of the de­struction of an intercalary cell or by the separation of two cells at a cross wall, the tips not at all or scarcely attenuated, the apices truncate. Cells longer or shorter than broad, 3-15 µ.long, the protoplasm homogeneous, sometimes pseudovacuolate, the cross walls covered on either side with a layer of granules. Terminal cell cylindrical, the outer membrane depressed-hemispherical or broadly depressed-conical, becoming thickened in age. Sheath material hyaline, not or only in part becoming blue when placed in chlor-zinc-iodide. Plant consisting of long or short naked trichomes or of trichomes in a homogeneous or laminose mucus or of one or few or many trichomes within a more or less discrete cylindrical, often branched, sheath. One isolate was studied and is described below. Microcoleusirriguus (Klitzing) Drouet Figs. 87-88. Isolate 35 The plant-mass is macroscopically arachnoid. At 14X, many separate, non-coil­ing filaments are seen, but only a few bundles of filaments occur (Fig. 87). The color of the plant-mass is medium olive-green at 2 and 4 weeks. The cells (Fig. 88) are 5.0-8.0 µ.wide and 4.0-6.0 µ.long. The terminal cell is slightly and shallowly pointed, and the outer wall may be thicker than other walls. Dark granules occur along both sides of the crosswalls and some are scattered throughout the cell. The sheaths are 0.5-1.0 µ.wide, colorless, and firm. A key to these isolates of Microcoleus follows. A KEY TO SPECIES OF MICROCOLEUS IN CULTURE I. Dense protoplasm and granules extending along the lateral walls of each cell as well as along the crosswalls . . ... ... .................... M. lyngbyaceus. 1. Dense protoplasm and granules along the crosswalls only . ............ .. 2 2. Trichomes becoming at most slightly attenuated at the tips . .. .. .. M. irriguus. 2. Trichomes becoming conspicuously attenuated at the tips ... . ..M. vaginatus. KEY TO THE VARIETIES OF MICROCOLEUS LYNGBYACEUS 1. Plant-mass macroscopically arachnoid . .... .. ... ... ....... ....... . .. M. lyngbyaceus. I. Plant-mass macroscopically vermiform .. .... .. .... ... M. lyngbyaceus v. vermiformis. KEY TO THE VARIETIES OF MICROCOLEUS VAGINATUS I. Plant-mass macroscopically vermiform ........ .............. .. . ..... .. 2 I. Plant-mass otherwise ...... . . 9 2. Plant-mass brown .. .. .. .. ....... .. .. . 3 2. Plant-mass some shade of green ... .. .... . . . . . . . . ... . . .... . ......... 4 3. Plant-mass macroscopically consisting of curving, spreading bundles of fila­ments ....... ......... ... M. vaginatus v. fuscus. 3. Plant-mass macroscopically consisting of many coils of bundles of filaments M . vaginatus v. fuscorubens. 4. Plant-mass macroscopically consisting of bundles of trichomes having an undulate appearance ..... M. vaginatus v. undulatus. 4. Plant-mass macroscopically consisting of bundles of trichomes that are not undulate ...... ... ......... 5 5. Many solitary filaments present among the bundles of filaments, giving distinctive background color to the bundles . . .. ......... 6 5. Some solitary filaments present, but never enough to impart distinct color to the agar .. .. .. . . . .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. . .. .. .. . . .. .. . . .... 7 6. Bundles of filaments thick, generally radiating from the inoculum --------­ M. vaginatus v. funiformis. 6. Bundles of filaments thin, generally growing equally over the entire sur­face _______ --------------------------------------------------__ ______ M. vaginatus v. cyano-viridis. 7. Plant-mass dark olive-green, becoming some shade of yellow only after 6-8 weeks __ ---------------------------------------------__ _________ _________ ______ M. vaginatus v. conicus. 7. Plant-mass lighter shade of green, beginning (unless in low light) to turn yellow at 3-4 weeks ______________ .. ------------------------------------------------------------------------8 8. Trichomes without sheaths: usually 3.5-4.0 µ. (sometimes to 4.5 µ.) wide__ ____ ---------------------------------------------_____ __________ __ M. vaginatus v. glabrus. 8. Trichomes with sheaths, at least in part ; usually 5.0---6.4 µ. (to 7.0 µ.) wide -------------------------------------------------------------------------------------M . vaginatus. 9. Plant-mass with undulate bundles of trichomes radiating from the inocu­lum ------------------------------------------------------------------------------------M. vaginatus v. radiatus. 9. Plant-mass with straight bundles of trichomes radiating from the inoculum ----------------------------------------------___ ___________ M. vaginatus v. araneaformis. Porphyrosiphon notarisii (Meneghini) Kiitzing The species is delimited by Drouet by its rotund to conical terminal cells that are not thickened along the outer wall. The separation from P. kurzii (not repre­sented among these isolates) is based on the longer cells (3-15 µ.) in P. notarisii; cell length in P. kurzii does not exceed 4 µ.. Drouet ( 1968) described Porphysosiphon notarisii as follows: Trichomes blue-green, yellow-green, olive, brown, red, violet, or gray-green, cylindri­cal, commonly constricted (at least here and there ) at the cross walls, 3-40 µ. in diam­eter, here and there and in part increasing or decreasing in diameter, straight or curv­ing or spiraled, capable of indeterminate growth in length, breaking by means of the destruction of an intercalary cell or by separation of cells at a cross wall, at the ends evidently long-attenuated through several cells. Cells shorter or longer than broad, (2.0 µ.) 3-15 µ. long, the protoplasm homogeneous or granulose, sometimes pseudo­vacuolate, the cross walls not granulated. Terminal cell at first hemispherical, becom­ing obtuse-or acute-conical, the outer membrane not thickened. Sheath material at first hyaline, later often developing yellow, brown, red, violet, or blue pigments, mostly turning blue in chlor-zinc-iodide. Plant consisting of long or short naked trichomes, or of trichomes in a homogeneous or laminose mucus, or of one to many trichomes in a more or less discrete, often branched, cylindrical sheath. Three isolates, from freshwater and marine environments, were studied in cul­ture. They are described as two varieties below. Porphyrosiphon notarisii (Meneghini) Kiitzing. Figs. 89-91. Isolates 5, 39 The plant-mass is macroscopically extremely and irregular convoluted (Fig. 89). At 14X, the bundles of trichomes appear very smooth and among them soli­tary trichomes are seen growing both on and into the agar (Fig. 90). The color of the plant-mas.c; is very dark yellowish-green ( 138) at 2 weeks and dark olive­green ( 108) at 4 weeks. The cells (Fig. 91) are 4.0-5.0 µ.wide and 2.0-3.5 µ.long. The terminal cell may be conical or rounded; it often curves or hooks. The end few cells may be tapered (to 2.5 µ.wide). The trichomes are not constricted at the crosswalls. The cell contents are granular, but no dark granules are seen either centrally or at the crosswalls. Sheaths may occur in older ( 4-8 weeks) cultures where they are very soft, colorless (apparent only if stained with methylene blue or India ink), and never seem to be common. Porphyrosiphon notarisii (Meneghini) Klitzing v. canus var. nov. Figs. 92-94. Isolate 40 V arietas a specie typica diff erens eo quod massa plantarum sine microscopo observata fasciculos trichomatum in centro arcuatos convolutosque qui recti radia­tique fiunt, ha bet; cinereo-brunnea. The plant-mass macroscopically has curved and coiled bundles of trichomes in the center that become straight and radiate from the center of the plant-mass (Fig. 92). At 14X, the bundles are seen to be smooth and many solitary trichomes occur (Fig. 93) . The color of the plant-mass is dark gray-yellow ( 91) at 2 and 4 weeks. The cells (Fig. 94) are 5.0-6.0 p. wide and 3.0-4.0 p. long. The terminal cell is conical or rounded; the end few cells may taper (to 3.5 p. wide). The trichomes are not constricted at the crosswalls. The cell contents are granular; distinct trans­lucent and dark granules are seen throughout the cells. Sheaths have not been observed. These isolates may be distinguished as follows. KEY TO THE VARIETIES OF PORPHYROSIPHON NOTARISII I. Plant-mass green . ....... ...... . . . .... .. . . ........... P. notarissi. I. Plant-mass gray-brown ... P. notarissi v. canus. Oscillatoria lutea Agardh The species is distinguished by Drouet ( 1968) by the general occurrence of short cells and by the slight attenuation of the end of the trichomes. Several of the isolates herein described are frequently more than slightly attenuated at the ends, but they seem to be more like this species than any other. Drouet ( 1968) described the species as follows: Trichomes blue-green, yellow-green, olive, brown, red, violet, or gray-green, cylindri­cal, scarcely constricted here and there at the cross walls, 2.5-10 p. in diameter, here and there and in part increasing or decreasing in diameter, straight or curving or spiraled, indeterminate in growth in length, fragmenting by means of the destruction of an intercalary cell or by separation of cells at a cross wall, cylindrical or attenuating somewhat through one or more cells at tips. Cells as long as or shorter than broad, often very short, 1-7 long, the protoplasm homogeneous or granulose, rarely pseudovacu­olate, the cross walls not granulated. Terminal cell broadly truncate-conical, the outer­membrane at first thin, becoming thickened in age, convex-platelike. Sheath material hyaline, often turning blue in chlor-zinc-iodide. Plant consisting of long or short tri­chomes, naked or in a homogeneous or laminose mucus, or solitary or few or many in more or less discrete, often branched sheaths. The isolates of this species studied in culture were grouped as follows. Oscillatoria lutea Agardh Figs. 95-97. Isolate 23 The plant-mass is vermiform macroscopically with curved bundles of filaments. At 14X, the bundles of filaments appear smooth, and solitary filaments may also be seen growing both on and into the agar (Figs. 95-96). The color of the plant­mass is deep yellowish-green (132) at 2 weeks and dark olive-green (126) at 4 weeks. The cells (Fig. 97) are 5.0-7.0 µ.wide and 1.0-4.0 µ.long. The terminal cell is rounded or capitate, and the outer wall may be thicker than that in other cells. The end few cells taper frequently (to 4 .0 µ. wide) . In old cultures pointed terminal cells may occur. The trichomes are not constricted at the crosswalls. Gas vacuoles seem to be of sparse occurrence. Sheaths appear with age; they are colorless and to 2.0 µ.wide. Oscillatoria lutea Agardh v. auxotrophica var. nov. Fig. 98. Isolates 22, 24 Varietas a specie typica di ffer ens eo quod in cultura increscens vitamino "Bu" eget. The plant-mass form is the same as that of isolate 23 (0. lutea). The cells (Fig. 98) are 5.0-7.0 µ.wide and 1.0-3.0 µ. long. The terminal cell is capitate, rounded, or (rarely) rostrate; the outer wall is thicker than other walls. The