J. PROTOZOOL.11(3), 317-344 (1964). 317

Speciation and Mating Behavior in "

MELVIN GOLDSTEINt Department of Botany, Indiana University, Bloomington, Indiana

SYNOPSIS. A comparative morphological study was made of strains of a single species, a fourth group composed of strains 73 clones of Eudorina isoated from 44 natural populations and of another species, and a fifth group made up of strains of 3 grown under controlled environmental conditions. Utilizing different species. Strains with different zygote arrangements the information obtained in this study in association with the were found generally not to intercross. existing taxonomic and experimental literature concerning Eu- Intraspecific crosses indicated that a single pair of alleles controls the inheritance of mating types and that gene flow dorina a monographic treatment of the genus was prepared. occurs between strains representing different natural popula- ?\Tine species and 4 varieties are now recognized. tions. Interspecific crosses resulted in the formation of poly- Sexual compatibility was investigated with 22 heterothallic ploid or aneuploid F1 offspring which showed an abnormal pairs, 3 male strains and one female strain representing 4 spe- segregation of the mating types. Abnormal mating type segre- cies and one variety of Eudorina. Five partially or completely gation was also obtained in F2 crosses involving certain of sexually-isolated groups were found: 3 groups containing the hybrid polyploid offspring.

HE earlier investigations of Stein( 65) on Gonium and lakes or in mud samples collected from their edges. The T pectorale and Coleman( 13) on Pandorina morum mud samples were air-dried for at least 2 weeks, and then revealed that sexual isolation existed to a high degree a teaspoonful of soil was placed in a sterile petri dish and rewetted with sterile distilled water If resting stages of between natural populations of the same species. It Eudorina were present in the soil, colonies would usually ap- was of interest to investigate whether the same phe- pear within 2-4 days after rewetting. nomenon occurred in species of another colonial green Ten clones were isolated from each natural population to flagellate, Eudorina, which, unlike the isogamous assure the isolation of both mating types of heterothallic Gonium pectorale and Pandorina morum, produces strains. The colonies were isclated under a dissecting micro- scope with a fine glass pipette and placed in a watch glass morphologically differentiated sperm and eggs. After containing sterile distilled water. This washing procedure was having isolated a large number of sexual strains of repeated several times and then single colonies were placed Ezidorina and having attempted to identify these in tubes of Pringsheim's soil-water medium containing CaC03 strains with descriptions in the existing systems of (64). The tubes were then placed under illumination of 300 taxonomy, it became evident that a study of sexual ft.-c. intensity at 20°C for 2-3 weeks in which time good growth was usually achieved. Generally homothallic or parthenosporic isolation must be preceded by a monographic treat- strains could be detected in the soil-water tubes by the pres- ment of the genus. Taxonomic criteria in earlier sys- ence of thick-walled zygotes or parthenospores respectively. tems were based on information from natural popula- and a single clcne would be saved. To test for heterothallic tions, but experience with clonal cultures soon showed strains, colonies from one clonal isolate were mixed with that some of these criteria were unreliable, having been colonies from the remaining clonal isolates, and the one male and one female clone which gave the best mating reaction the result of some influence of the environment or of were saved. age. The present study therefore consists of two parts: Maintenance cf stock cultures and experimental work were I) a monographic treatment of the genus Eudorina carried out under controlled environmental conditions. The based on studies of species in culture, and 2) an in- light source was banks of cool-white standard fluorescent vestigation of reproduction and sexual isolation in tubes. The lights were automatically controlled by a clock species of Eudorina. device which allcwed 16 hr of light and 8 hr of dark daily. The temperature was kept at 20" % 1°C. The strains were maintained in half-pint (250-ml) milk bottles of soil-water MATERIALS AND METHODS medium and on slants of soil-extract agar or Volvocacean agar The strains of Eudorina utilized in this study were isolated (64) from 44 natural populations occurring in small farm ponds The following technique for mating heterothallic strains of Eudorina was published previously by Starr(64). 4ctively * This investigation is a portion cf a dissertation submitted growing male and female clones were mixed in equal amounts to the Graduate School of Indiana University in partial ful- fillment of the requirements of the Ph.D. The writer expresses (2-3 ml of each mating type) in watch glasses supported on sincere appreciation to Dr R. C. Starr for suggesting the prob- glass triangles in petri dishes. An cqual amount of fresh soil- lem and for constructive criticism and advice willingly given water supernatant was added to the watch glasses containing throughcut the research and writing; to Dr. A. Wilbois Cole- the two mating types. A 5% solution cf NaHCO.3 was added man for collection and isolation of certain strains; to Drs to the bottom of the petri dish(62) which was then covered R M. Johns, P. W. Cook, J. R. Stein and others for soil and kept at 250-350 ft.-c. intensity of illumination for 1-4 samples; and to Dr. Hannah Croasdale fcr preparation of the days. The watch glass mating was useful for morpholcgical Latin diagnoses During the last 3 years of this study the observations of the sexual process and for securing a large writer held a pre-doctoral fellowship from the National Insti- yield of zygotes for zygote germination. tutes of Health ; this support is gratefully acknowledged. t Present address. Department of Botany, McGill University, Two methods of zygote germination were employed. Method Montreal 2, Quebec, Canada. 1 was used by Starr(61) for . Actively growing 318 SPECIATIONAND SEXUALITYIN Eudorina bacteria-free male and female clones were mixed in sterile those ephemeral traits resulting from some undefined watch glasses and the resulting zygotes allowed to remain in influence of the physical or biological environment. the bright light for 5-i days. The zygotes tvere then removed from the natch glasses with a fine glass pipette and placed By modern culture techniques the phycologist has es- on 15% soil-extract agar plates. They were spread out over tablished unialgal or clonal populations of algae which the surface of the agar with a sterile bent glass rod and, if can be studied over a long period under known en- desired. inverted over a petri dish oi chloroform ior 30 sec to vironmental conditions, thereby enabling a better kill any regetative material. The agar dishes oi zygotes were judgment of taxonomic criteria. Although use of cul- then placed in a 37" oven for 2-3 days alter which time they were returned to 250-350 it.-c. of illumination at 20". To tures in taxonomic studies is not yet widespread, they observe and photograph z).gote germination, a coverslip was have been of decided value in the taxonomy of certain placed over the germinating zygotes or a hanging drop prepa- difficult genera such as Chlamydomonas( 2 1.24), ration was made. Scenedamus( 57), Chlorococcum and related genera Method 2 oi zygote germination was primarily for genetic ( 2 -4.16 .29,63) Closterium ( 15 ) , Gonium ( 6 6) , and analysis and employed non-bacteria-iree strains. Activell- . prowing male and female clones were mised in watch glasses many Xanthophycean algae( 77). and the resulting zygotes were allon-ed to mature under illu- During the last 5 years 73 clones of Eudorina repre- mination oi 250-350 it.-c. intensity for 7-10 days. The dishes senting 44 natural populations have been cultured at oi zygotes were then placed in the dark at room temperature the Indiana University laboratory. These populations ior a week. .\!ter a week the zygotes were allowed to dry cn were collected in the United States, except for 2 popu- the surface oi the watch glasses. Fol1owin.c 1-2 days oi drying at room temperature the zygotes were rewetted with sterile lations from British Columbia, Canada. and one from soil-water supernatant and placed in the light at room tem- Czechoslovakia. Initial observations indicated that the perature. .liter 2-3 days the initial supernatant was replaced 73 clones were not morphologically identical so an at- with fresh medium. and germination generally occurred within tempt was made to identify these strains with species 24 hr. Ii germination failed to occur the washing process was in the existing systems of taxonomy. It was found that continued at 2-3 day inter\-als until germination was observed. For some intercrosses good germination was obtained only by the strains could not be satisfactorily identified accord- using the sun or an incandescent lamp as the light source. ing to the systems of Smith( 58) and Huber-Pestalozzi Z?-gotes not being used immediately were stored drl- on the (30), except for Eudorina carteri a species distin- suriace oi the natch glasses in paper coin-envelopes and re- guished primarily on the basis of sexual morphology. mained viable ior at least one year. By Pascher's (48) treatment of the Volvocales they For mitotic chromosome counts, dividing colonies were fixed could be identified with Eudorina elegans or Eudorina in a 3:1 sclution oi absolute alcohol and glacial acetic acid saturated with ferric acetate and mere stained with aceto- illinoisensis ; however. Pascher was aware of the great carrnine(7). Colonies from 2-4 week old stock cultures \\.ere variation between forms included under the single transferred to Ivatch glasses containing fresh soil-water super- species, E. elegans. natant. A 5% solution of SaHC03 was added to the bottom .4 comparative morphological study of the Eudorina oi the petri dish in which the watch glass was suppcrted and placed under illumination of 250-350 it.-c. intensity shortly strains was undertaken to serve as a foundation for a beiore the 8-hr dark period. Dividing colonies were most nu- monographic treatment of the genus. The major ob- merous 3-4 hr after the onset of the light period. The material jective of this study was to reveal the most reliable ivas then picked up with a pipette. concentrated by centrifuga- vegetative morphological characters so that existing tion, and placed in the fixative. The colcnics were suitable species descriptions could be revised accordingly, and ior staining aiter 6 hr of fixation. but the best results were obtained with material fixed ior at least 24 hr. new species could be described if necessary. The sec- For staining. a small amount oi fised material was picked ond objective was to investigate the relationship be- up with a pipette and placed on a clean microscope slide. The tween Eudorina and Pteodoi-ina. since preliminary fixative \\-as allowed to evaporate until the material mas almcst observations by the writer substantiated those by dry and 2-3 drops oi acetocarmine were then added. .l cover earlier investigators who had questioned the concept glass \\-as placed carefully over the material and allowed to of the latter genus. stand at room temperature for at least 5 min. The slide was then heated over an alcohol lamp for 3 min, care being taken The most uniform populations of Eudorina could be not to let the stain I~oil. The cover glass was ringed with was obtained by transferring single colonies from stock and n.ac then read!. for observation. Preparations were not cultures into tubes of soil-water medium and allowing squashed because disruption oi cells obliterated the chromo- them to grow under 300 ft.-c. light. Colonies from some>. the resulting actively-growing populations were exam- RESL-LTS ined for all vegetative morphological traits including the construction of the colonial envelope, the size and I. Taxonomy of Eudorina shape of the colonies, and the arrangement of the Monographic studies of most algae have depended cells. The size, shape. and organelles of the cells were by necessity on naturally-occurring populations, but also noted. the information gained from such populations often The comparative study revealed that the chloro- has not been definitive enough to enable the investiga- plasts, eyespots, and contractile vacuoles were uniform tor to distinguish reliable taxonomic criteria from in morphology for all 73 clones of Eudorina. The size SPECIATIONAND SEXUALITYIN Eudorina 3 19 of the colonies and the presence or absence of pos- era1 pyrenoids associated with it ...... terior mammillations of the sheath were found to vary E. unicocca var. peripheralis 2b. with the age of the colonies within a clone and there- H. Sexual reproduction dioecious ...... fore were not considered reliable taxonomic characters. E. elegans var. elegans la. Certain variations of vegetative morphological char- H. Sexual reproduction monoecious ...... I acters proved to be stable within clones, and therefore I. Sperm packets formed by anterior tier of cells were considered reliable criteria for defining species in E. elegans var. carteri lb. Eudorina. The most distinctive of these was the degree I. Sperm packets formed at random within the and pattern of reduction in size of the anterior cells colony ...... E. elegans var. synoica lc. in older colonies. The different clones exhibited a J. Mature cells with a large central or basal graded series from no reduction in size of the cells pyrenoid which rarely divides into two ...... to a marked reduction in size of the anterior tiers of E. unicocca var. unicocca 2a. cells. A second distinguishing vegetative character J. Mature cells with a large central or basal was the construction of the colonial envelope. All pyrenoid often with one to several smaller strains possessed a double-layered colonial envelope in peripheral pyrenoids associated with it ...... which the outer layer was always confluent. However, E. unicocca var. peripheralis 2b. the clones differed in the construction of the inner K. Colonies 16- or 32-celled with the anterior tier layer which in the majority of the strains was con- of cells markedly reduced in size in 32-celled fluent while in other strains it was composed of distinct colonies ...... E. illinoisensis 5. individual gelatinous sheaths. The number and ar- K. Colonies 32-, 64-, or 128-celled with the ante- rangement of pyrenoids fell into two distinct catego- rior tiers or more markedly reduced in size ...... L ries: unipyrenoid and multipyrenoid. The last impor- L. Cells of anterior third or half, plus cells at tant vegetative character used for species separation random in posterior portion of colony re- was the shape of the colonies. In most strains the duced in size ...... E. sphaerica 7. colonial envelope varied from spherical to ellipsoidal L. Cells only in anterior third or half of colony while other clones were consistently cylindrical. The reduced in size ...... M modes of monoecious and dioecious sexual reproduc- M. Sexual reproduction dioecious ...... tion proved to be reliable taxonomic criteria as well, E. californica var. californica 6a. but were used exclusively at the varietal level due to M. Sexual reproduction monoecious ...... the rarity of sexually reproducing material in nature. E. californica var. tifanyi 6b.