end few cells may taper (to 3 .0 µ. wide). In old cultures pointed terminal cells may occur. The trichomes are only rarely slightly constricted at the crosswalls. The cells contain granules scattered throughout the protoplasm; vacuoles may occur in old cultures and in vitamin-deficient media. Gas vacuoles occur at alka­line pH's to a limited extent on agar (and much more commonly in liquid). The sheaths are narrow in young cultures ( 0.5 µ.), becoming wider (to 2.0 µ.) with age, and colorless. Isolates 22 and 24 differ only slightly morphologically from isolate 23 (more capitate cells, more sheaths, some constrictions at the crosswalls), but several physi­ological differences were also noted. The most important of these was that isolate 23, although possibly enhanced by addition of vitamin B12 to the medium, did not require it for continued growth in axenic culture as did isolates 22 and 24. Oscillatoria lutea Agardh v. contorta var. nov. Figs. 99-102. Isolates 25, 1386, I390. V arietas a specie typica differens eo quod massa plantarum sine microscopo observata vermiformis, amenta atque anfractus arctos fasciculorum filamentorum, praebens; magnificatione 14X observata, fasciculi leves apparent. The plant-mass is macroscopically vermiform with loops and tight coils of bundles of filaments (Fig. 99). At 14X, the bundles appear very smooth, and few solitary filaments are seen among them (Fig. 100-101 ). The color of the plant­mass is deep yellow-green ( 132) at 2 weeks and medium to dark olive-green ( 125, 126) at 4 weeks. The cells (Fig. 102) are 4 .0-7 .0 p. wide and 1.5-6.0 p. long. The terminal cell is rounded (some with thickened outer walls), capitate, or rostrate. In older cul­tures the terminal cells may be pointed. The end few cells may taper (to 3.0 µ. wide). The trichomes are at times slightly constricted at the crosswalls. The cells contain granules scattered throughout the protoplasm; gas vacuoles occur fre­quently. Sheaths are soft to firm, colorless, and in older cultures are to 4.0 p. wide. Oscillatoria lutea Agardh v. scabra var. nov. Figs. 103-104. Isolate 38 Varietas a specie typica differens eo quod massa plantarum sine microscopo observata conferte vermiformis; magnificatione 14X visa, fasciculi filamentorum asperi apparentes. The plant-mass is macroscopically densely vermiform. At 14X, rough curving and coiling bundles of filaments are seen; numerous solitary filaments also occur (Fig. 103). The color of the plant-mass is deep yellowish-green ( 118 ) at 2 weeks and very dark yellow-green ( 138) at 4 weeks. The cells (Fig. 104) are 3.0-5.0 p. wide and 2.0-4.0 µlong. The terminal cell is rounded to slightly conical; the outer wall may be thickened. The trichomes are not or only very slightly constricted at the crosswalls. Granules occur randomly dispersed throughout the cells. With age, trichomes become extremely contorted and twisted within the sheaths. The sheaths are narrow ( 0.2 p.) at first, becoming wider with age (to 1.0 p. wide), colorless, and frequently lamellated. The isolates of 0. lutea may be identified with the aid of the following key: KEY TO THE VARITIES 01'" OSCILLATORIA LUTEA 1. Plant-mass macroscopically vermiform with curved bundles of filaments . 1. Plant-mass macroscopically vermiform with many coils of filaments ........ 0. lutea v. contorta. 2. Curved bundles of filaments smooth -----·------------------------·-------------------------3 2. Curved bundles of filaments rough. ___ _____ ------·--_ ___ _____ __ __Q. lutea v. scabra. 3. Organisms B,, auxotrophs __ __ __ _ 0. lutea v. auxotrophica. 3. Organisms not B1 2 auxotrophs _ _ ____ __ _ _ 0. lutea. Physiological Studies Various physiological studies were performed both for the taxonomic aid they might give and for any contribution to the knowledge of blue-green algal physiol­ogy they might make. The selection of the basal medium, the comparison of uni­algal and axenic cultures, and changes in cultures due to aging are first discussed. Growth in various carbon and nitrogen sources, with and without vitamins, pro­duction of extracellular enzymes, and sensitivity to crystal violet dye, and pH changes are also included in this section. SELECTION OF THE BASAL MEDIUM Several media were employed in an attempt to find the one most suitable to the growth of the most isolates. These media included BBM-K, Shen-X, KMD, C~-10, 1N BBM, and 3N BBM; the formulae for these media are given in the Materials and Methods. Most isolates were originally grown on BBM-K, a Tris-buffered, more dilute variation of 1N BBM which Kantz and Bold ( 1969 ) in their investigation of Nostoc and Anabaena found to be the most satisfactory medium for the isolates studied. Cultures of Oscillatoriaceae grown in this medium had moderate-to­excellent growth during the first 2 weeks, but the colors of the algae were generally pale, and within 3 weeks definite yellowing began in the great majority. By 4 weeks, almost all were yellow or orange with only a small amount of green coloration left. Microscopically, the cells had numerous granules, both dark and translucent. The growth on agar was generally very diffuse; _plant-mass types in some isolates varied considerably from the 3N BBM type herein designated as the definitive one. Shen-X, another dilute culture medium, was also used in preliminary testing; it caused even more rapid change of color in isolates (from green to red-orange in most cases) than did BBM-K. Growth was minimal. By 2 weeks color change was complete in most isolates (isolate 27 was a noticeable exception; it remained green although there was no detectable growth). Th~ isolates were not dead, however, for when transferred to BBM-K or 1N BBM they became green and started to grow within a few davs. KMD (Kratz and Myers' Medium D) was also tested. Growth was moderate­to-good when the cultures were 2 to 4 weeks old. In flasks, growth tended to re­sult in long filaments which formed large, loose masses in the culture flasks. Growth in the other media usually resulted in a large number of clumps of filaments and a membranous layer on the surface of the liquid. KMD thus resulted in a more "natural" macroscopic appearance of many of the isolates. Microscopically, how­ever, no significant variation from the other media could be seen in the cells when the cultures were in a healthy state. The deterioration (color change, formation of hormogonia, dead cells) of most of the cultures after about 4 weeks was the prin­cipal drawback to use of KMD for long-term observations. Cg-10 proved to be an excellent medium for growth of single filaments at time of isolation (see Materials and Methods) and also supported good growth of many isolates without the early deterioration of cultures seen in KMD. The same type of growth was observed in lN BBM and 3N .BBM. It was found that IN BBM supported vigorous growth longer than BBM-K. This increased growth was suspected to be due to the increased amount of nitrogen, a view sup­ported by the results of doubling or tripling the nitrate stock of BBM-K. The use of 3N BBM further enhanced growth; cultures in tightly capped tubes remained green and continued to grow for several months. The amount of growth (macro­scopically observed) was greater in 3N BBM over a long period of time than in other media. Although color changes began at about 4-5 weeks, frequently cul­tures several months old were still-at least in part-green. Plant-mass in lN BBM had more definite form than in BBM-K (although in all media, plant-mass form was constant for the medium). Cell morphology and plant-mass form were not modified when the isolates were grown in Tris-buffered 3N BBM (BBMT) at an initial pH of 7.0-7.2. COMPARISON OF UNIALGAL AND AxENIC CULTURES Statements have been made, from time to time, that the presence of bacteria and fungi in cultures negates their morphological value or at least their morphological constancy. All of the axenic isolates used in this work, except 1621 and 27, were either received in unialgal condition or were isolated from field collections. Ob­servations thus were made on cultures of the same organisms in both the unialgal and axenic states. The presence of an excessive number of fungi in a culture can modify the plant-mass form and the morphology of the trichomes. In such a case, the fungus often kills the alga eventually. The presence of bacteria was not seen to have any effect on the morphology of the trichomes. To check this conclusion, 10 isolates were recontaminated with a bacterium isolated from isolate 38 and with Aerobacter aerogenes. Growth through several transfers showed no differences be­tween the axenic control and the newly contaminated culture except in isolate K107. There the difference was only in the amount of growth (K107 grew better when contaminated with the bacterium from isolate 38 than without bacteria or with Aerobacter aero genes) ; the plant-mass form and the morphology of the cells were unchanged. The use of axenic cultures is always preferable and is mandatory for most physio­logical studies. Such cultures are necessary for completely defining variation in any organism, but in the writers' experience, it did not appear that unialgal cultures in the laboratory were any more "abnormal" or subject to change than either axenic cultures or organisms growing in nature. They are, in fact, much less vari­able than the latter. COMPARISONS OF CULTURES AT VARIOUS AGES The most striking change in the oscillatoriacean isolates as they age is that in color. Most, when grown on BBM agar plates under standard conditions, are ini­tially yellowish-green, olive-green, or brown. Change in color in many cultures begins in about 3 weeks; the process may be hastened by excessive drying of the agar, by higher light intensity, and by reducing the nitrogen in the medium. Some of the isolates (among them K107, K58, 4) exhibit little, if any, change in color even if cultures are maintained for 2 or 3 months. Most, however, eventually ( 1-3 months) become some shade of reddish-brown, yellow, or brown. Table 2 sum- TABLE 2. Change in Color of 20 Isolates of Oscillatoriaceae on 3N BBM Agar Between 2 and I 2 Weeks' Growth No. 2 4 5 7 10 12 20 22 26 30 KSl K55 K107 K182 1386 1485 1597 1621 1622 11306 2wk 138 (dark yellowish-green) 138 (dark yellowish-green) 138 (dark yellowish-green) 132 (deep yellowish-green) 138 (dark yellowish-green) 118 (deep yellowish-green) 125 (medium olive-green) 132 (deep yellowish-green) 96 (dark olive-brown) 126 (dark olive-green) 132 (deep yellowish-green) 126 (dark olive-green) 138 (dark yellow-green) 138 (dark yellowish-green) 132 deep yellowish-green) 132 (deep yellowish-green) 132 (deep yellowish-green) 132 (deep yellowish-green) 126 (dark olive-green) 118 deep yellow-green) 12 wk 126 (dark olive-green) 138 (dark yellowish-green) 95 (medium olive-brown) 74 (soft yellowish-brown) 51 (deep orange) 74 (soft yellowish-brown) 125 (medium olive-green ) 126 (dark