Key to the Species of Eudorina Eudorina Ehrenberg, 1831, Monatsb. Akad. Wiss. Berlin, p. 78 and 152, taf. 11, figs. 10.4-1OD. A. Cells of colony equal in size or nearly so ...... B L. sensu Miiller, 1773, Vermtium Leu. et. jlztviatil. seu A. Cells of anterior tier or tiers markedly reduced animal infus. historia. Hauniae et Lipsiae (according to in size ...... K Printz, 1927). B. Cells surrounded by individual gelatinous Pandorina Bory sensu Dujardin, 1841, p.p. Hist. Xatur. des sheaths ...... C Zoophytes, p. 317 (according to Printz, 1927). Botryocystis Kutzing, 1843, p.p. Phyc. gen. (according to B. Cells not surrounded by individual gelatin- Printz, 1927). Kiitzing, 1849, Species Algarum, F. rZ. ous sheaths ...... D Brockhaus, Lipsiae, p. 208. C. Individual gelatinous sheaths spherical in Shaw, 1894, Botan. Gaz. 19, 282, pl. 27. shape and connected by slender strands ...... Colonies spherical, ellipsoidal to cylindrical in shape, with E. interconnexa 8. colonial envelope smooth in outline, with anterior or posterior C. Individual gelatinous sheaths hexagonal in mammillations or covered with short conical projections. Colo- nies of 16, 32, 64, or 128 cells peripherally arranged in distinct shape and not connected by slender strands .... tiers within a double-layered gelatinous envelope ; the outer E. conradii 4. layer confluent, the inner layer confluent or composed of indi- D. Colonies cylindrical ...... E. cylindrica 3. vidual gelatinous sheaths surrounding each cell. Cells of same D. Colonies spherical to ellipsoidal ...... E size throughout colony or of 'two different sizes with smaller E. Colonies covered with short conical projections cells usually in anterior tier or tiers. Cells spherical to ovoid 9. in shape with two apically borne flagella exiting the colonial E. echidna envelope through separate canals. Single radially-striated cup- E. Colonies without short conical projections ...... F shaped chloroplast per cell with one to many pyrenoids. Two F. Mature cells multipyrenoid ...... G apical and generally several randomly-distributed contractile F. Mature cells generally unipyrenoid ...... J vacuoles. Eyespots largest in the anterior tier of cells and G. Mature cells with 3 to many approximately progressively smaller in the subsequent posterior tiers or com- H pletely lacking in the posterior tiers or tier. equal-sized and randomly distributed pyrenoids Asexual reproduction with plakeal stage and inversion, form- G. Mature cells with a large central or basal pyre- ing daughter colonies. When cells of two sizes in colony, noid often with one to several smaller periph- smaller cells may or may not undergo division. 320 SPECIATIONAND SEXUALITYIN Eudorina

Sexual reproduction heterogamous with biflagellate eggs and by \\.’ille(75), but their importance as a taxonomic with biflagellate sperm forming in packets. character was not realized until later. 1. Eudorina elegans Ehrenberg, 1831. Monatsb. dkad. Cl’iss. Pascher (48) recognized that there were included in Berlin, p. 78 and 152. tai. 11, figs. 10.4-10D. the single species E. elegans several different forms, la. Eudorina elegans var. elegans (Figs. 1, 2, 3) all with cells in distinct tiers, but varying in the shape Ehrenberg, 1831, Monatsb. Akad. Wiss. Berlin, p. 78 of the colony, the number of pyrenoids per cell, and and 152, taf. 11, Figs. 10.4-1OD. Cohn, 1855* 2. Fiss. ZooE. the presence or absence of posterior mammillations. 1.1, 4. Heit. p. 436. Rabenhorst. 1868. Flora Europaea dl- Smith(58) was dissatisfied with this situation and as garuin Dzdcis et Subrnarinae, Sectio 111, Eduardum Rum- a result redefined the specific limits of E. elegans and merun, Lipsiae, p. 98-9. t\-ille, 1897, Chlcrophyceae. in at the same time created 3 new species. The diag- Eng!er, .4. and Prantl, R.. Die .Vatiirlichen Pflanzenjamilien 1st ed.. 1. Teil, rZbteilung 2.. LYilhelm Enpelmann. Leipzig. nostic characters of E. elegans according to Smith were p. 42, Figs. 16 and 19. Chodat, 1902. MafCriaitr pow la as follows: colonies spherical to ellipsoidal in shape, Flore Cryptogamiqw Sirisse, Vol. 1. Fascicule 3. S.-J.\Vyss. colonial envelope smooth in outline, cells multipyre- Berne. p. 152. Printz, 1927, Chlorophyceae, in Engler. .4. noid and not arranged in distinct tiers. In the most and Prantl. K., Die ‘Vatiirlichen Pflaizzenfanzilien, 2nd ed.. recent floristic treatment of Eudorina Huber-Pestalozzi 3. Band, Wilhelm Engelmann, Leipzig. p. 56-8. Figs. 16 and 20. Pascher, 1927. Die Siisswasser-Flora, Heit 4.. Gustav. (30) combined certain diagnostic characters o€ E. Fisher, Jena, p. 440-1, Figs. 394-401. Deducenko-Shchego- elegans according to Pascher(48) and Smith( 58). He leva, Matvienko, and Shliorhatov. 1959. Cblorophyta: \-ol- described E. elegans as having spherical to ellipsoidal vocineae, in Savich. \..P., Opredditel presnovodnikh oodo- colonies. a colonial envelope smooth in outline or roslei SSSR, Vipusk 8, Izdatelstvn .Uiadernii Sauk SSSR. slightly flattened or mammillate, and cells in distinct Moskva-Leningrad. p. 209-10. pl. 101. Fig. 1. Huber- Pestalozzi, 1961, Die Binnengewiisser, Band X1-1,5. Teil. p. tiers containing one to several pyrenoids. 640-1. tai. CXXXII. abh. 889. In the present study E. plusicocca Smith is placed Eztdorina stagnale \Voile. 1887. Fresh-water .-llgae of the in synonymy with E. elegans. According to Smith(58) l’nited States, Commenius Press. Bethlehem. Pa., p. 160. pl. E. plusicocca and E. elegans were both multipyrenoid. CI.11, Figs. 11-21, but E. plusicocca possessed posterior mammillations Eitdorina plitsirocca Smith, 1930. BirlJ. Torre! Botan. and cells in distinct tiers. The present study revealed Club 57. 362-3. that all 73 clones possessed cells in distinct tiers. Eirdorina elPganA Ehrenb. iorrna dlipsoidn sub. var. tubifera Akehurst. 1934. J. Roy. Mirrosrop. Sor. 54, 99-10.3. Although posterior mammillations can be observed in pla. I and TI. some strains and appear to be entirely absent in others, their use as a reliable taxonomic criterion is precluded Colonies spherical, ellipsoidal or ovate-ellipsoidal in shape. a with the colonial envelope smooth in outline, or at times the by their variability within any clone as function of posterior cnd flattened cr mammillate. Colonies of 16, 32. or age. As the colonial envelope expanded with age the rarely 64 cells arranged in distinct tiers with a confluent mammillations often became obliterated as was noted double-layered gelatinous mi-elope. Cells equal in size or the by Smith(58) for Eudorina unicocca (Figs. 1, 2). anterior tier of cells slightly reduced in size in 32-celled colo- Therefore. this variable trait within and between multi- nies. Mature cells spherical or ovoid in shape with a single pyrenoid clones is excluded as a distinct taxonomic radially-striated cup-shaped chloroplast with 3 to man!- ap- proximately equal-sized and randomly arranged pyrenoids. character. and E. plusicocca is placed in synonymy Eyespots largest in the anterior tier oi cells and progressively with E. rlegans. The rather ill-defined species Eudo- smaller in the subsequent posterior tiers or completely lacking Tina stagnale was retained in synonymy with E. elegans in the posterior tier. for it only differed from Ehrenberg’s description in .\I1 cells irith an equal potential to produce 32-celled colo- the size of the cells. nies; anterior tier of cells often delayd in division. Sexual reproduction heterogamous and dioecious. with all Lastly. Eudorina elegans form a ellipsoida sub. var. cells oi a colony developing into sperm packets cr eggs. Thick- tubifera was placed in synonymy with E. elegans. It walled parthenospores formed in some strains. was distinguished by the possession of ellipsoidal colo- Free-swimming colonies 8.1-156 p long and 63-83 p broad; nies in which the flagella exited the colonial envelope cells 12-20 p in diameter. through separate canals, but the observation that in Since Ehrenberg’s original description of Eudorina all strains examined in the present study the flagella elegans( 20) the circumscription of the species has were seen to exit the colonial envelope in a similar changed considerably. Ehrenberg described E. elegans manner would seem to eliminate this as a distinguish- as having a clear spherical to broadly ellipsoidal gelati- ing varietal criterion. nous envelope containing 3 2 peripherally arranged 4s a result of the present investigation, the species cells. each possessing a single eye-spot and flagellum. Eudorina elegans is distinguished from all other species His figures indicated that the cells occurred in tiers, by the combination of the following characters: The but it was Rabenhorst (54) who stated clearly that the mature cells are all equal in size or nearly so and cells were in distinct tiers and placed equidistant from contain 3 to many ca. equal-sized and randomly ar- one another. The presence of pyrenoids was first noted ranged pyrenoids (Figs. 1-3). The cells are arranged SPECIATIONAND SEXUALITYIN Eudorina 321

in distinct tiers within a confluent double-layered tion of the first tier of cells although all previous gelatinous envelope. The colonies are spherical, ellip- accounts in the literature on E. illinoisensis were con- soidal, to ovate-ellipsoidal in shape being either smooth cerned with the probable sterility of the anterior cells in outline or with a flattened or mammillate posterior rather than their sexual specialization. end. In the present study two strains were isolated into In asexual reproduction all cells are capable of pro- cultures which were identical in vegetative morphology ducing 32-celled daughter colonies, although often the and sexual behavior to E. carteri (Figs. 4, 5, 6). On anterior tier of cells is delayed in division. Sexual the basis of vegetative morphology alone these strains reproduction is heterogamous and dioecious as reported are indistinguishable from E. elegans; therefore, the earlier(25,26,58) and confirmed by the writer. In species E. carteri is reduced to a variety of E. elegans sexually active material, sperm packets and eggs are based on its unique mode of sexual reproduction. formed in separate colonies. As the penetration of Geographical distribution sperm packets into the female colonies proceeds, the Asia-India (Carter, 1858; Iyengar, 1933). packets break up into individual sperm which fertilize North America-United States (Goldstein, see Table 1). the female gametes within the confines of the maternal Strains-103hma and 104hma. colonial envelope. Depending on the strain the zygotes are found in tight cohesive clumps within the old Ic. Eudorina elegans Ehrenb. var. synoica nov. var. (Figs. 7, 8). maternal colonial enveIope or the maternal matrix Reproductio sexualis monoecia, 1-4 fasciculis spermatum breaks down and the zygotes are released singly. fortuito in colonia eff ectis, cellulis relictis functionem ovorum Thick-walled parthenospores are often formed in habentibus. strains lacking sexual reproduction, but as yet they Sexual reproduction monoecious, with 1-4 sperm packets have not been observed in sexual strains. formed at random in the colony and the remaining cells serving as eggs. Geographiral distribution A second mode of monoecious sexual reproduction Europe-Germany (Ehrenberg, 1831 ; Hartmann, 1921) ; Eng- land (West, 1904). was observed in culture for several clones of Eudorina Asia-India (Iyengar, 1933), Russia (Deducenko-Shchegoleva, identical in vegetative morphology to E. elegans. In Matvienko, and Shkorbatov. 1959). contrast to E. elegans var. carteri the cells which form Africa-Union of South Africa (Pocock, 1937). the sperm packets occur at random in the colony and North America-United States (Wolle, 1887 ; Smith, 1930; the remaining cells serve as eggs (Fig. 7). Upon matu- Prescott, 1951; Goldstein, see Table 1). Central America-Panama Canal Zone (Prescott, 1955). ration of the sperm packets the individual sperm dis- Strains-7mf, 9f, 13mf, 17m, ZZp, 34m, 35m, 40rnf, 44mf, sociate from the packet and move freely among the Slsmf, 56mf, 60mf, 62mf, 65mf, 66p, bfsmf, 7Smf, 79mf, eggs (Figs. 7, 8). After fertilization, the zygotes are and 95mf. found in tight cohesive clumps (strain 105) or the Ib. Eudorina elegans Ehrenb. var. carteri (Smith) Gold- colonial matrix breaks down and the zygotes are re- stein nov. comb. (Figs. 4, 5, 6). leased singly (strains 11, 16, 33, 38, and 63). Eudorina elegans Ehrenb. sensu Carter, 1858, Ann. Mag. Geographical distribution Nat. Hist., 111, 2, 237-53, pl. 8. Eudorina carteri Smith, 1930, Bull. Torrey Botan Club North America-United States (Goldstein, see Table 1). 57, 363, pl. 17, Fig. 5. Huber-Pestalozzi, 1961, Die Binnen- Strains-llhmr, 16hmr, 33hmr, 38hmr, 63hmr, and l05hmr. gewasser, Band XVI, 5. Teil, p. 641, taf. CXXXII, abb. 890. 2. Eudorina unicocoa Smith, 1930, Bull. Torrey Botan. Club Eudorina iElinoisensis (Kofoid) Pascher sensu Iyengar, 57, 363, pl. 17, Figs. 3 and 4. 1933, J. Linn. SOL.London, 49, 336-8, pl. 28, Fig, 1. 2a. Eudorina unicocca var. unicocca (Figs. 9, 10). Carter(6) described in E. elegans a monoecious Smith, 1930, Bull. Torrey Botan. Club 57, 363, pl. 17, form of sexual reproduction which was initiated by Figs. 3 and 4. Huber-Pestalozzi, 1961, Die Binnengewiissev, the formation of sperm packets by the anterior tier Band XVI, 5. Teil, p. 642, taf. CXXXII, abb. 892. Eudorina elegans Ehrenb. sensu Chodat, 1902, Matiriaux of cells of the colony. When sperm packet formation pour la Flora Cryptogamique Suisse, Vol. 1, Fascicule 3., was complete, the packets dissociated and the indi- K.-J. Wyss, Berne, p. 151, Fig. 76. vidual sperm moved to the posterior portion of the Colonies spherical, ellipsoidal to ovate-ellipsoidal ; colonial colony. The remaining posterior vegetative cells served envelope smooth in outline or at times with distinct anterior as eggs. Smith(58) considered this type of sexual or posterior mammillations. Colonies of 16 or 32 cells periph- reproduction sufficiently distinctive to erect a new erally arranged in distinct tiers within a confluent double- species E. carteri based on the alga described by layered gelatinous envelope. All cells are equal in size or an- Carter. Eudorina carteri was observed in nature by terior tier of cells slightly reduced in size in 32-celled colonies. Mature cells spherical to sub-pyriform in shape containing a ) Iyengar (3 1 who apparently was unaware of Smith’s single radially-striated cup-shaped chloroplast with a large description of E. carteri. Iyengar identified this mo- single central or basal pyrenoid; in older cells single pyrenoid noecious form as E. illinoisensis due to the specializa- rarely dividing into 2. Eyespots largest in the anterior tier 322 SPECIATIONAKD SEXUALITYIN Eudorina of cells and progressively smaller in the subsequent posterior marily on the basis of a single pyrenoid per cell and tiers or completely lacking in the posterior tier. the possession of posterior mammillations. In the All cells with an equal potential to produce 32-celled daugh- ter colonies; anterior tier of cells often delayed in division. present study unipyrenoid strains occur with or with- Sexual reproduction heterogamous and dioecious, with all Out posterior mammillations. However, the presence cells of a colony developing into sperm packets or eggs. Thick- Of posterior mammi~~ationsis quite variable within a walled parthenospores formed in some strains. clone for, as noted by Smith(%), when the colonial Free-swimming colonies 60-130 p long and 50-106 p brcad: envelope expands with age the posterior mammillations cells 5.5-18 p in diameter. often become obliterated (Figs. 9, 10). In addition, Eudorina unuocca Smith was distinguished pri- Smith(58) used Chodat’s figure (10, p. 151, Fig. 76)

Figs. 1-3. Eudorina elegans var. elegans. Fig. 1. Young of strain 104hma. X 430. Fig. 5. Early stage in sexual repro- vegetative colony of 56f with posterior mammillations. X 550. duction oi strain 104hma just aiter sperm-packet formation. Fig. 2. Mature vegetative colcny of j6f without posterior X 430. Fig. 6. .A late stage in sexual reproduction of strain mammillations. x 470. Fig. 3. Surface view of a mature 104hma in which all but one of the sperm packets has disso- vegetative colon?; of 56f showing multipyrenoid cells. Xote ciated. Sote that the sperm cells have moved to the posterior the radial striations of the chloroplast. X 470. Figs. 4-6. Ell- portion of the colony containing the eggs. X ,320. dovina elegans var. rartpri. Fig. 4. Mature vegetative colony SPECIATIONAND SEXUALITYIN Eudorina 323 of an interiorly mammillate form as the type specimen algae described the same three types of pyrenoid con- for E. unicocca which must also be considered in the ditions for the Volvocales and Protococcales, but con- present revision of the species description. sidered the intermediate type as multipyrenoid. How- Therefore, the present species concept of E. unicocca ever, since these strains are predominantly unipyrenoid includes the following distinguishing features: The throughout most of the vegetative phase of their life mature cells are all equal in size or nearly so and cycle they are included within the species E. unirorca. contain a large central or basal pyrenoid (Figs. 9, 10). but are recognized as a distinct variety because of this This unipyrenoid condition persists throughout the intermediate pyrenoid condition. entire vegetative phase of the life cycle, except for an Geographical distribution occasional older cell in which the single pyrenoid North America-Canada (Goldstein, see Table 1). United divides into 2. The cells are arranged in distinct tiers States (Goldstein, see Table 1). within a confluent double-layered gelatinous envelope. Strains-4smf, 5m, 72smf, 100mf, and 102mf. The colonial envelope is spherical, ellipsoidal to ovate- 3. Eudorina cylindrica Korschikoff (Figs. 13, 14). ellipsoidal in shape, and may be smooth in outline or Korschikoff, 1938, ViznaZnik prisnovodnich voclorskj, V.R. at times with anterior or posterior mammillations d. R. 4, Kiew. Deducenko-Shchegoleva, Matvienko, and Shkor- (Figs. 9, 10). batov, 1959, ; Volvocineae, in Savich, V.P., Ope- All cells possess an equal potential to produce 32- delitel presnovodnikh vodoroslei SSSR, Vipusk 8, Izdatelstvo celled daughter colonies although the anterior tier of Akademii Nauk SSSR, Moskva-Leningrad, p. 210-11, pl. 102. cells may be delayed in division. Sexually reproducing Huher-Pestalozzi, 1961, Die Binnengewiisser, Band XVI, 5. Teil, p. 642-3, taf. CXXXII, abb. 893. strains are heterogamous and dioecious with all cells Colonies cylindrical in shape, with colonial envelope smooth of a colony developing into sperm packets or eggs. in outline. Colonies of 16 or 32 cells arranged in distinct tiers When gamete-producing male and female clones are within a confluent double-layered gelatinous envelope. All cells mixed, the sperm packets immediately surround the equal in size or anterior tier of cells slightly reduced in size female colonies and penetrate the colonial envelope. in 32-celled colonies. Mature cells spherical in shape with a single radially-striated cup-shaped chloroplast containing a During the penetration process the packets break up large central or basal pyrenoid with one to several smaller into individual sperm cells which fertilize the biflagel- peripheral pyrenoids often associated with it. Eyespots largest late eggs within the confines of the maternal colonial in the anterior tier of cells and progressively smaller in the envelope. After fertilization the zygotes are found in subsequent posterior tiers or lacking in the posterior tier. tight cohesive clumps surrounded by the old maternal All cells with an equal potential to produce 32-celled daugh- ter colonies; anterior tier of cells often delayed in division. colonial envelope. In strains lacking sexual reproduc- Free-swimming colonies 60-130 p long and 50-106 p broad. tion thick-walled parthenospores are often formed. Cells 5.5-18 p in diameter. Geographical distribution Eudorina cylindrica Korschikoff (36) was originally Europe-Switzerland (Chodat, 1902). described as a 16-celled species with cyIindrically- North America-United States (Smith, 1930 ; Goldstein, see shaped colonies. In the present study 2 pairs of hetero- Table 1). thallic strains (47smf and 5hf) consistently produced Central America-Panama Canal Zone (Prescott, 1955). cylindrically-shaped colonies containing 16 or 32 cells Strains-lmf, 6p, 14p, Zap, 73p, 81mf, and 93mf. (Figs. 13, 14). The 32-celled colonies were further 2b. Eudorina unicocca Smith var. peripheralis nov. var. distinguished by a slight reduction in size of the an- (Figs 11, 12). terior tier of cells. Cellulae adultae pyrenoideum magnum centrale basaleve praebentes ; Admodum ante formationem coloniarum-fili- The first observations of sexual reproduction in E. arum unum ad aliquot pyrenoidae parva in parte chloro- cylindrica were made during the course of this study. plasti periferali visa. Strains 47smf and 50mf are heterothallic, heterogam- Adult cells with a large central or basal pyrenoid. Just ous, and dioecious. The reproductive process is typical before daughter-colcny formation one to several small of that observed in dioecious strains of E. elegans and pyrenoids appearing in the peripheral portion of the chloro- plast. E. unicocca as reported in the preceding pages. Fol- lowing fertilization the zygotes form tight cohesive In this variety of E. unicocca each cell in a colony clumps within the old maternal colonial envelope. contains a single pyrenoid which increases in size as Eudorina cylindrica possesses the following distin- the cells enlarge with age (Fig. 11). Just prior to guishing vegetative characters. Cylindrically-shaped daughter colony formation small pyrenoids arise de colonies containing 16 or 32 cells arranged in distinct novo near the periphery of the chloroplast giving rise tiers (Figs. 13, 14). The colonial envelope is confluent to an apparent intermediate condition between a and double-layered in structure. The cells are gen- typical unipyrenoid and a multipyrenoid condition erally unipyrenoid but there may be at times one to (Fig. 12). Chadefaud(8) in a comparative morpho- several smaller pyrenoids in the periphery of the logical study of the cytoplasm of the green and brown chloroplast. 324 SPECIATIONAND SEXUALITYIN Eudorina

Geographical distribution stantes ; lamella exteriore confluente, interiore e vaginis singulis Asia-Russia (Korschikoff, 1938) hexagonalibus gelatinosis unamquemque cellulam cingentibus North America-United State5 (Goldstein. see Table 1). conposita. Omnis cellula adulta magnitudine similis aut quasi Strains-47smf and 50mf. sirnilis, spherica ad ovatam, singulum chloroplastum poculi- formem radialiter striatum, aliquot ad multa pyrenoidea 4. Eudorina conradii nov. sp. (Figs. 15. 16, 17. 18). hahentem, continens ; Stigmata in strato cellularum anteriore Eztdorina elegans Ehrenb. sensu Conrad, 1913, Reczreil ZnAt. maxima, in stratis posteriorihus sequentibus progredienter Leo Errera 9, 321-43. Figs. 1-13. rninora, aut in strato postremo nulla. Coloniae sphericae, ad late ellipsoideas, involucro coloniae Omnk cellula ad colonias-filias e 32 cellulis compositas effi- forrna lebi aut extremitate posteriore applanata aut paululum ciendas aequliter potens ; tempus divisionis cellularum strati mammillate. Coloniae e 16. 32 raro 64 cellulis in stratis mani- anterioris saepe moratum. festis intra involucrum gelatinosum duplex ordinatis con- Reproductio sexualis heterogama et dioecia, omni cellula