olive-green) 94 (light olive-brown ) 95 (medium olive-brown) 51 (deep orange ) 95 (medium olive-brown) 94/126 (light olive brown/ dark olive-green) 7 4 (soft yellowish-brown) 126 (dark olive-green) 126/ 95 (dark olive-green/ medium olive-brown) 74 (soft yellowish-brown) 7 4 (soft yellowish-brown) 69 (deep orange-yellow) 7 4 (soft yellowish-brown) TABDE 3. Color of 10 I solates of Oscillatoriaceae at 2 and 4 Weeks on 3N BBM, BBM-K, and KMD Agar Media No. 3NBBM BBM-K KMD 2wk 4wk 1 118 132 4 138 138 5 138 108/84 IO 138 132 12 118 118 17 96 96 19 132 126 24 132 126 39 118 118 Kl07 138 138 68 = soft orange yellow 74 =soft yellowish brown 75 =deep yellowish brown 84 = soft yellow 85 = deep yellow 96 = dark olive brown 100 = deep greenish yellow I 02 = medium greenish yellow 108 = dark olive 2wk 117/84 138 118 120 125 96 118 132 118 138 4wk 2wk 4wk 74 102/84 125 138 138 138 85 137 84 74 120 125 84 120 68 96 96 96 85 137 84 84 117 125 84 118 108/ 100 75 138 126 117 =soft yellow green 118 = deep yellow green 120 = medium yellow green 125 = medium olive green 126 = dark olive green 132 = deep yellowish green 137 =dark yellowish green 138 =very dark yellowish green marizes color change in some of the isolates during 2 to 12 weeks gTOwth. Although no special effort to affect the colors of the isolates was made, it was found that the various media often resulted in different patterns, both of initial and of terminal color. Table 3 summarizes the colors at 2 and 4 weeks of 10 isolates grown on 3N BBM, BBM-K, and KMD media. As can be seen from the table, there are isolates which have the same color in all three media ( 17, 34, 4) . If colors and change in color arc to be used as a characteristic of the species or variety, it is necessary to define the culture conditions. Not only the medium used but also the form of the medium have a definite effect on the color. Growth in liquid media or on slants almost always results in slower change in color than in petri dishes, probably as a result of greater available moisture in the culture tubes. The necessity of defining the age of the culture when the plant-mass is described was emphasized by Groover and Bold ( 1969). The attributes of shinyness and dryness have not been used in the current study, so that this change, frequently ob­served in green microalgae, has been eliminated. Gross and complete changes were not observed in the plant-mass of these isolates from 2 weeks on. The plant­mass may continue to grow for 8 or more weeks, and as it does so, it not only be­comes much larger in diameter but also much thicker. Therefore, in many isolates there tends to be a blurring of some characteristics, as the originally distinct ropes of trichomes become more confluent, rather than a definite change. Thirty of the isolates were observed microscopically at weekly intervals for pe­riods up to 3 months. Several patterns of morphological changes in the trichomes and filaments were noted in these isolates grown in BBM and variants thereof. Variation in liquid and solid media was also observed (Figs. 108-109). The first type of change was characteristic of Schizothrix calcicola v. vermiformis (isolates 16, 21, K 18, K 44, K5 7). As the culture aged, crosswalls became less dis­tinct (beginning at about 6 weeks), until they were almost entirely invisible in many filaments at 10-12 weeks. The central bright area of the cell resolved into several granular inclusions. Short false branches were also formed as the trichomes continued to grow, but the sheaths apparently did not expand after about 6-8 weeks (Fig. 105). Cultures of Schizothrix calcicola v. glomerulata (isolates 1426, 1482, 1487, 1596, 1597 ) became almost entirely hormogonial, with trichomes mostly 2re10 cells long between 4 and 7 weeks of age (Fig. 106). Other isolates ( 1598, 1427, 7) that have the same plant-mass type and cell size and cell shape remain filamentous. The third type of change was apparent in isolates that were fairly large, fast­growing, and extremely motile (isolates 22, 23, 24, 25, I621, 1386). The sheaths at first were either not seen or were very thin and infrequent in occurrence. The terminal cells were rounded, capitate, or rostrate. The sheaths appeared at 2 or 3 weeks in either liquid or agar-solidified media and became thicker from that time on. By the time the culture was 6-8 weeks old, the sheaths were frequently one­half to two-thirds as wide as the trichome itself. Staining with methylene blue or India ink did not reveal lamellation. The terminal cells of some of these trichomes became extremely pointed (Fig. 107). Isolate 1622 developed pointed ( cuspidate) terminal cells, but sheaths were not observed. Isolate K55 appeared progressivdy more constricted at the crosswalls with age. Isolates 11270, 26, K55, and K27 varied in the distribution of granules in the cells (whether central or along the crosswalls or both), but no correlation with age of cultures was established; their presence or absence seemed to be quite random (Figs. 71-72) . The cells of the remaining isolates studied over an extended period of time ( 4, 6, 27, K62, K107, Kl82) did not show any noticeable morphological changes. It is interesting to note that many of the changes associated with age also ap­ peared in young cultures grown at higher pH's (8.0-9.0). Whether the changes could be retarded by maintaining a lower pH (in BBM the pH may change from 6.5 to 10.0 depending on the alga), and whether the effects at higher pH's are in­fluenced by the type of buffer, remain to be elucidated. EFFECTS OF vARIOUS CARBON SOURCES The assimilation of assorted carbon sources in light and dark in the blue-green algae has been investigated by numerous workers. Early work was in part stimu­lated by the isolation into pure culture of endophytic species and a subsequent de­sire to demonstrate the presence of heterotrophic growth as an operative system in these algae. Reports of heterotrophic growth in a blue-green alga ( N ostoc ) were first given by Bouilhac ( 1898). Pringsheim ( 1913) was unsuccessful, however, in attempts to grow Oscillatoria brevis, 0. tenuis, and Nostoc sp. in the dark. Harder (1917), Winter ( 1935), and Allison, Hoover, and Morris ( 1937) demonstrated hetero­trophy in several strains of N ostoc with glucose as the carbon source. Enhancement of growth in light with various carbon sources was reported in all the above-cited investigations. The main result of these studies was to implant widely the idea that blue-green algae are usually heterotrophic. As more species have been examined, heterotrophy has been found to be the exception, not the rule. Allen ( 1952) found only six isolates (including one previously known) to be heterotrophic. These included Oscillatoria, Lyngbya, Phormidium, Plectonema, and Nostoc species; growth was very slow on a glucose-yeast autolysate medium. Kratz and Myers ( 1955) grew Allison's strain of Nostoc muscorum in the dark, hut were not able to grow another strain of the same species or several other species heterotrophically. Further reports of heterotrophy continue to appear in a wide range of blue-green species ( Kiyohara, et al., 1960; Fay, 1965; Kantz and Bold, 1968) although the occurrence of any true heterotrophic growth in blue-green algae is also questioned (Holm-Hansen, 1967; Van Baalen, personal communication). The assimilation in light of various carbon sources has been investigated by Allison, et al. ( 1937), Hoare and coworkers ( 1965, 1967), Carr and Pearce ( 1966), Carr, et al. ( 1967), Pearce and Carr ( 1969), and Kantz and Bold ( 1969). Glucose and fructose have generally been found to increase growth, although many other compounds (acetate, ribose, galactose, etc.) can be assimilated by some or­ganisms. Failure to assimilate a compound occurring in one of the metabolic path­ways is generally thought to be due to permeability limitations (Kratz and Myers, 1955; Fogg, l 956b), but such limitations have been questioned following work with cell-free preparations in which assimilation did not occur (Holm-Hansen, 1968) . Comparative studies of carbon nutrition of several species have rarely been un­dertaken; in two such studies (Van Baalen, 1962; Kantz and Bold, 1969) differ­ent patterns of carbon utilization have been found in the same morphological species, emphasizing again that it is tenuous to base discussions of physiological attributes unquestioningly upon results concerning one or a few species, varieties, or strains. Clean culture tubes ( 100 mm X 10 mm) were soaked for 3 days in deionized water which was changed daily. The tubes were then dried, plugged with cotton, and autoclaved. The control medium was 3N BBMT: eight carbon sources were tested by preparing eight aliquots of media, each containing the equivalent amount of carbon of a 1 7o glucose solution. The compounds tested were glucose, fructose, D-and L-arabinose, ribose, xylose, pyruvate (sodium salt), and acetate (sodium salt). The carbon equivalents were, in each case, dissolved in 20 ml of 3N BBMT and added by Millipore filtration to 980 ml of autoclaved 3N BBMT. The media were pipetted into the tubes, which were allowed to incubate at room temperature for 3 days so that bacterial contamination could be detected. Two separate sets of tubes were prepared; eight of each medium were inoclulated with each axenic isolate for each set. The inocula were taken from the surface of 3-week-old 3N BBM agar slants. Sterilized mineral oil was added to half of the tubes to simulate anaerobic conditions. One duplicate set of aerobic and anaerobic tubes was placed in the culture room under standard conditions and incubated for 3 weeks. The other set was stored in the dark and incubated for a total of 6 TABLE 4. Growth of Certain Axenic Isolates of Oscillatoriaceae in Media With Various Carbon Sources in Light I sol ate No, Control Ribose Fructose Glucose 0-arab, L-arab. Xylose l'yruv. Ace tate a a a ~-~ G+ N F T N N G F­ l' T T T N N N N ~· ~v. auxotrophica G N G T N N E T F T F F N T N N Q. ~v. auxotrophica 24 F T N N F T T N G T T N N N N N ~· ~v. ~ 25 EN FT NN GF FT F T T T N N ~­ vaginatus 1621 T N E T F F G T T• N G N T N N N N N ~· vasinatus v, ~ 26 G N EF NN EF FT F N T T N N T N .?_.~ 21 GN E G+ G+ T• G+ T+ F N T N F N N ~ ~-~ Kl8 G N E G+ G F G+ F F N T N F N N N N N ~· ~ KH F• N E G• G F G F T N T N F N N N N N ~­ ~v. venifor•is KS7 G­N EG CF GT FN F­ N N N N N N N ~-~v.~ 27 EN N N N N E+ G N N N N F T T G G­ N ~· ~v, sriralis Kl07 E T GT GF GT EN N N G N N N ~· ~v, mucosa Kl82 F N G N F N F N F+ N T N F N N N N N ~· ~v, glo•erulata 1426 F• N E G G F G• T F N F N N N N N N N ~· ~v. f!lo•erulata 1427 F N E G+ G G G G F N F N N N T T N N ~· ~v, glo111erulata 1482 G N G E G• E G E F T­ T+ N N N N N T N ~· .Ł!..!....Ł_i~l.