Figs. i and 8. Eudorina elegans var. synoica. Fig. 7. Sexual showing posterior mammillations and unipyrenoid cells. X 400. reproduction in strain 10jhmr. Sote the plakeal stage and Fig. 10. Mature vegetative colony of If lacking posterior mam- sperm packets which are dissociating. X 225. Fig. 8. Late millations. X 400. Figs. 11 and 12. Eudorina unicocca var. stage in sexual reproduction in strain lO5hmr in which all peripheralis. Fig. 11. Mature vegetative colony of 4sm with the sperm packets haye dissociated, and the sperm cells are unipyrenoid cells. X 540. Fig. 12. Portion of mature colony surrounding the eggs. X 240. Figs. 9 and 10. Eudorina icni- of 4sm just prior to daughter colony formation with large COCC~var. unicucca. Fig. 9. Young vegetative colony of lf central pyrenoids and smaller peripheral ones. X 540. SPECIATIONAND SEXUALITYIN Eudorina 325 coloniae fasciculum spermatum aut ova efficiente. Fasciculi packets pursue the female colonies which soon settle spherici anteriori-posteriorem cellularum differentiationem prae- down to the bottom of the watch glass. Following bentes ; in dimidio fasciculi anteriore cellulae magnae sphercae virides, stigmata praebentes, et in dimidio posteriore cellulae this, the outer confluent sheath of the female colonies minores fusiformes dilute virides sine stigmatibus. Nonnullae expands resulting in a separation of the cells. The gentes parthenosporas membrans crassis praeditas formant. sperm packets break up and the individual sperm Coloniae 55-170 p long., 45-140 p lat. Cellulae ccloniarum penetrate the maternal matrix. Both types of sperm libere natantium 12-25 ,U diam. cells are found surrounding the eggs. The smaller Colonies spherical to broadly ellipsoidal, with the colonial envelope smooth in outline or the postericr end flattened or posterior sperm cells become spherical in shape and slightly mammillate. Colonies of 16, 32, rarely 64 cells ar- appear to be the most active male gametes. Gametic ranged in distinct tiers within a double-layered gelatinous en- union was not observed, but extremely thick-walled velope; the outer layer confluent, the inner layer composed zygotes were produced in loose clumps rather than the of hexagonal individual gelatinous sheaths surrounding each tight cohesive clumps described in E. elegans and E. cell. Adult cells all equal in size or nearly so, spherical to ovoid in shape containing a single radially-striated cup-shaped unicocca (Fig. 18). Considering the uniqueness of the chloroplast with several to many pyrenoids. Eyespots largest colonial envelope and, in addition, the mode of sexual in the anterior tier of cells and progressively smaller in the reproduction this is described as a new species, Eu- subsequent posterior tiers or completely lacking in the pos- dorina conradii. terior tier. All cells with an equal potential to produce 32-celled daugh- GeographicaE distribution ter colonies; anterior tier of cells often delayed in division. Europe-Belgium (Conrad, 1913). Sexual reproduction heterogamous and dioecious with all North America-United States (Goldstein, see Table 1). cells of a colony developing into sperm packets or eggs. Sperm Strains-29p and 70hd. packets spherical with anterior-posterior cellular differentia- tion ; large, spherical, green cells with eyespots in the anterior 5. Eudorina illinaisensis (Kofoid) Pascher (Figs. 19, 20, 21). half of the packet and small, spindle-shaped, pale-green cells Pascher, 1927, Die Sirsswasser-Flora, Heft 4., Gustav Fisher, lacking eyespots in the posterior half of the packet. Thick- Jena, p. 443-4, Figs. 464-5. Prescott, 1951, Algae of the West- walled parthenospores formed in some strains. ern Great Lakes Area, Cranbrook Inut. Sci. Bull. 30, p. 77, pl. Colonies 55-170 iu. long and 45-140 p broad. Cells of free- 2, Fig. 3. Palik, 1955, Rev. Algol. 1, 146-51, pls. I and 11, Figs. swimming colonies 12-25 @ in diameter. 1-21. Deducenko-Shchegoleva, Matvienko & Shkorbatov, 1959, Chlorophyta.Volvocineae, in Savich, V.P., Opredelitel pres- Conrad (14) described as Eudorina elegans colonies novodnikh vodoroslei SSSR, Vipusk 8, Izdatelstvo Akademii in which each cell was surrounded by a distinct hexa- Nauk SSSR, Moskva-Leningrad, p. 211-12, pl. 101, Fig. 2. gonal individual gelatinous sheath. The individual Huber-Pestalozzi, 1961, Die Binnengewasser, Band XVI, 5. Teil, p. 643-4,taf. CXXXIII, abb. 894. gelatinous sheaths were in turn surrounded by an outer Pleodorina dlinobensis Kofoid, 1898, Bull. Ill. State Lab. confluent sheath. In the present study all 73 clones Nat. Hist. 5, 273-93, pls. XXXVI and XXXVII. Merton, 1908, were carefully examined for presence or absence of 2. wiss. Zool., 90, 445-77, text Figs. 1 and 2; pls. 27 and 28. individual gelatinous sheaths and only 2 strains (29p Grove, 1915, New Phytol. 14, 169-82, Figs. 1-11. Printz, 1927, and 70 hd) were found to possess them. Conrad in- Chlorophyceae, in Engler, A. and Prantl, K., Die Natiirlkhen Pfianzenfamilien, 2nd ed., 3. Band, Wilhelm Engelmann. Leip- dicated that fixation and staining were necessary zig, p. 58, Fig. 3PA. to demonstrate the individual gelatinous sheaths, Colonies spherical to elipsoidal in shape, with the colonial but in strains 29 and 70 they are clearly evident envelo'pe smooth in outline or with posterior mammillations. with or without fixation and staining (Figs. 15, 16). Colonies of 16 or 32 cells arranged in distinct tiers within a Sexual reproduction was not observed by Conrad, confluent double-layered gelatinous envelope. Anterior tier of cells markedly reduced in sue in mature 32-celled colonies. but in strain 70 a homothallic dioecious condition Mature cells spherical in shape containing a single radially- was found; strain 29 proved to be parthenosporic. striated cup-shaped chloroplast with one to several pyrenoids. Strain 70 produces sperm packets unlike any previ- Eyespots largest in the anterior tier of cells and progressively ously described or observed for a heterogamous or smaller in the subsequent posterior tiers or completely lacking oogamous member of the . These unique in the posterior tier. Anterior tier of cells in 3Z-celled colonies may or may not sperm packets possess a clear anterior-posterior cellu- divide ; if division occurs 32-celled daughter colonies never lar differentiation with large, spherical, green cells with produced. eyespots in the anterior half of the packet and small, Sexual reproduction heterogamous and dioecious, with bi- spindle-shaped, pale-green cells lacking eyespots in the flagellate sperm in packets and biflagellate eggs. posterior half of the packet (Fig. 17). As described Colonies 46-200 p long and 38-175 $L broad. Anterior cells 7.1-11.9 p in diameter, cells of second tier 11.9-19 p in diameter. by Pocock (49) for sperm packets of Vohox rousseletii and V. capensis inversion is complete in strain 70 and Kofoid(3 5 ) described Pleodorina illinoisensis in a gelatinous envelope surrounds the sperm packet (Fig. which the anterior tier of cells in 32-celled colonies 17). The resulting sperm packets are therefore spheri- was markedly reduced in size and usually did not par- cal in appearance rather than plate-like as is normally ticipate in reproduction. Chodat( 10) considered P. found in Eudorina. In sexual material the sperm illinoisensis as a physiological form of E. elegans and 326 SPECIATIONAND SEXUALITYIN Eudorina placed it in synonymy with this species. Reexamining and 3) the otherwise similar morphology to E. elegans, Kofoid’s 1898 description and subsequent accounts Palik(46) working with a natural collection of E. of this alga(27.41), Pascher(48) believed it to be a illinoisensis from Hungary, and Waters (71) working distinct species. but belonging to Eudorina rather than with E. illinoisensis from Minnesota (Culture Collec- Pleodorina. This opinion was based on the following tion of Algae at Indiana University #807 and 808) evidence: 1) the anterior cells had not lost their confirmed the above 3 observations. asexual reproductive capacity ( 2 7,3 5.4 1 ) ~ 2 ) even in Additional information confirming the change in Eudorina elegans the 4 anterior cells often divided generic status of E. illinoisensis has been obtained in more slowly than the remaining posterior cells( 28), a study of sexual compatibility with Eudorina to be

Figs. 13 and 14. Eudorina cylinduica. Fig. 13. 16-celled lateral optical section of a sperm packet of strain 70hd. Note colony of 47f. X 380. Fig. 14. 32-celled colony oi 4if. Sote the hollow spherical nature of the packet which consists of the slightly reduced anterior tier of cells and the cylindrically large anterior cells and small posterior cells. In addition, note shaped colonial envelope characteristic of this species. X 380. that the packet is surrounded by a gelatinous envelope. X 1680. Figs. 15-18. Eudorina conradii. Fig. 15. Surface vicw of strain Fig. 18. Zygotes of strain 70hd. Note that they are arranged 70hd shcwing the individual gelatinous sheaths around the in a loose clump within the confines of the old maternal matrix cells. X 630. Fig. 16. .4nother view of strain 70hd showing rather than tight cohesive clumps as is more typical for the the construction of the colonial envelope. X 630. Fig. 17. A other species in the gcnus. X 320. SPECIATIONAND SEXUALITYIN Eudorina 327 discussed in detail in a later section. Eudorina illi- Band, Wilhelm Engelmann, Leipzig, p. 58. Prescott, 1951, noisensis (808mf) was found to be reciprocally com- Algae of the Western Great Lakes Area, Cranbrook Inst. patible with certain strains of E. elegans and E. cylin- Sci., Bull. 30, p. 77, pl. 2, Fig. 1. Ueducenko-Shchegoleva, Matvienko & Shkorbatov, 1959, Chlorophyta:Volvocineae, drica. Also the male clone of E. illinoisensis was in Savich, V. P., Opredelitel presnovodnikh vodoroslei SSSR, compatible with a female E. unicocca, and the female Vipusk 8, Izdatelstvo Akademii Nauk SSSR, Moskva-Lenin- clone of E. illinoisensis was compatible with 2 male grad, p. 212, pl. 101, Fig. 4. Huber-Pestalozzi, 1961, Die clones of E. unicocca var. peripheralis. Furthermore, Binnengewiisser, Band XVI, 5. Teil, p. 649, taf. CXXXIV, the reciprocal intercrosses involving E. illinoisensis abb. 898. (808mf) and E. elegans (56mf) resulted in polyploid Eudorina indica Iyengar, 1933, J. Linn. Soc. London 49, 369, text Fig. 3, p1. 28, Figs. 2, 12, and 13. Doraiswami, progeny which is a common occurrence in interspecific 1940, J. Zndiana Botan. Soc. 19, 113-39, pl. I1 and 111. crosses in the plant kingdom. Huber-Pestalozzi, 1961, Die Binnengewiisser, Band XVI, 5. Sexual reproduction was first reported for E. illi- Teil, p. 644-6, taf. CXXXIII, abb. 895. noisensis by Merton (4 1 ) , and later by Grove (2 7) , Pleodorina westii Tiffany, 1935, Arch. Protistenk. 85, Palik( 46), and Waters ( 7 1) . All the above accounts 142-3, Fig. 1. indicated that the species was heterogamous and dioe- Colonies spherical to ellipsoidal in shape, with the colonial cious Sperm packets were formed in separate male envelope smooth in outline or at times with posterior mam- millations. Colonies of 32, 64, or 128 cells in distinct tiers colonies, and according to Grove( 27) and Waters( 71) within a confluent double-layered gelatinous envelcpe. Cells even the anterior tier of cells in 32-celled colonies could of two different sizes in mature colonies, small cells in the form sperm packets. Biflagellate eggs resided in sepa- anterior % or of the colony and large cells in the posterior rate female colonies, and a single observation by Palik half of the colony. Mature cells spherical in shape, with one (46) indicated that the anterior tier of cells could pyrenoid in the small cells and one to many in the large pos- terior cells. Eyespots largest in the anterior tier of cells and serve as eggs. progressively smaller in the subsequent posterior tiers or com- In the present study the above observations have pleteIy lacking in the posterior tiers. been confirmed except for the participation of the an- Small anterior cells may or may not participate in asexual terior tier of cells in 32-celled colonies in gamete for- reproduction ; their participation in sexual reproduction not mation. The sexual process is essentially the same as observed. Sexual reproduction heterogamous and dioecious, with bi- in dioecious strains of E. elegans and E. unicocca. flagellate eggs and with biflagellate sperm formed in packets. Following fertilization the zygotes are found in tight Colonies 175-300 p in diameter. Small cells 12 p in diameter, cohesive clumps surrounded by the old maternal colo- large cells 2-3 times larger in diameter. nial envelope. The distinguishing characters of E. illinoisensis are In 1894 Shaw described as a new genus and species as follows. The colonies are composed of 16 or 32 Pleodorina californica in which the spherical colonies cells, with the anterior tier of cells markedly reduced were composed of 64 or 128 cells, the anterior % or in size in 32-celled colonies (Fig. 19). The cells are being reduced in size and presumably somatic. arranged in distinct tiers within a confluent double- Chodat (10) questioned the genus and placed it in layered gelatinous envelope. When daughter colony synonymy with Eudorina, however, subsequent investi- formation occurs, the small anterior cells may remain gators( 17,30,48,51,53,59) have retained the genus and vegetative or may divide. If division occurs, it is species P. californica. always delayed, and the resulting daughter colonies Recently Gerisch (23) provided evidence indicating possess 4, 8 or 16 cells (Figs. 20, 21). that P. californica might best be considered a species of Eudorina. He experimentally demonstrated that Geographical distribution the small, presumably somatic, anterior cells of P. Europe-Germany (Merton, 1908), England (Grove, 1915), californica had not lost their reproductive capacity and Hungary (Palik, 1955). were capable of asexual reproduction. It was the Asia-Russia (Deducenko-Shchegcleva, Matvienko S. Shkor- batov, 1959). somatic nature of the small anterior cells and not the Sorth itmerica-United States (Kofoid, 1898; Prescott, 1951 ; reduced size alone which distinguished P. calif ornica, Waters, 1959; Goldstein, see Table 1). for small anterior cells have been reported for species Central America-Panama Canal Zone (Prescott, 1955). of the genus Eudorina as we11(31,48,58). Strains-808mf and 97mf. In 1933 Iyengar described a new species Eudorina 6. Eudorina californica (Shaw) Goldstein nov. comb. indica with 32- and 64-celled colonies in which the 6a. Eudorina californica var. californica (Figs. 22, 23, 24). first 2 tiers of cells were reduced in size and pre- Pleodorina californica Shaw, 1894, Botan. Gaz. 19 279- sumably somatic. On the basis of vegetative morphol- 83, pl. 27. Chatton, 1911, Bull. Sci. France et Belgique, VII, ogy he felt that this species was intermediate between 44, 309-31, Figs. 1 and 2, pl. 7. Pascher, 1927, Die Siisswas- E. and P. californica. However, 32-celled ser-Flora, Heft 4., Gustav Fischer, Jena, p. 446-50, Figs. illinoisensis 407-12. Printz, 1927, Chlorophyceae, in Engler, A. and colonies identical with E. indica have been observed Prantl, K., Die Natudichen Pflanzenfamilien, 2nd ed., 3. in natural populations of Eudorina (Pleodorina) cali- 328 SPECIATIONAND SEXUALITYIN Eudorina

fornica by West K! Kest(74) and Tiffany(70) and with E. californka. and therefore, belongs in syn- in clonal cultures of E. californica (198hd, 809hd, and onymy with the species. 112hd) by the writer (Fig. 22). In addition. 64-celled Tiffany( 70) described Pleodorina westii based on a colonies were frequently found in the clones of E. description of West & West(74, p. 506, pl. 6, Fig. 14) ralifornica with only the first 2 tiers of cells reduced of P. californica with posterior mammillations. In as in size. Considering the above observations as well much as posterior mammillations are not considered as those of Doraiswami( 18) who noted that the small a reliable taxonomic character, the writer considers anterior cells of E. indica had not lost their repro- P. westii as a synonym of Eudorina (Pleodorina) ductive capacity. it is felt that E. indica is identical californica.

Figs. 19-2 1. Eudorina illinoisensis. Fig. 19. Segetative col- Figs. 22-24. Eudorina californica var. calijornica. Fig. 22. 32- ony of 808m. Note the markedly reduced anterior tier of cells. celled colony of strain 198hd with the 1st two tiers of cells X 360. Fig. 20. Early stage in daughter colony formation in reduced in size. X 200. Fig. 23. 64-celled colony of strain 808m. Sote the undivided anterior cells and the dividing pos- 198hd with the 1st three tiers of cells reduced in size. X 200. terior cells. X 590. Fig. 21. Late stage in daughter colony Fig. 24. A young 128-celled colony of strain 198hd with the formation in 808m with the anterior cells in the 4- and begin- cells in the anterior half of the colony being reduced in size. ning of the 8-celled stage of division. Sote that the posterior X 200. cells have already completed division and inversion. X 510. SPECIATIONAND SEXUALITYIN Eudorina 329

In the present study Eudorina calijornica is distin- Bloomington, Indiana. Numerous observations of sex- guished primarily by 32-, 64-, and 128-celled colonies ual reproduction have been made on these and other in which the cells in the anterior third or half of the sexual strains of Eudorina, and a change in the mode colony are reduced in size, and the remaining posterior of sexual reproduction has never been observed. cells are larger in size (Figs. 22, 23, 24). Daughter Therefore, it was felt that the dioecious colonies found colony formation is generally confined to the large by Tiffany represented a population of E. californica posterior cells, but the small anterior cells retain the while the monoecious colonies represented a new vari- potential to divide. ety which is designated as E. calijornica var. tiflanyi. In this new variety sperm packets, eggs, and zygotes Geographical distribution occur within the same colony. Europe-France (Chatton, 1911) and England (West and West, 1905; West, 1916). Geographical distribution i\sia-Russia (Swirenko, 1926). North America-United States (Shaw, 1894; Mottier, 1894; North America-United States (Tiffany, 1935) Clinton, 1894; Prescott, 1951; Goldstein, see Table 1). 7. Eudorina s,phaerica (Iyengar) Goldstein nov. comb. Central America-Panama Canal Zone ,Prescott, 1955). Pleodorina sphaerica Iyengar, 1933, J. Linn. SOC. London Strains-l12hd, 198hd, and 809 hd. 49, 370, text Fig. 4; pl. 28, Figs. 4 and 5. Iyengar and Ramana- than, 1951, Phytomorphology 1, 215-24, pl. I, Figs. 1-12. The preceding portion of this study has dealt with Huber-Pestalozzi, 1961, Die Binnengewusser, Band XVI, 5, those species and varieties of Eudorina which have Teil, p. 649-50, taf. CXXXV, abb. 899. been studied in culture. The remaining portion of Colonies spherical to ellipsoidal in shape, with the colonial this monograph concerns 3 previously described spe- envelope smooth in outline. Colonies of 64 or 128 cells ar- ranged in tiers near the periphery of the colcnial envelope. cies and one new variety of Eudorina, material of Cells of 2 different sizes in mature colonies, small cells in the which has not been examined by the writer. However, anterior % or K of the colony, small cells interspersed among it is hoped that in the future these tana mi2ht be large cells in the posterior half of the colony; small cells pre- isolated into culture in order to perform a dctailed sumably scmatic. Mature cells spherical in shape with one comparison of their distinguishing taxonomic f2atures. pyrenoid in the small cells and many in the large cells. Eye- spots largest in the anterior tier of cells and progressively 6b Eudorina californica (Shaw) Goldstein var tiffanyi smaller in the subsequent poslteroir tiers. nov. var. Sexual reproduction heterogamous and dioecious, with bi- flagellate sperm formed in packets and with biflagellate eggs. Pleodorina californica Shaw sensu Tiffany, 1935, Arch. ProtistenR. 85, 141-2, Fig. 4. Free-swimming colonies 180-320 ,u in diameter. Small cells Reproductio sexualis monoecia, coloniae aut praecipue up to 12 p in diameter, large cells up to 28 p in diameter femineae, solum 1-4 fasciculis spermatum praeditae, aut praecipue mascular, solum 1-2 gametis femineis praeditae. Pleodorina sphaerica was described by Iyengar (31 ) Sexual reprc duction monoecius, colonies either predomi- as having 64- and 128-celled colonies with small cells nantly female with only 1-4 sperm packets or predomi- occupying not only the anterior or of the colony nantly male with only 1-2 female gametes. but also interspersed among larger cells in the pos- terior portion of the colony. Colonies similar in mor- During the summer of 1933 Tiffany( 70) examined phology have been observed in unhealthy populations populations of E. californica from a marshy pond on of strain 198hd of Eudorina calijornica which may Middle Bass Island in Lake Erie. The colonies ob- served in early July were dioecious while those col- indicate that E. sphaerica is only a physiological form of E. calijornica. However, until more conclusive lected in the latter part of the month were monoe- proof is obtained, the species will be retained but cious. Tiffany felt that these 2 sexual forms were representative of a single population and that the placed in the genus Eudorina along with the closely change in sexuality was associated with a change in related species E. californica and E. illinoisensis. environmental conditions. Geographical distribution Observations of sexual reproduction have been made Asia-India (Iyengar, 1933 ; Iyengar &- Ramanathan, 1951) for 3 strains of E. calijornica (112hd, 198hd, and 8. Eudorina interconnexa Prexott 809hd) indicating a dioecious condition which was Prescott, 1955, Ohio J. Sci. 55, 100, pl. \'I, Fig. 3. constant for each strain. It is possible that Tiffany The following description was taken from Prescott(51) : may have observed 2 different strains appearing in Colony oval, the gelatinous sheath wide and sometimes the same pond at different times, but giving the im- slightly produced to form lobes posteriorly; cells spherical ; pression of a single population with 2 different modes 32 in number, bearing a pair of prominant gelatinous canals of sexual reproduction. Similar observations were anteriorly through which the flagella extend ; cells inclosed in individual gelatinous sheaths which are interconnected with made when a single male strain of E. elegans(l7m) neighboring cells by slender strands so that a fenestration is and a monoecious strain of E. elegans var. synoica formed within the colonial mucilage; cells 10-18.5 p in di- (16hmr) were collected from the same pond near ameter ; colony 89 p in diameter. 330 SPECIATIONAND SEXUALITYIN Eudorina

Eudorina interconnera was originally found in a sexual reproduction. With the aid of clonal cultures plankton sample from Gatun Lake in the Panama grown under prescribed environmental conditions it Canal Zone and has not been reported since its dis- is now possible to investigate thoroughly the asexual covery in 1955. In one respect it is similar to E. con- and sexual reproductive potential within the genus radii for both species possess individual gelatinous Eudorina-a task impossible with natural collections sheaths. In contrast. E. interconnexa differs from E. due to the rarity of reproducing material. conradii by possessing prominent gelatinous canals dsexual reproduction. The mode of asexual repro- through which the flagella extend. individual gelati- duction in Eudorina, a phenomenon common to all nous sheaths which are interconnected by slender colonial green flagellates in the family Volvocaceae, strands forming a fenestration within the colonial involves a number of successive cell divisions resulting mucilage. and spherical rather than hexagonal indi- in the formation of a curved plate or plakea which vidual gelatinous sheaths. subsequently undergoes inversion to form a daughter colony. Geographical distribution In all species of Eudorina observed in the present Central .\merica-Panama Canal Zone (Prescott. 1955) study each cell of a vegetative colony is potentially 9. Eudorina echidna S\virenko capable of asexual reproduction. In species such as Swirenko, 1926. .4rch. Protistenk. 55. 191, Fig. X. Printz, E. clegans and E. unicocca, with all cells equal in size 1927. Chlorophyceae. in Engler, A. Sr Prantl, K., Die .V'atiir- or nearly so, the division of the anterior tier of cells lichen Pflaizz?n.familien, 2nd ed., 3. Band. \Vilhelm Enpelmann, Leipzig. p. 58. Pascher. 1927, Die Szmwiisser-Flora, Heft 4.. often lags behind the divisions in the posterior cells Gustav Fisher, Jena, p. 442-3, Fig. 403. Smith, 1930. Bull. as noted by Hartmann( 28). In strains of E. i2linoisen- Torrey Botan. Club 57, 363. Deducenkc-Shchegoleva, Mat- sis( 27,35) and E. californica(23), with the anterior vienko & Shlorbatov. 1959, Ch1orophyta:Yolvocineae. in Sa- tier or tiers of cells reduced in size, division may fail vich. Y. p.. Opredelitel presnovodnikh uodoroslei SSSR, \.ipusk to occur in the small cells; if it does occur, the lag 8, Izdateistvo Akademii Sauk SSSK, Mcskva-Leningrad, p. 211. pl. 101, Fig. 3. Huber-Pestalozzi, 1961, Die Binnengewiis- is pronounced. Correlated with the reduced size of ser, Band XVI, 5. Teil, p. 646. taf. CXLYII, abb. 896. the cells in the anterior tier or tiers the resulting The folloming description is after Swirenko(68) : daughter colonies contain fewer cells than colonies Colonies ellipsoidal, 160-185 @ long ; 16 cells, gelatinous produced by the posterior cells. envelope covered with sharp, conical projecticns. 8-10 p long. Daughter colony formation in Eudorina involves 4 Individual cells spherical. 19-20 in diameter. anterior pole g to 7 successive bipartitions occurring parallel to the hyline bearing 2 flagella 40 1-1 long. Cells with a single chloro- plast containing 1 eyespot and a pyrenoid. longitudinal axis of the cell resulting in a curved plate of cells or plakea (Fig. 26). As viewed from the an- Pascher (48) recognized Eudorina echidna but stated terior end of the mother cell the plane of the first divi- that further observations of the species would be of sion generally bisects the cell between the points of value. More recently Huber-Pestalozzi (30, p. 703-5) attachment of the parental flagella. The remaining suggested that E. echidna was only a pathological form successive divisions occur in a regular pattern as of E. elegans. According to Evens(22) the conical illustrated (Fig. 25). projections were due to secretions or excretions from The successive nuclear divisions are best observed 2 species of rotifers, -4splanchna priodonta and Asco- in material stained with acetocarmine. The phases of morpha saltans: however, Huber-Pestalozzi's observa- mitosis are like those reported by Cave & Pocock (7) tions indicated that the pathological condition arose through the effect of epiphytic bacteria. Until such I 1 I observations can be experimentally demonstrated the I I I species E. echidna, distinguished by short conical pro- I I I jections covering the surface of the colony. must be _- I retained. +-i

Geographical distribution Asia-Russia (Swirenko, 1926). Europe-Belgium (Evens. 1946).