•. v, si:loaerulata 1487 G N E G+ G+ F G+ F F N F T T N N N N N ~· ~v. glo•erulata 1597 (; N E G G F+ G F F• N F• N F N N N _!. ~v. 1lo•erulua 1598 G N E E G F• G+ T G­N G­ N N N N N 1485 F N E G+ G G F T F N F N F­ N N N E • Excel lent growth (covering the sides of the culture tube •nd surf•ce of the mediu•) G • Good arowth F • f•ir growth T • Trace growth N • No growth all values based on •acroscopic observations acrowth of the isolates in darkness was very li•ited: 23, glucose, an, T•; 22, glucose, an, T; 25, glucose, a, T, an, F; kJ8, ribose, a, F, fructose, a, F ; K44, ribose, a, T•; KS7, ribose, a, T•, fructose, a, T•, glucose, a, T•; 1427, fructose, T ; 1482, D-arab., an, F. Tests not discussed in this dissertation showed that only 22 in glucose survived a second-transfer. weeks. Table 4 summarizes growth of the isolates as macroscopically determined at the end of 3 weeks. Growth in the control medium ( 3N BBMT) was rated as "trace" to "excel­lent" in comparison to growth in other media; most isolates were considered to show "good" growth (Fig. 110-112). There was no growth in any isolate in 3N BBMT in the dark, and in the light under anaerobic conditions only K107 showed a trace amount of growth. Growth of all but four of the isolates ( 23, 24, 25, K107) was enhanced by at least one of the carbon sources. Ribose increased growth in isolates 21, 26, 1621, K18, K44, K57, K182, 1426, 1427, 1482, 1485, 1487, 1597, and 1598 under aerobic conditions. Anaerobic conditions in the presence of ribose resulted in in­creased growth in all of the above except 26, I621, and K 182. Growth in the dark in ribose-supplemented medium was observed for Kl8, K44, K57 (aerobically) , 1487 (anerobically-a minute amount), and I598 (both aerobically and aner­obically. Glucose enhanced growth in isolates 22, 27, 1426, 1427, and anerobically in 1482. Fructose increased the amount of growth in isolates 1426, 1427, and aner­obically in 1482. Isolate 23 grew slightly anaerobically, and isolate K57 grew slightly aerobically in glucose in the dark. Isolate 22 continued growth in the dark after a transfer to new media. KS 7 showed a "trace" amount of growth in fructose aerobically in the dark. Isolates 22, 23, 24, 25, and 26 were entirely inhibited in fructose, and isolate 1621 grew only slightly. Both D-and L-arabinose did not increase growth, or inhibited it, in most iso­lates in the light, although all grew with these sources except for K 107 in L-ara­binose. There were only "trace" amounts of growth in five of the isolates ( 22, 23, 25, 26, I582 in D-arabinose; 22, 23, 24, 25, 1487 in L-arabinose), and no growth of any occurred in darkness. Xy!ose supported at least some growth in three-fourths of the isolates, and in Kl 07 growth was rated "good." Isolates 22, 25, 26, 27 grew slightly anerobically in xylose. No growth of any of the isolates occurred in darkness. Pyruvate and acetate both supported growth in isolates 26 and 27. Isolate 27 grew quite well aerobically in acetate and anerobically in pyruvate: isolate 26 grew only slightly. Isolate 1427 grew slightly in pyruvate both anerobically and aerobically, and isolate 1482 grew slightly aerobically in acetate. No other isolates grew at all in either medium in light and none grew in the dark. The inhibition of certain of the isolates by fructose and the enhancement of many by ribose to a far greater degree than by either glucose or fructose are the most distinctive patterns of carbon utilization seen in these isolates. Heterotrophy is definitely not a pronounced attribute in any, if indeed, it is present at all. The several isolates of a species (as Kl8, K44, 21-Schizothrix calcicola; 1482, 1487, 1597-S. calcicola v. glomerulata; 22, 24-0scillatoria lutea v. glomerulata) showed identical or very similar patterns. Elfects of Various Nitrogen Sources Investigations of blue-green algal nitrogen metabolism have been made for over 50 years. Since the confirmation of the occurrence of nitrogen fixation in some blue-green algae (Drewes, 1928), the majority of work has been related to this phenomenon; reviews of the subject have been made by Fogg ( 1956a, 1962), Nicholas ( 1963), and Stewart ( 1966). Economic aspects have not been ignored either (Singh, 1961 ) . Reports of nitrogen fixation in the Oscillatoriaceae {Cope­land, 1932) have since been disproven, although there has been recent interest in Trichodesmium ( = Oscillatoria) as a possible nitrogen fixer (Dugdale, Goering, and Ryther, 1964) ; the capacity for nitrogen fixation seems to be found only in heterocystous genera. Fay et al. ( 1968) are of the opinion that the heterocyst may actually be the location of nitrogen fixation in blue-green algae. Pringsheim ( 1913), in the first investigation definitely known to use axenic cultures of blue-green algae, studied the effects of a number of organic nitrogen sources on Oscillatoria brevis, 0. tenuis, and Nostoc sp. and found that a wide range supported growth. Maertens ( 1914), working with various species, and Harder ( 1917), using an endophytic Nostoc punctiforme, also successfully grew their isolates with many nitrogen sources . . .\lien ( 1952) studied the effect of various cultural conditions upon a large num­ber of blue-green algae, including several Oscillatoriaceae (Oscillatoria, Phor­midium, Lyngbya). She grew isolates on a variety of nitrogen sources and found NO:i-and NH,+ to be used by all, some amino acids by most, and casein by many. The prevalence of N03-and NH,+ as good nitrogen sources has been confirmed by many studies since then (Magee and Burris, 1954; Kratz and Myers, 1955; Pintner and Provasoli, 1958; Van Baalen, 1962; McLachlan and Gorham, 1962; Volk and Phinney, 1968; Kantz, 1968). Urea has been found to be a good source of nitrogen, though rarely as good as N03-, by Kratz and Myers ( 1955 ), Van Baalen ( 1962), McLachlan and Gorham ( 1962), and Kantz ( 1968), while Volk and Phinney ( 1968) found urea inhibited growth in a strain of Anabaena spiroides. These investigations have also pointed out that some of the amino acids support growth, although more seem not to, and results vary widely with the organism used. In addition to these studies on the effect of various nitrogen sources on the growth of some of the blue-green algae, a few have also been made in relation to ecological or morphological characteristics. Nitrogen fixation studies have, of course, been the subject of most. The limitation of growth due to available nitrogen in the coccoid Microcystis aeruginosa was demonstrated by Gerloff and Skoog ( 195 7) . Nitrogen cycles, primarily in nitrogen fixing species, in freshwater areas are discussed by Billaud ( 196 7 ) . Fogg ( 1949) , in his study of heterocysts in Anabaena cylindrica, found that the formation of heterocysts was inhibited by NH,+ and that heterocyst formation was usually inhibited by the presence of much available combined nitrogen. The same general results were obtained by Mickel­son, Davis, and Tischer ( 1967). The effect of inorganic nitrogen sources on mor­phology of A nabaena doliolum was investigated by Singh and Srivasteva ( I 968 ) . The possible use of differential growth in a range of nitrogen sources as a sup­plementary taxonomic attribute has been considered by several investigators of the green and blue-green algae (Cain, I 966; Smith and Bold, I 966; Groover and Bold, I 969; Kantz and Bold, I 969). Growth of the axenic isolates used in this investigation in various inorganic and organic nitrogen sources was tested to see if results of such tests might be of taxo­nomic significance, and to ascertain what the effects of the various nitrogen sources on the morphology of the isolates might be. Clean culture tubes ( 100 mm X 10 mm) were soaked for 3 days in deionized water which was changed daily. The tubes were then dried, plugged with cotton, and autoclaved. The control medium was IN BBMT; an amount of nitrogen equal to that of the NaN03 in IN BBM was added to BBMT-N in preparing the other media. Eleven other sources, including gelatin, were tested as well as growth in the absence of combined nitrogen. The nitrogen equivalents were, in each case, dissolved in I0-25 my deionized water and added by Millipore filtration to the sterile BBMT-N made up to 975-990 ml. The pH of the BBMT-N was 7.2; Table 5 gives the initial pH of each medium. The media were pipetted into the tubes and allowed to incubate at room temperature for 3 days so that bacterial con­tamination, if any, could be detected. Two sets of duplicate tubes were inoculated. The inocula were grown in BBM-K, which contains half as much NOa as IN BBMT, in an attempt to minimize the effects of residual nitrogenous compounds. That this method does, indeed, reduce carry-over effects into the new media was seen in the test run with no combined nitrogen source. Some cultures transferred from 3N BBM to BBM-N showed fair growth at first although color changes soon began, and growth appeared to have ceased in all cultures after 1 week. Cultures transferred from BBM-K, however, showed no or only an extremely minute amount of growth for the first day or two. Where only "fair" or "trace" growth was recorded, subsequent transfers on these media might result in eventual death of the isolate; such transfers were not made. The amounts of growth in the various media as macroscopically determined, and the pH (except for gelatin and in the absence of nitrogen) at the end of 3 weeks' growth under standard conditions are summarized in Table 5. All of the isolates grew well with NaN03 as the nitrogen source; the pH of all the media increased during growth. With the exception of isolate KI 07, the vari­ous pH increases appear relative to the amount of growth; KI 07 grows more slowly than the other isolates, yet the terminal pH (8.3) was the second highest of the group. Microscopic examination revealed that the trichomes appeared gener­ally to have a low percentage of abnormal or irregularly shaped cells and few distinct granular inclusions. They corresponded in morphology to the descriptions of the isolates given earlier in this report. Gelatin also was a good nitrogen source for all the isolates tested. All cultures showed growth, graded as "good" or "excellent," both throughout the tube and on its surface. Further discussion of growth in gelatin is def erred to a later section. Ammonium nitrate and ammonium sulfate, lX urea, and lOX urea supported growth rated as "good" or "excellent" in most isolates. In many, however, the cell morphology appeared quite different from that in NaNOa in that numerous gran­ules and irregular cells frequently occurred. One group of isolates (21, Kl8, K44, KS 7, 27) did not show much difference in morphology in any of these media as compared to NaNO". The pH in NH4N03 and (NH4)2S04 was lowered some­what in all but isolate Kl82, where there was no change; in urea, there was usu­ally an increase in pH, although a decrease was found in some isolates, and in some the pH remained stable. All isolates grew in NaN02, but except for isolates 24 and !482, macroscopic growth was less-sometimes substantially so-than in NaNO". Some large num­bers of cellular inclusions (isolates 22, 23, 24) ; others were indistinguishable from the set grown in NaNO" (21, !426, !482) . Uric acid supported "excellent" growth in two isolates, "good" growth in nine, and "fair" growth in nine. Cells were frequently irregular, however; many hormo­gonia were seen, and the cells were often brownish-green or somewhat bleached. Growth in casamino acids, succinamide, and uracil was graded as "good" to "no" growth. Casamino acids supported "fair" growth in six, "trace" growth in eleven, and "no" growth in three cases. With succinamide, five isolates were con­sidered to show "good" growth, eight "fair," and seven "trace" growth. The cul­tures were in many cases (see Table 5 ) yellowing at the end of 3 weeks in these media. Microscopically, they also appeared to have aged prematurely in that cross­walls were frequently indistinguishable; many hormogonia and dead cells were present; and false branches were seen in those isolates which had been found to produce them with age. Dense granular inclusions were of rare occurrence. Hydrolysis of Gelatin The use of gelatin hydrolysis as a supplementary attribute has been a common test for some time in bacteriological studies; application of the test to a wide variety of algae by several workers has shown that many algae, freshwater and marine, secrete an extracellular gelatinase (Tanner, 1923; Pringsheim, 1951; Allen, 1952; Tazawa and Miwa, 1953; Kessler and Czygan, 1967; Groover and Bold, 1969; Kantz and Bold, 1969). The gelatin medium was prepared by first melting 100 g Difeo gelatin in 400 ml deionized water, then pouring the gelatin into dialyzing tubes and soaking them in deionized water for 24 hr with four changes of water to remove soluble nitrogenous TARLE 5. Growth of Certain Axenic Isolates of Oscillatoriaceae in Media with Various Nitrogen Sources Isolate No . (NH ) 2so,f7.1J cas aeid(7.2J uric acid(7 , 2) uraci I (7 . 4) succina­111ide (7. 4) urea-IX( 7. 3) urea­lOX ( 7 . 3J urea ­lOOX ( 7. 3J gel -N 23 E(7. 9J b G(7. SJ E (6 . 8J E (6. 9) T• (7. OJ G ( 7. l J F( 7 . 2) G(7. 2J E(7 . 3) T( 7. 2J N(7. 2) 2.• ~v . auxotrophica 22 E(7. 7J F( 7. SJ E (6. 8) G(6 . 9) T• (7 . OJ E(7. 1) T+• (7. 1) F" (7 . 2) G(7. 3) G( 7. 7) N(7. 3J .2_. ~v . a uxotrophica 24 E(7. 8J E (7. 6J E (6 .8) G(6. 8J F" (6. 9) G(7. O) F> (7. lJ F•(7.0J G(7. 2J E(7 .1) N (7 .1 J 2.·~v.~ 2S E(7. SJ T(7. 2J T(6. 8J T(6. 9J F• (6. 9J G(7 .OJ c• c1. o) c• (7. OJ F (7. 2J T( 1. 1) N(7 .1) t!_. vaginatus 162 1 E (7. 7J T(7. 2J T(6 . 9J T(6. 9) T(7. 0 F (7. 0) F• (7 . 0) F(7. lJ G ( 7. 2) N ( 7. I) N(7. 2) t!_. vaginatus v , fuscus 26 E(7. 8J T(7. 2J T(6.8) F (6. 8) N(7. 2J E*(7. 1) G(7 . lJ G-(7. lJ F (7. 3J T(7. 1) N(7. 2J ! • calcicola 21 E (7 . 7J G+ (7. SJ E(6. 8) G+ (6. 9J T(7. OJ G• (7 . OJ G• (7 .OJ T(7. lJ E (7. 4) E(7 .4) N(7. 2) ! • calcicola Kl8 E (7. 7J G(7. 6J E (6 . 8) E(6 . 9) T(7. OJ F (7. lJ F• (7. 2) F" (7. I) E(7. 3J E(7. 4) N(7. 2J N ! · calcicola K44 E(7. 9J G(7. SJ E (6. 7) E(6. 4J T(7. OJ F (7. 2) F• (7. 2J F" (7. lJ E(7. SJ E(7. 3) N(7. 3) !· calcicola v. vermifor11is KS7 E(7. 6J G(7. SJ F (6 .8J F(6. 9J N(6. 9J G(7 .0) F(7. 1) T(7. 0) E• (7. SJ G(7. 7) N(7. I) N !· calcicola v . ~ 27 E(7. 9J F(7. 6J E(6. 8J E(6. 8) T (7 .OJ G(7 . I) G(7. lJ G(7 .·2) E(7. 6J E (7. 8) T (7. 3J !· calcicola v. spiralis K107 E (8. 3J T(7. 2) T(7 .O) T(7 .1) T• (7 . 1) G• (7. I) c• (7. lJ F • (7. 2) T(7. 1) G(7. 4) N ( 7. IJ !• calcicola v. ~ K182 G(7 . SJ F (7 . 4J G(6 . 9J G(7 . 0J N(7. I) F (7. IJ G(7. 2J F(7. 2J F (7 .6J F (7. I) N(7. 3) N !· calcicola v . glomerulata 1426 E(7 . 9J T(7. 4J G(6. 8) E (6. 8) F(7. 0) F(7. 2J F(7. 2) T(7. 2) G(7. 4) G(7. 3) N(7. 2) N !· calcicola v. glomerulata 1427 E(8. OJ F( 7 . 6J G(6·. 8J E(6. 7) T(7 . OJ F (7. 1) F(7. 2) T(7 . I) G(7 . 8J G(7 . 9) N(7. I) ! · calcicola v, glomerulata 1482 E(8. OJ E(7 . 7J G(6. 7J E (6. SJ F( 7. I J G(7. IJ c· c1.1) c• c1. 2J E(7. 7J G(7. 2) N(7. 3J !· calcicola v. glomerulata 1487 E (7. 9J F (7. 6) G(6. 8) E(6 . 8J T(7. lJ F (7 . 1) T(7. IJ T (7. l J G(7. 4) G(7. 3) N(7. 2) !· calcicola v. glomerulata 1597 E(7. 9J F (7. SJ G(6. 8J E(6. 9J T(7. OJ F(7. lJ T(7. lJ T(7. 1) G(7. 4) G(7. 3) N(7. 3) !• calcicola v . glomerulata 1598 t:(8,S) G(7 . 8) E (6. 8J E(6. 3J F" (7 . oJ G(7 . lJ F" (7 . 2) F"(7 .1J E (7. 4) E(7. 2J N(7. 3) !· calcicola v. amorpha 148S E(7.9J T (7. S) G(6. 8) G(6 . 9) F (7 .OJ F(7. 2) F• (7. lJ T(7 . 1) G(7. 8J G(7. 2) N(7. 2J E = Excellent growth (covering the sides of the culture tube and surface of the medium) G • Good growth F • Fair growth T • Trace growth N • No grow t h al 1 values based on macroscopic observ ations : ~!: ~~ ~~= ::~!~: =~t!~m~ :!e!~0 ~~~=!~0:nder standard conditions indicates yellowing or redness of alga by end of 3 wee ~ - substances in the solution. The BBM stocks for 1 liter (except for NaN03) were added to deionized water to make 600 ml of solution. The dialyzed gelatin was added to this BBM, melted, and poured into tubes. The latter were then autoclaved and allowed to resolidify. Deep stab inoculations were made of each of the 20 axenic isolates. Duplicate tubes were inoculated for two separate tests. Inocula were taken from agar slants. All of the isolates grew well in the medium, using gelatin as the sole nitrogen source. Isolates K 107, Kl8, and K182 entirely hydrolyzed the medium within 6-9 weeks. Isolate 1485 showed slight hydrolysis at 13 weeks, as did isolate I598 at 14 weeks. At 18 weeks, isolates 1426, 1427, and I487 showed hydrolysis at the top of the tube. No further change was observed in any isolate after 18 weeks. Isolates 1426, 1427, 1482, 1485, 1487, I597, I598, K107, and K182 secreted a blue pigment into the medium. Isolates K 44 and K5 7 secreted a blue-green pig­ment, and isolate 26 secreted a purplish-red pigment. These pigments were also secreted when the isolates were grown on nutrient agar or proteose-peptone agar, although apparently always in lesser quantities. Secretion of pigments on complex organic media was also noted by Kantz ( 1968) in Nostoc and A nabaena. Here, as in his isolates, appearance does not signify a breakdown of cells. Isolates 22, 23, and 24 all grew well throughout the tubes, but in 3-4 weeks iso­late 23 became colorless. Isolates 22 and 24, which are very similar morphologi­cally to 23, remained green and continued to grow for several months. Hydrolysis of Starch The presence or absence of extracellular amylasic activity has been used as a supplementary taxonomic criterion by several investigators of the Chlorophyco­phyta and Cyanophycophyta (Mattox and Bold, 1962; Bischoff and Bold, 1963; Cain, 1963; Brown and Bold, 1964; Wiedeman, 1964; Smith and Bold, 1966; Groover and Bold, 1969; Kantz and Bold, 1969). Harder ( 1917) found that starch could be used as a carbon source in both light and dark by an endophytic Nostoc punctiforme. The digestion of a soluble starch into diffusible compounds is the result of enzymatic reactions of the a-and fl-amylases; these amylases are secreted by many microorganisms. It is a matter of some question whether the en­zymes and mechanisms are really the same in all cases. Soluble potato starch ( 0.1 g/ liter) was added to 3N BBM and solidified with 1.5% agar. The autoclaved sterile solution was poured into sterile petri dishes, which, after solidifying, were inoculated in the center with a small amount of each of the 20 axenic isolates. Duplicate plates were inoculated for three separate tests. After 2 weeks' growth under standard conditions, the plates were flooded with a dilute I2-KI solution, followed by washing with deionized water. All the isolates showed at least a small colorless zone. In some cases the size of the zone appeared to be correlated with the amount of growth; thus, the agar on which isolates 26 and I621 grew was completely colorless as they entirely covered the plate while that of K107, which grew only slightly, was deep blue except for a narrow halo around the plant-mass. Other isolates did, however, seem to show differential responses. Isolates K18, K44, and 21 showed complete hydrolysis of the starch. Isolate K57, which is identical morphologically to K18, K44, and 21 although it has a slightly different plant-mass form and color-change pattern, showed a wide halo, but also a clear band of unhydrolyzed starch. While isolates 22, 23, 24, and 25 grew well on the starch agar, they showed only slight hydrolysis. Isolate 27, which is not fast-growing on the starch media, nevertheless showed a wide halo. Isolate K182, 1426, 1427, 1482, 1485, 1487, 1597, and I598 all showed clear halos. The presence of amylases reacting similarly to those of green algae in the blue­green algae is not unexpected as cyanophycean starch has been shown to have the same properties as does amylopectin of "higher" plants. Sensitivity to Crystal Violet The differential inhibitory effect of adding various concentrations of crystal violet to standard media has been used as a supplementary taxonomic attribute by Bold and coworkers for a number of species of green algae with varying degrees of success (Mattox and Bold, 1962; Brown and Bold, 1964; Groover and Bold 1969). The crystal violet plates were prepared by adding 10 different concentrations of crystal violet to 3N BBM. A stock solution was made by dissolving 1 g crystal violet (Difeo) in 100 ml deionized water. The stock was used to prepare media with the following concentrations of crystal violet: 0.00001 c;'r, 0.00002 % , 0.000045 % , 0.0001 %, 0.0002 % , 0.00045 % , and 0.001 % Three additional solutions were made by weighing the necessary amounts of crystal violet and adding them directly to the 3N BBM to give final concentrations of 0.002 % , 0.0045 % , and 0.01 % per liter. The solutions were solidified with 15 g agar, autoclaved, poured into small sterile petri dishes, and inoculated at once with the axenic isolates. They were then placed in the culture room and allowed to grow for 2 weeks. Tests were done twice in duplicate. Table 6 summarizes the growth of the isolates after 2 weeks. The blue-green algae tested here appear on the whole to be somewhat more sen­sitive to crystal violet than the green algae which have been heretofore investigated. The greatest degree of resistance (growing on 0.002 % CV agar) was shown by iso­lates 22 and 24 (Fig. 116), considered on all criteria to be duplicate isolates of Oscillatoria leuta v. auxotrophica. The only isolates ( 23 and 1482) that grew on 0.001 % CV agar and not above were different species ( Oscillatoria lutea and Schizothrix calcicola v. glomerulata) . The most sensitive organisms (Fig. 115) were isolates 21 , 26, 27, K57, and K107, none of which grew above a concentra­tion of 0.0001 % ; these isolates (except for 21 and K5 7) are all quite distinct from one another in several other respects. Seven isolates grew on 0.002 % CV agar, but not above it (Fig. 114) : four grew on 0.00045 ?'c CV agar, but not above it (Fig. 