IT. Reproduction in Eudorina L-nderstanding of the nature of colony differentia- tion as exhibited by certain species and varieties in I I 1 i i the genus Eudorina as well as the sexual compatibility Fig. 25, Pattern of successive cleavage planes in formation and genetic relationships between species demands a of the plakeal stage in Eudorina. 1st division =, 2nd divi- sion ~, 3rd division ----- , 4th division --a a- , 5th divi- thorough knowledge of the processes of asexual and sion - - - *. SPECIATIONAND SEXUALITYIN Eudorina 33 1 for several members of the Volvocaceae. There is a the flagellar condition in normally biflagellate cells, marked synchronization of nuclear divisions during for when colonies are viewed from certain angles the the formation of the daughter colony. As reported by cells often appear uniflagellate. Cave & Pocock(7) the nucleolus is apparent in early While young daughter colonies are still within the prophase for the first 2 or 3 divisions, but is not evi- old matrix, eyespots arise de now in the cells, and dent in the later divisions because of the decreased thus the anterior and posterior poles of the colony can size of the nucleolus. be distinguished. As noted above, the parental eyespot When division is complete, the plakea is cup-shaped and the points of attachment of the parental flagella in appearance. Observing the concave side of the are located in the peripheral cells of the plakea. After plakea the parental eyespot is found in one of the inversion the old eyespot can be seen in a cell of the newly formed cells at the periphery of the plakea, last two posterior tiers of the young daughter colony; and the 2 parental flagella are attached to peripheral the parental flagella are also found attached to cells cells on opposite sides of the curved plate as noted in the posterior tiers. Therefore, it appears that the by Gerisch(23) or to a single peripheral cell of the posterior tiers of cells correspond to the peripheral plakea observed by Doraiswami( 18). The nucleus of cells of the plakea while the anterior tiers of cells in each newly-formed cell lies near the concave side of the the young daughter colony correspond to the centraI plakea which faces the surface of the parental colony. plakeal cells( 23). This knowledge of the develop- To complete daughter colony formation the concave mental sequence in daughter colony formation will side of the plakea reverses itself by a process called serve as an important tool in solving the problems of inversion. The once concave side of the plakea be- cellular differentiation in Eudorina and other members comes convex as the peripheral cells of the plakea are of the Volvocaceae. drawn in the reverse direction until the corners meet Sexual reproduction. The various modes of sexual resulting in the formation of a closed sphere. As a reproduction in the different species and varieties were consequence of inversion the nucleated ends of the discussed in the preceding taxonomic section of this daughter cells are situated to the exterior of the study. However, certain aspects of the sexual process daughter colony. which are of no apparent value from a taxonomic At the onset of inversion each cell of the plakea standpoint but of importance in understanding the produces a new pair of flagella. When the flagella first sexual potential of Eudorina will now be discussed. appear they are equal in length, but as inversion pro- In the past several years strains of 6 species and 3 ceeds one flagellum of a pair often elongates to more varieties of Eudorina have been isolated from 44 natu- than twice the length of the other. When inversion is ral populations and include 12 homothallic strains, 24 complete a new colonial envelope is secreted around heterothallic pairs, and 8 parthenosporic strains. Four the cells and the shorter flagellum is embedded in the of the 44 natural populations yielded only male strains matrix (Fig. 27). Similar observations have been and one of the popuiations yielded a single female made by the writer for 2 other colonial green flagel- strain. All species were found to be heterogamous lates, Pandorzna morum also in the family Volvocaceae and either monoecious or dioecious. Biflagellate sperm and Astreplzomene gubernuculifera a colonial form were formed in packets, and the eggs were biflagellate lacking inversion and thus placed in a separate family, and generally fertilized within the confines of the colo- the Astrephomenaceae. nial envelope. All monoecious strains of Eudorina Generally when the young daughter colonies escape were homothallic as exemplified by the two varieties from the maternal colonial envelope each cell possesses examined in this study, E. elegans var. carteri and E. 2 flagella of equal length. However, when water elegans var. synoica, in which gametic union occurred mounts of dividing material are made, the young between sperm and eggs formed within the same col- daughter colonies may escape prematurely due to the ony. A homothallic dioecious condition was observed pressure of the cover glass. Thus young free-swimming in two species of Eudorina, E. conradii and E. cali- colonies may be observed with apparently uniflagellate fornica. The majority of the dioecious species such as cells, which may explain why Ehrenberg( 20) described E. elegans, E. unicocca and E. cylindrica were hetero- uniflagellate cells for Eudorina. In addition, these thallic possessing separate male and female clones. observations may account for the description of Mas- However, it was observed that within a heterothallic tigosphaera a little-known genus of the Volvocaceae male clone the sperm packets may surround and pene- with uniflagellate cells. This is supported by the ob- trate a vegetative male colony simulating the process servations of Jane (see 30, p. 650) who reported the of fertilization. In male clones this process does not occurrence of Mastigosphaera or a morphologically usually result in zygote formation, but in 5 male clones similar member of the Volvocaceae with flagella of isolated from nature (E. elegans 51sm and 67sm; E. unequal length. On the other hand it is equally unicocca var. peripheralis 4sm and 72 sm; and E. plausible that both Ehrenberg and Jane misinterpreted cylindvica 47 sm) a small number of zygotes are pro- 332 SPECIATIONAND SEXUALITYIN Eudorina duced. When these male clones are placed in watch tative cells (Figs. 30, 31). Generally several sperm glasses under those conditions inducing the sexual are found gliding slowly over the surface of each egg process the majority of the colonies form sperm pack- and often visiting several eggs within the same colony. ets while several colonies produce eggs which are The actual union of gametes was not observed in this subsequently fertilized. To distinguish these clones study, but other investigators have reported that the they are referred to as “selling males.” However. for male gametes fuse with the females by their anterior all practical purposes they are considered heterothallic ends(46,50), posterior ends(32), and laterally(42). because when crossed with compatible female colonies. Although gametic union was not observed by the thousands of zygotes are produced. writer, both mating types segregated at zygote germi- Sexual reproduction may be observed in homothallic nation for certain heterothallic strains, indicating that strains within 7-10 days after being transferred to true fertilization had occurred. However, asexual fresh soil-water bottles placed under illumination of strains of E. elegans, E. unicocca, and E. conradii have 250-350 ft.-c. intensity. Under similar conditions male been isolated from nature which produce thick-walled, heterothallic clones produce sperm packets in the ab- zygote-like parthenospores as noted by Mainx( 40) in sence of a compatible female clone. The sequence of E. elegans, and these have not been checked geneti- events in sperm packet formation is similar to those cally. As noted by Pascher(48) unfertilized eggs may in daughter colony formation. In all species of Eudo- undergo daughter colony formation ; sperm cells were rina except E. conradii (70hd) inversion is only par- found by the writer to be incapable of reproducing tially completed and the sperm packet lacks a gelati- asexually. nous envelope. The packet is a curved platelet of 16. Following fertilization relatively immobile, quadri- 32. or 64 spindle-shaped cells; each cell possesses a flagellate zygotes are produced often bearing 2 eye- single anterior eyespot, nucleus, a pair of apical flag- spots. The dark-green zygotes move slowly within the ella of equal length, and a yellow-green chloroplast confines of the gelatinous envelope soon losing their which occupies the lower half of the cells (Fig. 28). flagella and secreting a smooth thick wall. In strains In contrast, the sperm packet of E. conradii (70hd) of most species studied in culture the zygotes formed undergoes compete inversion to form a hollow sphere tight cohesive clumps bound by a persistent maternal of cells surrounded by a gelatinous envelope as de- matrix (Fig. 32) while in other strains the gelatinous scribed by Pocock(49) for Volvox rousseletii and V. envelope becomes watery and expanded, and finally capensis. However, in the 2 species of Volvox the breaks down releasing the zygotes singly (Fig. 33). sperm cells are all identical in morphology. whereas After one week under illumination of 300 ft.-c. in- in E. conradii the anterior half of the packet consists tensity the zygotes lose their dark-green color and turn of large. spherical, green cells with eyespots and the golden-orange. posterior half of the packet is composed of small, Zygote germination. The morphological details of spindle-shaped, pale-green cells lacking eyespots ( Fig. zygote germination in E. elegans were first observed 17). by Otrokow(45), and confirmed by Shreiber(S5). When actively growing compatible male and female More recently Palik( 46) and Waters ( 7 1) have oh- clones are mixed under those conditions known to served the germination process in zygotes of E. iZZi- induce sexual reproduction the sexual process can be noisensis. Generally a single viable biflagellate gone observed within 1-4 days. Initially the sperm packets escapes from the zygote; less frequently there are 2 escape from the old colonial envelope and swim rap- or 3 gones. Waters( 7 1) reported that 2 viable prod- idly among the female colonies. n’hen contact is made ucts were most frequently observed. As yet the cyto- with female colonies the packets surround the colonies logical events have not been observed in Eudorina, and rupture the outer colonial envelope by an active but genetic evidence to be presented in more detail “burrowing process” (Fig. 29). During the penetra- later indicates that meiosis occurs at zygote germina- tion process the packets break up into individual sperm tion as observed in other members of the Volvocaceae cells which enter the female colony (Figs. 30. 31). (13.65). \Thile in the packets the sperm cells are spindle- In the present study zygote germination was ob- shaped. but once they are released they undergo fur- served for 2 heterothallic strains of E. elegans (40mf ther morphological differentiation as noted by Iyengar and 62mf). The process is initiated by the appearance ( 3 1 ) . Generally the anterior portion of the sperm cell of a crescent-shaped hyaline area adjacent to the wall becomes slightly attenuated while the posterior portion of an otherwise translucent orange zygote (Fig. 34). of the sperm cell becomes attenuated into a fine hya- The wall adjacent to the hyaline area becomes dis- line tail-like structure. The sperm cells move slowly tended into a thin-walled protuberance separating it in a corkscrew-like fashion within the confines of the from the protoplast in this area. Subsequent to this female colonial envelope and are soon attracted to the process the protoplast itself begins to bulge into the eggs lvhich cannot be visibly distinguished from vege- space formed by the thin-walled protuberance. As SPECIATIONAND SEXUALITYIN Eudorina 333 this occurs a portion of the now slightly distended is released into the medium (Figs. 38, 39, 40). This protoplast is pinched off and comes to lie between the appears to be the only functional gone produced at thin wall and protoplast (Figs. 35, 36). This is fol- germination for the non-flagellate portion of the lowed by the formation of a pair of flagella which protoplast remains in the zygote wall and was not slowly increase in length and may soon be seen beating observed to undergo any further development (Figs. within the space separating the protoplast and thin- 39, 40). walled protuberance (Fig. 37). When the protoplast The unicellular biflagellate gone whkh escapes from has bulged out to where it is almost adjacent to the the zygote wall lacks a rigid wall for its shape varies thin wall it divides in half and the flagellated portion wh le moving over the surface of the agar (Figs. 39, apparently under pressure ruptures the thin wall and 40). The gone is granular in appearance, orange-

Figs. 26, 27. Asexual reproduction. Fig. 26. Two, 4, 8, and 62m. X 550. Fig. 29. Sperm packets of 62m surrounding a 16-celled plakeal stages in E. elegans var. carteri (104hma). female colony of 62f. X 380. Fig. 30. A later stage in sexual Note the two 16-celled plakeas in the center. X 430. Fig. 27. reprcduction showing a female colony of 62f still surrounded Young 32-celled daughter colony of E. conradii with appar- by sperm packets and individual sperm cells within the con- ently uniilagellate cells. X 1680. Figs. 28-31. Sexual reproduc- fines of the female colonial envelope. X 380. Fig. 31. .4 very tion in Eudorina elegans. Fig. 28. Intact sperm packets still late stage in the sexual process showing many sperm cells within the confines oi the old male colonial envelope of strain surrounding the eggs. X 400. 333 SPECIATIONAND SEXUALITYIN Eudorina brown, and often contains a large refractive oil globule sexual compatibility has been confined to heterothallic (Fig. 40). In liquid the single-celled gone may swim strains of the same or different species isolated from for several hours, then settle down and divide. It diverse geographical localities in the United States and undergoes 4 or 5 successive divisions forming a colony Canada. At present 22 heterothallic pairs, 3 male as in asexual reproduction (Fig. 41 ). The oil globule strains. and one female strain representing 4 species, generally remains undivided and is incorporated at E. elegans, E. unicocca, E. cylindrica, and E. illinoisen- random into one of the light green cells of the 16- or sis and one variety E. unicocca var. peripheralis have 32-celled gone colony (Fig. 42). been included in the study (see Table 1). Almost Sexual Compatibility in Eudorina. The study of half of the strains were collected in the vicinity of

Figs. 32, 33. Sexual reproduction in Eiidorina elegans. Fig. into a thin-walled protuberance. Note the distended portion 32. Clumped zlgotes of strain 56mf. X 380. Fig. 33 Scattered of the protoplast which will soon be pinched off. X 830. Fig. zygotes of strain 40mi. X 380. Figs. 34-37. Zygote germina- 36. A% small portion of the protoplast has been pinched off and tion in Eudorina elegans (strains 40mf and 62mf). Fig. 34. 1s lying in the space between the thin-walled protuberance and Initial stage in qgote germination showing the crescent-shaped the protoplast. X 830. Fig. 37. Flagella have been formed hyalinr area adjacent to the wall of the ngote. X 830. Fig one of which can be seen within the space between the proto- 35. The mall adjacent to the hyaline area has become distended plast and thin-walled protuberance. X 830. SPECIATIONAND SEXUALITYIN Eudorina 335