113). The differences in growth by one concentration of duplicate isolates of a variety or a species ( K 18 and 21, 1482 and 1487, etc.) are almost certainly not sig­ TABLE6. Inhibition of Axenic Oscillatoriaceae on Crystal Violet Agar at Di!f erent Concentrations Concentr1tion ill Isolate No. • 0000 I • 00002 • 00004 5 • 0001 • 000:? ,00045 , 00 I .002 • 0045 .01 Ł· ~ 23 G­ T T Ł· b!!!.!. v. auxotro2hica 22 T T T­ Ł. ~v. auxotro~hica 24 G­ T T T­ T · T­ T­ Ł . .1..2.!!.! v . ~ 25 G+ T T T ~· vag inatus 16:?1 G­ G­ T T ~· vaginatus v. ~ 26 T T 2-· ~ 21 C+ T 2_. ~ K18 G+ T T­ 2_. ~ K44 C• T T­ 2_. ~v. vertni form is KS7 2-· ~v. !l.!!..!!.!..!. 27 C+ T 2-· ~v. sp i r a Iis K 107 T 2_. ~v. ~ K 182 G+ G­ T - 2-· ~v. ,i:lomerulata 1426 G+ T T­ 2-· ~v . g l omerulata I 427 C+ T T­ 2_ . ~v . Elomerulata 1482 C+ C­ T T­ T­ 2_. ~v. llo11e r u l ata 1487 G+ C+ T T T­ 2_. ~v. ~lomerulata 1597 T T 2_. ~v. R. lomerulata 1598 T T T­ 2_. ~v. a111orph a 1485 G­ T T­ E . Exce 11 ent ,1?:rowth--same as control (3N BB•) G . Good ~rowth T .Trace growth; T-indicates origi nal inoculum rerna i ned green . but that no or only .very few tricho11es grew aw av from it No growth ' . nificant. The only obvious separation seems to be that most of the larger isolates grew at slightly higher concentrations than did the smaller ones. Isolates 26 and !482 are exceptions to this generalization. Effect of Vitamins Three vitamins, B12, thiamine, and biotin, have been shown to be growth factors required singly or in combination by a number of algae. Reviews of auxotrophy in the algae have been made by Lewin ( 1961 ) and Droop ( 1962) . In the blue-green algae, reports have been mainly of studies of marine species ( Pintner and Provasoli, 1958; Van Baalen, 1961, 1962). Van Baalen's study of 15 isolates revealed a B12 requirement in more than half of them. Kantz and Bold ( 1969) reported an isolate of Anabaena utermohli in which growth was greatly enhanced by the addition of a vitamin mixture, but whether this represented an absolute requirement was not determined. Preliminary tests were made to establish whether any of the axenic isolates had a vitamin requirement. The cultures were transferred from 3N BBM + B12 + Eagle's vitamin mixture to 3N BBM without vitamins. Successive transfers were then made to the vitamin-free medium. For the first month and a half no differ­ences in growth were noted. By the end of 3 months, during which three transfers had been made, three isolates, 22, 24, and 25, showed only minute amounts of growth on agar plates. Microscopic observation showed that even filaments in 1­or 2-week-old culture<; contained numerous granules and vacuoles and had very thick sheaths. The isolates were transferred again to four sets of media: 3N BBM, 3N BBM + B12, 3N BBM + Eagle's, 3N BBM + B12 + Eagle's. Both liquid and agar-solidified media were used. After this transfer, isolates 22, 24, and 25 were no longer viable on 3N BBM without vitamins. The other isolates showed no difference on agar with any of the four media (Fig. 117). Isolates 22, 24, and 25 showed greatly increased growth in 3N BBM + B12 and in 3N BBM + B12 + Eagle's. Reciprocal transfers were then made, and good growth of the isolates continued only on those media containing B12 (Fig. 118). After two successive transfers on 3N BBM + B12, the cultures were once again flourishing. Growth in liquid media showed more differences than that on agar. After 2 weeks, isolates Kl8 and K44 showed slightly more growth in all enriched media than in 3N BBM. Isolates K107, 22, 24, 25, 26, and 1621 grew better in B12 and in B12 + Eagle's than in other media. Isolates 1426, 1427, 1482, 1485, 1487, 1597, and 1598 showed slightly more growth in 3N BBM + B12 + Eagle's than in the other media, but as growth was very good in all, macroscopic determination of differences was difficult. Some of the unialgal isolates were tested on 3N BBM without vitamins, 3N BBM + B12, and 3N BBM + B12 + Eagle's during the course of this investigation. Isolates 5, 1386, and 1390 did not show significant growth after three to four suc­cessive transfers not containing B12. Whether some of the other unialgal isolates might have some vitamin requirement, or at least show increased growth with vitamins, if they were in axenic culture or had different contaminants in the uni­algal state is impossible to determine without further work. The results from these tests are similar to other, previous ones with blue-green algae in that no absolute requirement for any vitamin except B12 was demonstrated, although something in the Eagle's mixture enhances growth in liquid media of several of the isolates. Continued growth of three isolates (22, 24, and 25) in axenic culture was impossible to maintain without the addition of B12, but which analogues are necessary is not known. Effects of Varying the pH One of the classical tenets of culturing blue-green algae has been the require­ment for alkaline growth conditions. For some organisms, a pH as high as 10.0 has been considered optimal (Allen, 1952; Kratz and Myers, 1955; Gerloff and Skoog, 1957). More recently, the use of better buffer systems has allowed for lower pH ranges to be tolerated by some of the same organisms ( McLachlan and Gorham, 1962 ). Since the basal medium for the morphological studies in this investigation was 3N BBM, the pH of which falls in the range of 6.5-6.8, and since blue-green algae are encountered in a wide range of habitats, all of which certainly do not continually have high pH values, comparative studies of some of the isolates at various pH's are of interest. Seven sets of media were prepared with initial pH's of 4.0, 5.0, 6.0, 6.5 (3N BBM ), 7.0, 8.0, and 9.0. Those at pH 4.0 and 5.0 were citrate-buffered; those at 7 .0, 8.0, and 9.0 were Tris-buffered. The control, 3N BBM, and the 6.0 medium were phosphate-buffered. Terminal pH's (taken after 3 weeks growth) in most cases were only slightly higher than the initial pH in the citrate-and Tris-buffered medium, while those of the phosphate-buffered media often rose to 8.0-9.0. Twenty isolates, 10 axenic and I 0 unialgal, were selected; they were isolates 3, 4, 18, 23, 25, 26, 27, 43, Kl8, K44, K55, K57, K58, K107, K182, 1427, 1487, I617, I621, and I622. These isolates included representatives of a wide range of morphology and also had been isolated from a wide range of habitats (see Table 1). Duplicate flasks were inoculated for two separate tests and incubated under standard conditions. Macroscopic and microscopic observations were made at the end of 3 weeks. Table 7 summarizes the appearance of the flasks after 3 weeks. As can be seen, despite the good growth normally seen in 3N BBM, growth in many isolates was as good or better at pH 7.0-9.0 as at 6.5 (isolates 4, 1487, K44, K57, K107, K182 ). Others ( 18, 25, 26, 27, 3, K18, K58) had excellent growth at points anywhere between 5.0 and 7.0-9.0. A few (K55, 23, 43, I621, I622) were sharply delimited at one or two pH's very close to that of 3N BBM. K58 survived in all but the lowest pH, but growth was slow in all, and differences could not be seen within 2-3 weeks. I617 grew excellently at 5.0, 6.0, and 6.5 and only enough to be considered "fair" above 6.5. Macroscopically, the plant-mass varied regularly with pH in some of the isolates. Growth in K44 and K18 was filamentous with a thin layer of filaments forming on the surface of the media and on the sides and bottom of the flask at pH 6.0-7.0, hut discrete clumps of the algae occurred at 8.0 and 9.0 (Fig. 120). Isolate I6 l 7 was reddish-brown at 5.0 and 6.0, but distinctly gray-brown at 6.5. Isolate I622 was bright green at 6.5 and 7 .0, olive-brown at 8.0 and 9 .0. Th!'.se changes in color were accompanied by changes in the cell or trichome morphology. The original inocula of isolates 18, K44, and K57 remained green at pH 4.0, but no growth occurred. Isolate 3 was the only one which grew even slightly at 4.0 in the 3-week period. It had been isolated from a bog soil sample. While growth was better at the higher pH's, there was tolerance of the acidic media as well as of the alkaline ones. There were numerous microscopic differences observed in some of the isolates at different pH's. No visible differences, however, were observed in isolates 18, K44, Kl 8, K58, 27, and 43 in any of the media. TABLE 7. Growth of Certain Isolates of Oscillatoriaceae at Various pH's at 3 Weeks Ailsie F. Baker and Harold C. Bold Isolate !.:.!!. s.o 6.0 6.S 7.0 8.0 9.0 3 T E E E E G F 4 N T T T G E 18 N T E E E E E N N N G E T T 25 N N N G G F F 26 N N N G F F 27 N N N E E E 43 N T E G G G G K18 N N T E E E E K44 N T G G E KSS N T T E G T T KS7 N N T F G F G KS8 N G G G G G F K107 N N N G G E E K182 N N T T G G G 1427 N N F G G G 14 87 N N T G G E E 1617 N E E E F F F 1621 N N N E G N N 1622 N N N E F F E = excellent G = good. F = fair T s trace N = no growth as macroscopically and microscopically determined with reference amount of growth, plant-masi color, and morphology Isolate 1427 at 6.0 (the lowest at which it grew) had many terminal, short, curving branches one to three cells long (Fig. 119). These branches were also seen in 1426 in ammonium sulfate medium, but they were not elsewhere observed. Isolates 1427 and 1487 had many crooked trichomes and irregularly shaped cells in lower pH's while at higher ones cells were very regularly arranged. A striking characteristic of isolates 23 and 25 was the presence of gas vacuoles in alkaline media and their almost complete absence below 7.0 (Figs. 121-122). Isolate I621 was characterized by dark granules along the crosswalls in 3N BBM and at 7.0, but at 8.0 and 9.0 numerous granules were scattered throughout the cells (Figs. 123-124). In I617 granules along the crosswalls occurred at 5.0 and 6.0, but not in 3N BBM in this experiment-3N BBM contained dark granules scattered throughout the cells. Isolate K55 showed the normal morphology at 6.5 and 7 .0, but in other media cells were constricted at the crosswalls; sheaths were quite thick; and vacuoles occurred at 8.0 and 9.0. Isolate 4 at 6.5 was almost en­tirely hormogonial; at 7 .0 it was filamentous, and the cells were generally black from numerous granules. At 8.0, about half the cells had dense granules, and at 9.0 only a few cells were densely granulate (Fig. 125). Isolates which did not grow well at the higher pH's generally appeared pre­maturely aged; granules, vacuoles, copious sheaths, and extensive hormogonia formation were characteristic. Discussion This report summarizes an investigation of variation in certain Oscillatoriaceae, an investigation primarily concerned with coordinating variation with taxonomy. The data herein summarized have shown that constant characteristics may be ob­tained under constant conditions. The problems encountered in trying to classify members of this family accord­ing to the classical system of Gomont ( 1892 ) were alleviated by the revision of the family by Drouet in 1968, but a whole new set of difficulties was precipitated at the same time. There are fewer choices to make, fewer answers to arrive at, and fewer chances of deciding that the organism in question really corresponds to the description, since the descriptions allow for almost every conceivable variation. An organism, however, can frequently be collected and identified quite rapidly as one of the 23 species of Oscillatoriaceae recognized by Drouet. If the isolate is merely kept in the laboratory or if it is grown in culture, and if it is observed repeatedly, changes may occur in the characteristics which were apparent when the identifi­cation was made. If these variations can occur in the laboratory, it is only reason­able to assume that they also can occur somewhere in the field. It would thus seem somewhat irrational to dismiss summarily all cultures that vary from the "natural" states as revealing abnormalities and unnatural phenomena, a conclusion voiced by some phycologists. Some cultures continue to look exactly as they did when col­lected; some seemingly grow better, since some collections are certainly made from what must be less than optimal habitats. Some cultures do deteriorate, for there is no one common best laboratory habitat any more than there is one common out­door habitat. That differences are not necessarily found under different conditions, however, is seen in the example of isolate 5 and 39 ( Porphyrosiphon notarisii). Isolate 5 was collected in a pool in Austin, Texas; isolate 39 was growing on a beach in Monterey, California. They looked alike when observed at the time of collection, and the similarities were maintained in culture, both on agar (3N BBM) and in liquid ( 3N BBM or VSE-von Stosch's enrichment of sea water). On the other hand, two isolates ( 12 and 34), Microcoleus lyngbyaceus v. vermi­formis and M. vaginatus v. fuscus, respectively, represent in culture very different forms from those originally collected. When collected, many trichomes were in a single sheath; isolate 12 was identified as M icrocoleus (sensu Gomont), and iso­late 34 were identified as Schizothrix mexicana (sensu Gomont and also accord­ing to Drouet). In these cultures in the laboratory only one trichome per sheath was ever observed, and in the case of S. mexicana, there were cytological changes. It will be of interest to determine what culture conditions are necessary to maintain the multiple-trichome forms of Gomont's genera Microcoleus, Schizothrix, Sym­ploca, etc. Also unknown is whether other isolates identical in culture to 34 ( 17, 26, 11270) would, if transplanted to field locations, assume the "typical" Schizo­thrix mexicana form. It has been noted elsewhere (Cox and Bold, 1966; Kantz and Bold, 1969) that the current interest in soil algae, which rarely appear in the natural state in great enough quantity to be identified by classical methods, has led to a need for some additions to methods of classification. A corollary to this idea is seen in that enrich­ment cultures could easily give rise to a "bloom" of a species described (as in many of the descriptions given in Geitler's work) as occurring only as single cells or fila­ments. Drouet has overcome the need for using habitat as a key characteristic in all genera except Oscillatoria, and it is, of course, possible that 0. erythraea (de­scribed as "marine planktonts, forming water blooms") is a well-defined species confined to the marine plankton. If it is, it seems possible that there are other such habitat-limited species as well, which possibility does not seem to have been empha­sized by Drouet. It is not now considered unreasonable to have to follow a chrysophycean, chloro­phycean, or phaeophycean alga through its life history or to have to find numerous stages of the carporporophytic spore development of a red alga in order to deter­mine the order, family, genus, or species of the organism. Even though in the blue­green algae, especially in the homocystous ones, there is little to the life history, it appears quite probable from the current work that it might be wise to observe a blue-green algal isolate over a period of several weeks before finally deciding what species it is. What part physiological studies could come to have in blue-green algal taxon­omy is at present unknown. Most physiological investigations have been made with one or a few isolates and have often been concerned with basic processes that have been shown to be quite similar in all. The presence of nitrogen-fixing strains of several species is one of the few exceptions. The possibility of using supplementary physiological and cultural characteristics, as in the Actinomycetes, for example, has been considered only rarely. Such aspects of taxonomy will most probably not be explored to any noticeable extent until blue-green algal taxonomy is thought of less in terms of vascular plants and more in terms of microorganisms. The physiological studies summarized herein represented no great departure from established data on similar organisms. The various physiological tests em­ployed were of little taxonomic value in themselves, but they were considered as further support for the reality of some of the varieties that had been erected on the basis of morphological data. The several differences between the autotrophic Oscil­latoria lutea (isolate 23) and the auxotrophic variety, 0. lutea v. auxotrophica (isolates 22 and 24), were the best indications that physiological strains exist and must be considered in laboratory studies. A method of classification based on cultures, while no doubt a great asset to the experimental botanist, would leave the field botanist somewhat at a disadvantage, unless or until correlation of characteristics and their relations to the environment are worked out. Such correlations will have to be based not only on laboratory cul­tures and field observations, but also upon transplants of known cultures to the field in a manner similar to that of Cox and Bald's ( 1966) investigation of Stigeoclo­nium. In order to be a practical tool, a future classification (and definitely an "ideal" one) must be established that combines both field characteristics from pre­liminary determination and characteristics of organisms grown in the laboratory under specified conditions. Summary Eighty-two isolates of Oscillatoriaceae, representing the genera Schizothrix, Microcoleus, Porphyrosiphon, and Oscillatoria, were obtained from soil samples, air samples, marine, and freshwater habitats. They were studied in culture in sev­eral defined media; the basal medium selected for further morphological and phy­siological studies was 3N BBM. Comparisons of unialgal and axenic cultures showed bacterial contamination had no effect on the morphology of the trichome, filament, or on the plant-mass type, but that extensive fungal contamination had detrimental effects. The isolates studied exhibited definite patterns of aging (color change, sheath and trichome changes) associated with alteration of the medium. Cultures were grown on basal medium supplemented with 11 alternate nitrogen sources and with nine carbon sources. Slight heterotrophic growth was initially seen in eight isolates. Growth of the isolates was enhanced in the light by addition of carbon compounds, most frequently by ribose or glucose. All the axenic isolates hydrolyzed starch to varying degrees, but only eight hydrolyzed gelatin. Sensitivity to crystal violet of the axenic isolates was also tested. Three of the axenic isolates exhibited a B12 requirement, and growth of several others was enhanced by addi­tion of vitamins to the basal medium. Several unialgal isolates also appeared to have a vitamin requirement. Varying the initial pH of the basal medium resulted in great variation in plant-mass type and in trichome/ filament morphology. Many of the changes seen at higher pH's were comparable to those seen as cultures grown in 3N BBM age. On the basis of morphological observations, the type of plant-mass, form of the sheaths, and morphology of the terminal cell of the trichomes were determined to be the most constant characteristics, while type of granulation and vacuolation, cell size and shape were quite variable. All morphological attributes were seen to vary under differing conditions, but the plant-mass type, sheath, and terminal cell were constant under each set of conditions, although the mature terminal cell type was frequently somewhat difficult to find. The other attributes did not appear con­stantly under any of the tested conditions. The diverse isolates included in each large species were classified as 41 varieties mainly on the basis of the plant-mass type. Keys to the isolates studied in culture have been prepared and the cultures of the organisms herein discussed have been deposited in the Culture Collection of Algae at Indiana University. Herbarium specimens have been deposited at the Chicago Field Museum of Natural History. Literature Cited ALLEN, M. B. 1952. The cultivation of Myxophyceae. Arch. f. Mikrobiol. 17: 3+-53. ALLEN, M. M., and R. Y. STANIER. 1968. Growth and division of some unicellular blue-green algae. J. gen. Microbiol. 51 : 199-202. ALLISON, F. E., S. R. HoovER, and H. J. MoRRIS. 1937. Physiological studies ,,·ith the nitrogen-fixing alga Nostoc muscorum. Bot. Caz. 98: 433-463. BILLAt..:D, V. A. D. 1967. Aspects of the nitrogen nutrition of some naturally occur­ring populations of blue-green algae. U.S. Dept. Interior, Federal Water Pollu­tion Control Administration, Corvallis, Ore. pp. 35-53. BISCHOFF, H. W., and H. C. BoLD. 1963. Phycological studies. IV. Some soil algae from Enchanted Rock and related algal species. The Univ. Texas Pub!. No. 6318. BoL"ILHAC, R. 1898. Sur la n~getation d'une plante verte, le Nostoc punctiforme, a l'obscurite absolue. C.R. acad. sci. Paris 126: 1583-1586. BROWN , R. M., and H. C. Bow. 1964. Phycological studies. V. Comparative studies of the algal genera Tetracystis and Clzlorococcum. The Univ. Texas Pub!. No. 6+17. CAIN, BRO. J. 1963. The morphology, taxonomy, and physiology of certain Chlamy­domonas-like algae. Ph.D. dissertation, The Cniv. of Texas, Austin. 1966. Nitrogen utilization in 38 freshwater chlamydomonad algae. Can. J. Bot. +3 : 1367-1378. CANABAEUS, L. 1929. Dber die Heterocysten und Gasvakuolen der Blaualgen und ihre Beziehungen zueinander. Pflanzenforschung 13: 1-48. CARR· N. G., and J. PEARCE. 1966. Photoheterotropism in blue-green algae. Bio­chem. J. 99: 28P. , G. ExELL, V. FLYNN, M. HALLAWAY, and S. TALUKDAR. 1967. Minor q uinones of some M yxophyceae. Arch. biochem. biophys. 120: 503-507. Cox, E. R., and H. C. BoLD. 1966. Phycological studies. VII. Taxonomic investi­gations of Stigeoclonium. The Univ. Texas Pub!. No. 6612. COPELAND, J. J. 1932. Nitrogen fixation by some Myxophyceae. Amer. Jour. Bot. 19: 84+. (Abstract). CRow, W. B. 1923. The variation and taxonomy of the genus Microcystis in Ceylon. New Phytol. 