TABLE1. Straws of Eudorina TABLE1. Contintted The strains isolated from each natural population were given a separate numher which is followed by a postscript indicat- Z or P ar- ing the mode of sexual reproduction. m = male strain, sm = Strain Species rangement Source selfing male, f = female strain, sf = selfing female, mf = heterothallic and dioecious strain, hd z homothallic and di- 6Omf E. elegans Scattered Lake sample oecious strain, hma = honiothallic and monoecious strain in Brown Co., Ind. Khich the anterior tier of cells forms sperm packets, hmr = G2mf E. elegans Scattered Pond sample homothallic and monoecious strain in which the sperm pack- Monroe Co., Ind. ets are formed at random in the colony, and p = partheno- sporic strain. 63hmr E. e legans Sea ttcred Mud sample var. synoaca Monroe Co., Irid. Z or P ar- 65mf E. elegans Clumped Mud sample Strain Species rangement Source Monroe Co., Ind. 66p E. elegans Scattered Lake sample lmf E. unicocca Clumped Pond sample (same as 60mf) Monroe Co., Ind. 67smf E. elegans Scattered Pond sample 4smf E. unicocca Clumped Pond sample (same as G2mf) var. peripheralis Monroe Go., Ind. 70hd E. conradzz Clumped Pond sample 5m E. tinicocca Clumped Ind. Baldwin Co., Ala. var. peripheralis 72smf E. unacocca Clumped Pig wallow GP E. unieocca Clumped Lake sample var. perapheralis Wastenaw Co., Blich. Monroe Co., Ind. 73P E. unacoeca Clumped Mud sample 7mf E. elegans Scattered Pond sample Chlum, Czech. Monroe Go., Ind. 75mf E. elegans Scattered Mud sample 9f E. elegans Scattered Pond sample Monroe Co., Ind. Monroe Co., Ind. 79lllf E. elegans Scattered Mud sample llhuir E. elegans Scattered Mud sample Owen Co., Ind. var. synoica Monroe Co., Ind. 81nif E. tinzcocca Clumped Rock pool 13mf E. elegans Clumped Mud sample Llano Co., Tex. Tulare Co., Calif. 93mf E. tinacocca Clumped Tractor rut 1411 E. uuicocca Clumped Pond sample Pranklin Co., Ohio Barnstable Co., Mass. 95n1f E. elegans Clumped Mud sample l6limr E. elegans Scattered Mud sample Bexar Co., Tex. var. synoica Monroe Co., Ind. 97mf E. tllanoasensrs Clamped Mud sample 1 'im E. elegans Scattered Mud sample Henriepin Co., Minn. (same as 16hmr) l0Ornf E. unzcocca Clumped Roadside ditch 20 E. unicocca Clumped Pond sample var. perapheralis B.C., Canada Douglas Co., 111. 102mf E. unacocca Clumped Cow track 2211 E. elegaws Clnmped Lake sample var. pertpheralis B.C., Canada Brown Co., Ind. 103hma E. elegans Clumped Mud sample 29p E. conradii Clumped Pond sample Tar. carten Bexar Co., Tex. Hubbard Co., Minn. 104hma E. elegans Clumped Mud sample 3311nir E. elegans Scattered Mud sample Tar. carterz Washington Co., Ky. var. synoica Polk Co., Iowa 105hmr E,elegans Clumped Mud sample 34m E. elegans Clumped Mud sample var. synoaca Wilson Co., Tex. Polk Co., Iowa 112hd E. calzfornzca Scattered Mud sample 35m E. elegans ? Yo10 Co., Calif. (same as 79mf) 37P E. unicocca Clumped Mud sample 198hd E. calzfornica Scattered Quarry pool (same as llhmr) Brown Co., Ind. 38hmr 3.elegans Scattered Mud sample 808mf E. tllanoasenszs Clumped Mud sample var. synoica Monroe Co., Irid. Anoka Co., Minn. 40inf E. elegans Scattered Mud sample 809hd E. calzfornzca Scattered Pond sample Monroe Co., Ind. Monroe Go., Ind. 44nif E. elegans Clumped Pond sample Monroe Co., Ind. Bloomington, Indiana, and the remaining strains were 47smf E. cylindrica Clumped Mud sample collected in Iowa, Minnesota, Ohio, Alabama, Texas, Polk Co., Iowa California, and British Columbia, Canada. 50mf E. cylindrica Clumped Mud sample Polk Co., Iowa To test for sexual compatibility, actively growing male and female clones were mixed in all possible 51Siilf E. elegans Scattered Mud puddle Tuscaloosa Co., Ala. male-female combinations in watch glasses under those conditions known to induce the sexual process. Each 56mf E. elegans Clumped Mud sample Putnam Co., Ind. of the heterothallic pairs was mated as well to serve 336 SPECIATIONAND SEXUALITYIN Eudorina as a control. Only the presence of zygotes could be the Groups are for the most part sexually isolated used as an indication of cross compatibility, for poten- from one another and therefore distinguished on this tial eggs were indistinguishable from vegetative cells basis. Only 2 of the 104 intercrosses involve strains and sperm packets were generally present in the male with different zygote arrangements, indicating almost clones when they were mixed with the females. Even complete sexual isolation between these forms. the penetration of female colonies by sperm packets More than half of the heterothallic strains utilized could not be used as a measure of cross compatibility in this study belonged to the species E. etegans. because this process was often observed without sub- Strains of this species compose Groups I, 11, and I11 sequent zygote formation. The matings were first entirely and Group IV in part. As indicated in Table performed in duplicate in watch glasses and the re- 2 the strains of Groups I and I1 possessed zygotes sulting zygotes were generally kept for genetic analy- in the scattered arrangement and were completely sis. The matings were then repeated in triplicate using sexually isolated from those strains of E. elegans in 3-depression spot plates. Groups I11 and IV which possessed zygotes in tight Initially the strains were arranged in numerical clumps. order according to the species. but this arrangement The strains of E. elegans making up Groups I and failed to show any clear pattern of sesual compatibility I1 were all collected within a 20 mi. radius of Bloom- between clones of the same or different species. It will ington. Indiana, except for strain 5lsmf from Tusca- be recalled that in Eudorina there are 2 distinct zy- loosa, Alabama. The 2 Groups were sexually isolated gote arrangements. Following fertilization the zygotes escept for several unidirectional intercrosses involving either formed tight cohesive clumps bound by a per- female clones of Group I and male clones from Group sistent maternal natriv or the zygotes were released 11. In regard to the unidirectional crosses 2 of the singly when the maternal envelope broke down. Bear- heterothallic pairs of Group I1 (62mf and 67smf) ing these facts in mind the strains of each species were isolated from the same pond on the same day were rearranged placing those strains with similar in order to compare their sexual behavior. As illus- zygote arrangements together as shown in Table 2. trated in Table 2. both pairs were not only recipro- cally compatible with each other, but also with strains 40mf and 5lsmf of Group 11. However. the pattern of sexual compatibility exhibited by the 2 male clones 62m and 67sm differed in relation to 7f in Group I. Zygotes were produced in the mating of 62m with 7f while 67sm x 7f failed to yield zygotes.

6ROUP The strain which displayed the most unusual pattern r 11 111 IY Y of sexual compatibility in regard to Groups I and I1 was E. elegans 17m. Although this single male strain was placed in Group I1 it was almost equally com- patible with members of both Groups I and 11. It is unfortunate that a complementary female clone was not isolated with strain 17m for its behavior might have shed more light on the relationship of Groups I and I1 of E. elegans. Group I11 consisted only of 2 heterothallic pairs and a single male strain of E. elegans all with zygotes in tight cohesive clumps. Within this Group, 441 from Bloomington, Indiana, crossed with 13m from Lemon Cove. California, while 13f crossed with 34m from Iowa. In addition the female clone 13f was found to be compatible with 2 male clones from Group =\s a consequence of this arrangement not only did a V. E. unicocca 93m and E. unicocca var. peripheralis clearer view of the data emerge. but the strains were 100m. It is unfortunate that a complementary female now organized in such a manner that 5 relatively dis- clone was not isolated with strain 34m for its pattern tinct intercrossing Groups could be recognized. of sexual compatibility might have indicated a closer \Tithin each Group the members possess the same relationship between the strains of Group 111. zygote arrangement and are capable of intercrossing Group IV consisted of 3 heterothallic strains of E. freely with each other. Occasional successful inter- elegans, 2 heterothallic strains of E. cylindrica, and crosses can be found between strains belonging to one heterothallic pair of E. illinoisensis-all with different Groups. .4lthough these crosses do occur. zygotes formed in tight cohesive clumps. All reciprocal SPECIATIONAND SEXUALITYIN Eudorina 337

intra- and interspecific matings within this Group strains of E. unicocca var. peripheralis and one hetero- yielded zygotes except for 2 combinations; 95f X 47sm thallic pair of E. unicocca 93mf which cross in all and 95f x 50m. However, as noted earlier, the strains possible male-female combinations. Just a single inter- of E. elegans in Group IV were completely sexually cross, E. unicocca 93f X 81m, links these 2 subgroups. isolated from those strains of E. elegans in Groups, I, Inheritance of mating types. After examining the 11, and 111. Disregarding the reactions with E. uni- sexual compatibility results, it was of interest to de- cocca If to be discussed shortly, no strains of Group termine whether genetic compatibility existed as well IV intercrossed with members of other Groups except for the intra- and interspecific crosses. The resulting for E. illinoisensis 808f with E. unicocca var. periph- zygotes of certain crosses were germinated in order eralis 4sm and lOOm from Group V. An analysis of to analyze the inheritance of mating types. At ger- the inheritance of mating types has been performed mination generally a single viable product or gone for both intra- and interspecific crosses in Group IV escaped from the old zygote wall. The biflagellate gone and will be discussed in detail later. was capable of swimming up to several hours after Group V is composed of 3 heterothallic strains of which it settled down, and by a series of 4 to 5 suc- E. unicocca and 5 strains of E. unicocca var. periph- cessive bipartitions followed by inversion gave rise eralis. One member of this Group, E. unicocca If, to a gone colony. displayed the most unique pattern of sexual compati- To check for the inheritance of mating types, gone bility observed in this study. In light of these results colonies were isolated singly into test tubes of soil- a more careful examination was made of all the ap- water medium. When sufficient growth was achieved, parent nonreciprocal crosses involving this female they were backcrossed to the parents to identify their clone with the male clones of Group V, as well as those mating type. Backcrosses were performed in 3-depres- representatives of Groups 11, 111, and IV. In the sion Pyrex spot plates in the following manner. An cross E. unicocca If x lm the zygotes were formed aliquot of the population was placed in each of the in typical tight cohesive clumps which was also true 3 depressions, then mixed with the female parent in for the reciprocal intercrosses involving E. unicocca one depression and the male parent in another depres- lmf with E. unicocca 81mf. However, in other inter- sion. The third depression contained only the gone actions involving strain If, particularly with those population to check for selfing. When selfing was ob- male clones from Groups 11, 111, and IV, only a small served, subclones of the gone population were made number of zygote-like structures were produced in a to check for the presence of a complementary mating very atypical fashion. The “zygotes” remained within type. In addition to the segregation of mating types, the confines of the colonial envelope, but rather than 3 other factors proved to be excellent indicators of forming tight cohesive clumps they were arranged genetic compatibility in Eudorina: 1) ability of the near the periphery of the colonial envelope similar biflagellate, single-celled gone to divide and form nor- to vegetative cells. The most striking difference in mal gone colonies, 2) $% survival of isolated gone these reactions was the fact that often the colonies colonies, and 3) chromosome number of the F1 containing these zygote-like structures were sur- progeny. rounded by a “halo” of many sperm seemingly in- This investigation concerned intra- and interspecific capable of penetrating the colonial envelope (Fig. 43). crosses involving 4 heterothallic pairs of E. elegans This same atypical sexual reaction occurred in the (40mf, 62mf, 56mf, and 65mf) and one heterothallic mixtures of If x 17m and If x 62m which were the pair of E. illinoisensis (808mf). only 2 cases observed in which strains with different The results obtained for intraspecific crosses will be zygote arrangements appeared to cross. Numerous examined first. Strains 40mf and 62mf were 2 hetero- attempts have been made to germinate these zygote- thallic pairs of E. elegans collected from nearby ponds like structures in order to prove genetically whether in the vicinity of Bloomington, Indiana (Table 2. fertilization had occurred or not, but so far the at- Group 11). At zygote germination in the reciprocal tempts have been unsuccessful. Therefore, until such crosses (40f X 40m; 62f X 62m; 40f X 62m; 62f X time that proof is available to deny or validate the 40m) the biflagellate gones settled down forming presence of true non-reciprocal crosses involving E. mostly 16- and 32-celled gone colonies. Two hundred unicocca If, these apparent intercrosses must remain or more gone colonies were isolated for each of the questionable. 4 crosses and a high percentage of these survived and Excluding the above mentioned interactions involv- were checked for mating types (Table 3). In general ing E. unicocca If, Group V appears to be subdivided there was a 1: 1 segregation for the mating types with into 2 almost completely sexually isolated subgroups. the exception of one cross 62f X 62m. However, this One subgroup consisting of E. unicocca Imf and 81mf cross has been checked again more recently (Table 3) which cross reciprocally in a normal fashion and a and a 1:l segregation of the mating types was ob- second larger subgroup consisting of 5 heterothallic tained. It is possible that the excess of female off- 338 SPECIATIONAND SEXUALITYIN Eudoyina

TABLE3. Iiilrrritcciice of iiiotitt!/ t!/pcs for itttrctspccific crosscs of Euclorinx

Ability of single- So. gone Ratio of cellccl golies to coloiiies (k survival of mating types Cross form gone colonies isolatctl gone coloiiies F:M

E. de.//clcr/t.s 40f x 40111 escrllell t 310 93.5 100 : 8i I. ., 62 ,500 - - 1.20 : 93 t ,. ,. x 6'7m 40f x 62111 7'75 68.S 76 : i9 I. ., * ., .. 62f x 4Idin 3"t - 1.27 : 124 .Stif x 56111 2011 i.2 79 : 69 ,, >. ., .. Gfx ti5111 loll 9i 46 : 51 36f (i.7111 70.5 i0 : 71 ,, ,* x "00 (iSf x 56ni 2lMl 71 6i : i5 E. illitioisricsis 8Cl8f x 808111 ?no 90..i 96 : 85 - F = Fe111:1lcs,JI malt^.. * Jlaiiy more goiic~colonics siirrirecl than wrre eliwked for iii:tting types, btit the prrcrirt sw- viwl mis not recorded. t == I)ne to the sigiiificairt twess of feni:ile progeny o1)tninrd for this VYOSS, 60 inore goiie colonies were isolnted froni a second vross made iiiore recently. Fifty of the goiic colonies isolated siin-ircd and wre clircked for iuntiiig tTpcs. A ratio of 23 feiiiales : 27 niales was olrt:kincd inilicatiug a 1 : 1 segregatio~rof the iii:itiiig t-pes. spring initially obtained for the cross 62f X 62m was for this cross. Excellent survival was obtained as well due to selection of those gone colonies which appeared as a 1 : 1 segregation of the mating types (Table 3). most viable. These colonies could very well have The inheritance of mating types has been studied represented mainly females for the writer often noted for the reciprocal intercrosses involving E. elegans that the female clones generally grew better in culture 56mf and E. illinoisensis 808mf (Table 2, Group IV). than the male clones. However. one cannot overlook It will be recalled that these 2 species are distinguished the possibilities of induced parthenospores or some primarily by the size and reproductive potential of mechanism of differential survival at zygote germina- the anterior tier of cells in 32-celled colonies. In E. tion operating in favor of the female offspring. ckgans all cells are equal in size or nearly so with Strains 56mf and 65mf were 2 heterothallic pairs of each cell possessing an equal potential to produce E. elegans also collected in Indiana (Table 2. Group 32-celled daughter colonies. In contrast. the anterior IY): however. strain 65mf was collected in Blooming- tier of cells in E. illinoisensis is markedly reduced in ton. and strain 56mf was obtained from Greencastle, size and may or may not undergo division. If the ca. 40 miles away. As in the above crosses the bi- small anterior cells do divide. the resulting daughter flagellate gones produced mainly 16- and 32-celled colonies contain 4, 8 or 16 but never 32 cells. gone colonies. 200 of which were isolated for each of As indicated in Table 3, 74% of the gone colonies the crosses 56f x 56m. 56f X 65m. and 65f X 56m isolated for the cross 561 X 56m survived giving a and 100 for the cross 65f x 65311. The gone colonies 1 : 1 segregation of the mating types. In the cross 808f .showed excellent survival with a 1:l segregation of 808m. 90.5y of the gone colonies isolated survived the mating types (Table 3). x A third intraspecific cross involved a single hetero- and a 1: 1 ratio of the mating types was also obtained. thallic pair of E. illinoisensis (808rnf) collected in In contrast, of 208 gone colonies isolated for the cross Minneapolis. Minnesota (Table 2. Group IY). Two 56f x 808rn, only 23.5Cj, survived and could be hundred 16- and 32-celled gone colonies were isolated checked for mating type (Table 4). More striking,