22: 59-66. 1924. Variation in species in Cyanophyceae. J. genetics. 14: 397--1-24. 1925. Variation in the hormogones of Lyngbya nigra Ag. J. roy. micr. soc. 45: 37-42. 1928. The morphology of the filaments of Cyanophyceae. Ann. de protist. 1 : 19-36. DEMETER, 0. 1956. Dber Modification bei Cyanophyceae. Arch. f. Mikrobiol. 24: 105-133. DROOP, M. R. 1962. Organic micronutrients. In Lewin, R. A., ed., Physiology and biochemistry of the algae. Academic Press, Inc., New York. pp. 141-159. DREWES, K. 1928. Dber die Assimilation des Luftstickstoffs durch Blaualgen. Centralbl. Bakt. 2: 88-102. DROUET, F. 1962. Gomont's ecophenes of the blue-green alga, Microcoleus vagi­natus ( Oscillatoriaceae) . Proceedings of the Academy of Natural Sciences of Philadelphia 114: 191-205. 1963. Ecophenes of Schizothrix calcicola (Oscillatoriaceae). Ibid. 115: 261-281. 1964. Ecophenes of Miclwcoleus chtonoplastes. Rev. Algol., N.S., 7: 314-324. 1968. Revision of the classification of the Oscillatoriaceae. Mono­graph 15: The Academy of Natural Sciences of Philadelphia. 370 pp. , and W. A. DAILY. 1956. Revision of the coccoid Myxophyceae. Butler Univ. Bot. Studies 12: 1-218. DUGDALE, R. C., J. J. GOERING, and J. H. RYTHER. 1964. High nitrogen fixation rates in the Sargasso Sea and in the Arabian Sea. Limnol. Oceanog. 9: 507-510. EcHLIN, P., and I. MoRRIS. 1965. The relationships between blue-green algae and bacteria. Biol. Rev. 40: 143-187. FAY, R., W. D. P. STEWART, A. E. WALSRY, and G. E. FoGG. 1968. Is the hetero­cyst the site of nitrogen fixation in blue-green algae? Nature 220: 810--812. FoGG, G. E. 1949. Growth and heterocyst production in Anabaena cylindrica Lemm. II. In relation to carbon and nitrogen metabolism. Ann Bot. N.S., 13: 241-259. 1956a. Nitrogen fixation by photosynthetic organisms. Ann. Rev. Plant Physiol. 7: 51-70. 1956b. The comparative physiology and biochemistry of the blue­green algae. Bacteriol. rev. 20: 148--165. 1962. Nitrogen fixation. In Lewin, R. A., ed., Physiology and bio­chemistry of the algae. Academic Press, Inc., New York. pp. 161-170. GEITLER, L. 1932. Cyanophyceae. Kryptogamen-Flora von Deutschland, Oster­reich, und der Schweiz. ed. L. Rabenhorst. Akademische Verlagsgesellschaft m. b. H . Leipzig. 1196 pp. GERLOFF, G. C., and F_SKOOG. 1957. Nitrogen as a limiting factor for the growth of Microcystis aerugi11osa. Ecology 38: 556-561. GoLUBIC, S. 1965a. Ober die Variabilatat zweier mariner Oscillatorien aus der Sektion "Margaritiferae." Schweiz. Z. Hydrolbiol. 27: 233-237. 1965b. Dber Konvergenzerscheinungen bei Standortsformen der Blaualgen unter extremen Lebensbedingungen. Ibid. 207-217. -----, and E. KANN. 1967. Zur Klarung der taxonomischcn Beziehungen zwischen Tolypothrix distorta Klitzing und T . penicillata Thuret (Cyanophyta) . Ibid. 29: 145-160. GoMONT, M. 1892. Monographie des Oscillariees (Nostocacees homocystees) . Ann. Sci. nat. 7. Bot. 15: 263-368, 16: 91-264. GoRHAM, P. R., J. MCLACHLAN, U. T. HAMMER, and W. K. K1M. 1964. Isolation and culture of toxic strains of Anabaena flos-aquae (Lyngb.) de Breb. Verh. Internat. Verein. Limnol. 15: 796-804. GROOVER, R. D., and H. C. BoLD. 1969. Phycological Studies VIII. The taxonomy and comparati\·e physiology of the Chlorosarcinales and certain other edaphic algae. The Cni\·. Texas Pub!. 6907. HARDER, R. 1917. Ernahrungsphysi0logische lJntersuchungen an Cyanophyceen, hauptsachlich dem endophytischen N ostoc punctiforme. Z. Botan. 9: 145-242. HOARE, D. L., and R. B. MooRE. 1965. Photoassimilation of organic compounds by autotrophic blue-green algae. Biochem. biophys. Acta 109: 622--625. -----, S. L. HOARE, and R. B. MooRE. 1967. The photoassimilation of organic compounds by autotrophic blue-green algae. J. gen. Microbiol. 49: 351­ 370. HoLLERBACH, M . M. 1928. Sur la morphologic de Tolypothrix elenkinii Holl. clans Jes conditions d'habitation naturelle et de laboratoire. Arch. russ. protistol. 2: 159-1 i8. HOLM-HANSEN, 0. 1967. Recent advances in the physiology of blue-green algae. In Environmental requirements of blue-green algae. U.S. Dept. Interior, Federal Water Pollution Control Administration, Corvallis, Ore. pp. 87-96. 1968. Ecology, physiology, and biochemistry of blue-green algae. Ann Rev. Microbiol. 22: 47-70. jAAG, 0 . 194·3. Scytonema myochrous (Dillw.) Ag., Formenkreis und Variabilitat einer Blaualge. Boissera 7: 437-454. KANTZ, P. T. 1966. Some south Texas soil algae with special reference to the genus Nostoc. M.A. Thesis, The Univ. of Texas, Austin. 1968. Taxonomic, morphological, and physiological studies of the algal genera Nostoc and Anabaena. Ph.D. dissertation, The Univ. of Texas at Austin. -----,and H. C. BoLD. 1969. Phycological Studies IX. Morphological and taxonomic investigations of Nostoc and Anabaena in culture. The Univ. Texas Pub!. 7604. KESSLER, E., and F. C. CzYGAN. 1967. Physiologische und biochemische Beitrage zur Taxonomie der Gattungen Ankistrodesmus und Scenedesmus. I. Hydrogenase, Sekundar-Carotinoide und Gelatine-Verfliissigung. Arch. f. Mikrabiol. 55: 320­ 346. KIYOHARA, T., Y. FUJITA, A. HATTORI, and A. WATANABE. 1960. Heterotrophic culture of a blue-green alga, TolyjJothrix tenuis. J. gen. appl. Microbiol. 6: 176­ 182. KOMAREK , J. 1958. Die Taxonomische Revision der planktischen Blaualgen der Tscheceslowakei. In Komarek, ]., and H. Ettl, Algologische Studien. Verlag der Tscheceslowakischen Akademie der Wissenschaften. Prag. 358 pp. KosTER, J. T. 1966. Some remarks on the taxonomy of the Cyanophyceae. Acta Bot. Neer!. 15: 57-62. KRATZ, W. A., and J. MYERS. 1955. Nutrition and growth of several blue-green algae. Amer. Jour. Bot. 42 : 282-287. LEWIN, R. A 1961. Growth factors: metabolic factors limiting growth. Phyto­flagelbtes and algae. Ency. of Plant Physiol. 14: 401-417. MAERTEN S, H. 1914. Das Wachstum von Blaualgen in mineralischen Nahrlosun­gen. Beitr. z. Biol. d. Pflanzen 12 : 439-496. MAGEE, W. E., and R. H . BURRIS. 1954. Fixation of nitrogen and utilization of combined nitrogen by Nostoc muscorum. Amer. Jour. Bot. 41: 777-782. MATTOX, K. R., and H. C. BoLD. 1962. Phycological studies. III. The taxonomy of certain ulotrichacean algae. The Univ. Texas Pub!. No. 6222. Y!cLACHLAN, J., and P. R. GORHAM . 1962. Effects of pH and nitrogen sources on growth of Microcystis aeruginosa Kutz. Can. J. Microbiol. 8: 1-11. ----, U . T. HAMMER, and P.R. GoRHAM. 1963. Observations on the growth and colony habits of ten strains of Aphanizomenon fios-aquae. Phycologia 2: 157-168. MEE1.JSE, B. J. D. 1962. Storage products. In Lewin, R. A., ed., Physiology and biochemistry of the algae. Academic Press, New York. pp. 289-313. MICKELSON, J. C., E. B. DAVIS, and R. G. TISCHER. 1967. The effects of various nitrogen sources upon heterocyst formation in Anabaena fios-aquae. Jour. Experl. Bot. 18: 397-405. NICHOLAS, D. J. D. 1963. The biochemistry of nitrogen fixation. In Symbiontic associations. 13th Symposium of the Society for General Microbiology. University Press, Cambridge. PADMAJA, T., and T. V. DESIKACHARY. 1967. Trends in the taxonomy of algae. Symposium on Newer Trends in Taxonomy 34: 338-364. PEARCE, J., and N. G. CARR. 1969. The incorporation and m~tabolism of glucose by Anabaena variabilis. J. gen. Microbiol. 54: 451-462. PEARSON, J., and J. M. KINGSBURY. 1966. Culturally induced variation in four morphologically diverse blue-green algae. Amer. Jour. Bot. 53: 192-200. PEAT, A., and B. A. WHITTON. 1967. Environmental effects on the structure of the blue-green alga Chlorogloea fritschii. Arch. f. Mikrobiol. 57: 155-180. PINTNER, J. J., and L. PROVASOLI. 1958. Artificial cultivation of a red-pigmented marine blue-green alga, Phormidium persicinum. J. gen. Microbiol. 18: 190-197. PRINGSHEIM, E. G. 1913. Kulturversuche mit Chlorophyll-fuhrenden Mikro­organismen. Ill. Zur Physiologie der Schizophyceen. Beitr. z. Biol. d. Pflanzen 12: 49-108. ------1946. Pure cultures of algae, their preparation and maintenance. University Press, Cambridge. 119 pp. 1951. Ober fiirblose Diatomeen. Arch. f. Mikrobiol. 16: 18-27. 1967. Phycology in the field and in the laboratory. J. Phycol. 3 : 93-95. ----1968a. Bakterien und Cyanophyceen. Obereinstimmungen und Un­terscheide. Ost. Bot. Zeit. 114: 324-340. 1968b. Cyanophyceen-Problemc. Planta 79 : 1-9. SCHWABE, G. H. 1960. Zur Morphologie und Okologie einiger Plectonema-arten. Nova Hedwigia 2: 243-268. 1964. Ober Oscillatoria acutissima Kuff. Rev. Algol., n.s., 7: 296--303. SHARP, J. H. 1969. Blue-green algae from Bermuda waters: ecologically-selected variations of a single species. J. Phycol. 5: 53-57. SHEN, E. 1966. Morphogenetic and cytological investigations of Chara contraria and C. zeylanica. Ph.D. dissertation, The Univ. of Texas, Austin. SINGH, H. N., and B. S. SRIVASTAVA. 1968. Studies on morphogenesis in a blue­green alga. I. Effect of inorganic nitrogen sources on developmental morphology of Anabaena doliolum. Can. J. Microbiol. 14: 1342-1346. SINGH, R. N. 1961. Role of blue-green algae in nitrogen economy of Indian agri­culture. Indian Council of Agricultural Research, New Delhi. 175 pp. SMITH, R. L., and H. C. BoLD. 1966. Phycological studies. VI. Investigations of the algal genera Eremosphaera and Oocystis. The Univ. Texas Puhl. No. 6612. SORENSEN, L. 0 ., and J. T. CONOVER. 1962. Algal mat communities of Lyngbya confi:rvoides (C. Agardh) Gomont. Pub!. Inst. Marine Science (Port Aransas) 8: 61-74. STARR, R. C. 1964. The culture collection of algae at Indiana University. Amer. Jour. Bot. 51: 1013-1044. STEIN, J. R. 1963. Morphological \·ariation of a Tolypothrix in culture. Brit. Phycol. Bull. 2: 206-209. STEWART, W. D. P. 1966. Nitrogen fixation in plants. The Athlone Press, London. SToscH, H. VON. 1964. Wirkungen von Jod und Arsenit auf Meersalgen in Kultur. In Proc. 4th Int. Seaweed Symposium (Biarritz, Sept., 1961). The MacMillan Co., New York. pp. 142-150. TANNER, H. 1923. Le polymorphisme du Tetraedon minimum. Bull. Soc. Bot Geneve 15: 115-146. TAZAWA, Y., and A. MiwA. 1953. Ober Algenproteinase. Botan. Mag. (Tokyo) 66: 77-80. VAN BAALEN, C. 1961. Vitamin B, 2 requirement of a marine blue-green alga. Science 133: 1922. 1962. Studies on marine blue-green algae. Bot. Mar. 4: 129-139. 1965. Quantitative surface plating of coccoid blue-green algae. J. Phycol. 1: 19-22. 1967. Further observations on growth of single cells of coccoid blue­green algae. J. Phycol. 3: 154-15 7. VoLK, S. L., and H. K. PHINNEY. 1968. Mineral requirements for the growth of Anabaena spiroides in vitro . Can. Jour. Bot. 46: 619-630. WIEDEMAN, V. E. 1964. Some aspects of algal ecology in a waste-stabilization pond system. Ph.D. dissertation, The Univ. of Texas, Austin. -----, P. L. WALNE, and F. R. TRAINOR. 1964. A new technique for obtain­ing axenic cultures of algae. Can. Jour. Bot. 42: 958-959. WHITTON, B. A., and A. PEAT. 1969. On Oscillatoria redekei Van Goor. Arch. f. Mikrobiol. 68: 362-376. WINTER, G. 1937. Ober die Assimilation des Luftstickstoffs