T.\in.F. 4. IiiTjerif(ii!cc of iii(ttiit

Ability of single- So. g011c Ratio of mating celled golies to c.olonies % survival of types c'ross foriii gone colonies isolated gone eoloiiirs F : 31 : 831 : SF : HD

~ ~~__.___ FI geiitwtion 3if x 8O8m poor '708 23.5 11 : 20 : 18 : - : - 808f X 56ni 1ti ti 33 .1:53:-:-:- F2 genvration Diploid selfiiig male 84sni good 36 (i3 10 : -: 13 : -: - 1)il)loiilfrniale 88f x 84~111 escelleiit 2IlCI 80 108 :13 :30 : 4 : 6

~~ __- - 3' 1Females, 11 = Sales, SN = Selfing mnlca, SP = Srlfing fenialcs, aiid HD = Homoth:11- lie dioecious. Sotr: Thc three classes of segregants SJI, SF, and HD are all lio~i~otliallicand are cate- gorized 011 thv h:rsis of their selfiiig renetions a~ldthe reaetions wit11 the tester stocks 56f and 80Rm. SPECIATIONAND SEXUALITYIN Eudorina 339

however, was the inability of many gones to divide the usual 16 or 32 cells (Fig. 46). However, some once they settled down. Failing to divide, these gones 16-celled gone colonies were produced by single-celled would enlarge in size followed by lysis of the cell gones, but no 32-celled gone colonies were found as contents (Fig. 44). Those gones capable of division was common in the intraspecific crosses. In addition, often gave rise to atypical colonies, resulting from an 3 rather than 2 classes of segregants were produced in extremely abnormal sequence of divisions in gone the cross 56f X 808m; males, females, and a third colony formation (Fig. 45). The resulting atypical class of segregants referred to as selfing males (Table colonies contained odd numbers of cells rather than 4). These selfing males were similar to those isolated

Figs. 38-42. Zygote germination in Eudorina elegans (strains derived from a single-celled gone. Note the one enlarged cell 40mf and 62mf). Fig. 38. Flagellated portion of the proto- containing the oil globule. X 830. Fig. 42. Gone colony de- plast rupturing the thin wall. X 423. Fig. 39. Division of the rived from a single-celled gone. Note the one enlarged cell protoplast completed with flagellated half outside zygote wall containing the refractive oil globule. X 830. Fig. 43. Sexual and unflagellate portion within the old wall. X 1584. Fig. 40. compatibility in Eudorina. Fig. 43. Halo effect produced in The functional single-celled gone is now completely separated the mixture of E. unicocca If and E. elegans 62m. Note the from the old zygote wall while the non-functional spore re- many sperm surrounding the colonies that are seemingly mains in the old zygote wall. Note the granular appearance incapable of penetrating the female colonial envelopes. X SO. of the single-celled gone. X 1232. Fig. 41. Four-cell plakea 340 SPECIATIONAND SEXUALITYIN Eudorina from nature lor they produced an abundance of sperm 84sm and all possessed a chromosome number com- packets and mated well with a complementary female parable to this parental strain. Strain 84sm was also clone, yet produced a small number of zygotes within crossed with a polyploid female strain 88f also derived the clone. However, unlike the selfing males isolated from the cross 56f x 808m. The biflagellate gones from nature, zygote germination was obtained. the developed normally and 80% of the 200 gone colonies results of which will be discussed shortly. isolated survived and were checked for mating type In the reciprocal interspecific cross 808f X 56m, using clones 56f and 808m as tester stocks in the 166 gone colonies were isolated, and 33% survived backcrosses. The mating type segregation obtained and were checked for mating type. As in the cross for this cross was unusual in 2 respects: 1) 5 classes 56f X 808m. many gones failed to divide and died of segregants were produced, females, males, selfing when the cell contents lysed: or if the gone underwent males. selfing females, and homothallic clones, and 2) division, atypical gone colonies generally resulted. the female offspring outnumbered the remaining classes However, only male and female progeny resulted from of segregants by more than a 2: 1 ratio (Table 4). this cross with a large excess of male offspring The homothallic clones crossed with the tester stocks (Table 4). as indicated by the number of zygotes produced. Back- In light of the observed mortality or inability of crosses of polyploid F1 progeny to the haploid parental the single-celled gones to produce typical gone colonies. clones were attempted, but survival of the offspring the low percentage of survival of gone colonies iso- was so poor that these data were not recorded in this lated. and the abnormal segregation of mating types study. for the reciprocal interspecific crosses, a cytological in- vestigation was initiated. Mitotic chromosome counts DISCCSSION AND GENERAL CONCLUSIONS made for at least 14 of the F1 progeny derived from The taxonomic and general biological significance each of the reciprocal interspecific crosses, 56f X 808m of sexual and genetic isolation within a species was and 808f x 56m, revealed a polyploid or aneuploid reviewed in detail by Sonneborn(60) for several spe- condition. The counts ranged from 24-28 chromo- cies of ciliated protozoa, particularly in Paramecium, somes while the 4 parental clones all had an approxi- Tetrahymena, and Euplotes. The most extensively mate chromosome number of 14 (Figs. 47, 48). In analyzed species is Paramecium aurelia obtained from addition chromosome counts were made for 10 progeny approximately 300 natural populations widely dis- derived from each of the following crosses, 56f X 56m tributed throughout the world. Investigation of these and 808f x 808m, and no increase in chromosome strains has revealed 14 distinct genetically isolated number was observed (Fig. 49). groups or syngens (Sonneborn, pers. commun.). The Morphologically the F1 progeny derived from the term syngen designates those strains of an organism reciprocal interspecific crosses were also distinct. Gen- which share a “potentially common gene pool.” Each erally the F1 clones were composed of colonies with syngen of P. aurelia is composed of two complemen- 16 cells or less, but occasionally with 32 cells. Often tary mating types between which gene exchange can one to several cells died at random within the colonies. occur. Six of the syngens were found to be completely Four of the F1 clones produced 32-celled colonies in sexually isolated from the other syngens while among quantities sufficient for measurements. It was found the remaining 8 syngens there were varying degrees that the colonies and cells were on the average larger of compatibility. The intersyngenic reactions included than those of the parental strains: the shape of the cases in which the animals adhered briefly but never colonies and cells was extremely variable in these and formed true conjugant pairs, while in other cases true all other hybrid F1 clones. Lastly. in the F1 hybrid conjugant pairs were formed. However, in the latter clones producing 32-celled colonies the anterior tier cases the exconjugants either failed to yield viable of cells was markedly reduced in size as in the parental Fl offspring or if the exconjugant clones did survive clones 808f and 808m. a viable F2 generation was not produced at autogamy. The genetic analysis of mating types was then ex- The geographical distribution of the different syngens tended for certain crosses to the F2 generation. has also been studied and certain of the syngens ap- Thirty-six F2 progeny were obtained from one of pear to be world-wide in distribution whereas others the selfing males (84sm) derived from the cross 56f are localized in their geographical range. In this regard x 808m. The biflagellate gones developed normally it is clear that temperature is a major factor influenc- into 16- and 32-celled gone colonies and 63% of the ing the range of a syngen on the continent where it 36 gone colonies isolated survived and were checked occurs ( 60). for mating type. Two classes of segregants were pro- Sexual isolation has been reported in Chlamydo- duced; 13 selfing males and 10 females (Table 4). wzonas, Gonium, and Pandorina. Schrieber( 55) re- Mitotic chromosome counts were made for 8 of the ported complete sexual isolation among 70 clones of F2 progeny derived from the polyploid selfing male Chlamydomonas representing 6 species, all of which SPECIATIONAND SEXUALITYIN Eudorina 341

were collected in one small area in Germany. More in Indiana. When these strains were tested in all recently Stein (65) reported almost complete sexual possible combinations, a total of 18 intercrosses was isolation, except for 7 intercrosses, among 11 hetero- obtained, all involving strains with similar zygote thallic pairs of Gonium pectorale, approximately half aggregations or arrangements. Based on this study of which were collected within a 20-mile radius of Coleman recognized 15 sexually isolated groups or Bloomington, Indiana. The most extensive study of syngens (in sensu Sonneborn) with the Indiana strains sexual isolation within a single algal species was per- represented in 8 of the 15 sexually isolated groups. formed with Pandorina morum by Coleman( 13). Her However, Coleman(l3) had to qualify the use of the study involved 22 heterothallic pairs and 9 “hetero- term syngen for Pandorina morum because she lacked thallic” clones of Pandorina morum collected mainly genetic proof that gene flow occurred between sexually

Figs. 44-46. Hybrid offspring derived from the cross E. ploid and haploid strains of Eudorina. Fig. 47. Prometaphase elegans 56f X E. illinoisensis 808m. Fig. 44. Enlarged single- in 84sm, a polyploid selfing male derived from the cross 56f X celled gone undergoing lysis. X 830. Fig. 45. Formation of 808m. X 1900. Fig. 48. Metaphase in 808m. X 1900. Fig. 49. gone colonies. Note the abnormal sequence of divisions in Metaphase in 98m, a strain derived from the cross 561 X 56m. plakea formation. X 690. Fig. 46. Abnormal gone colony. X 1900. X 830. Figs. 47-49. Mitotic chromosomes in a derived poly- 342 SPECIATIONAND SEXUALITYIN Eudorina compatible strains within a single syngen. mating types, and most important the formation of In the present study sexual isolation was observed polyploid or aneuploid F1 offspring. These results among the strains of Eudorina elegans. Based on their provided two important facts concerning the future pattern of sexual compatibility the 12 heterothallic application of syngens in the genus Eudorina: 1) pairs, 2 male strains and a single female were arranged sexual compatibility alone cannot be used as a cri- into 4 partially or completely sexually isolated groups. terion to determine strains which share a potentially Ability to intercross in E. elegans was definitely related common gene pool, and 2) the formation of F1 poly- to the zygote arrangement as noted by Coleman( 13). ploids or aneuploids associated with an abnormal for sexual isolation was complete between strains segregation of the mating types provided a good cri- with different zygote arrangements. However, sexual terion to distinguish syngens in the genus. The isolation also occurred to a lesser extent between validity of this hypothesis will be tested more ade- strains with similar zygote arrangements. Sexual iso- quately in the future when the remaining interspecific lation was also observed between the strains of E. crosses in Group IV are analyzed genetically. unicocca and the strains of the newly-described variety Interspecific hybridization is known to occur in E. unkocca var. peripheralis. all with zygotes in tight representatives of almost every major group of the cohesive clumps. Therefore, it may be concluded that plant kingdom. Until the present, its occurrence in sexual isolation does exist within at least two species the algae was known for only two genera; the unicellu- of Eudorina, but in comparison to the two isogamous lar green flagellate Chlamydomonas and the marine species. Gonium pectorale and Pandorina morum, brown alga Fucus. Both natural and experimental there is a considerably greater tendency for outbreed- hybrids have been produced in several interspecific ing among strains of a single morphological species of combinations in Fucus( 5,34,69), but only experimen- Eudorina as indicated by the fewer intercrossing tal hybrids have been produced in crosses of morpho- groups. logically distinct species of Chlamydomonas(47,67). Vntil the present study no attempt has been made Pascher(47) made chromosome counts for the F1 to establish the existence of true syngens in an algal hybrid offspring produced in Chlamydomonas, but no species. An initial atempt was made to do so through increase in chromosome number was observed. There- the analysis of the inheritance of mating types for the fore. it appears that the crosses involving E. elegans reciprocal intraspecific crosses involving E. elegans 56mf and E. illinoisensis 808mf represent the first 40mf and 62mf (Group 11) and E. elegans 56mf and case in the algae of interspecific hybridization resulting 65mf (Group IV). .4s observed in other haploid or- in polyploid or aneuploid F1 offspring. ganisms including the unicellular green flagellate Associated with the increased chromosome number Chlamydomonas(39) and the isopamous colonial certain of the F1 progeny derived from the cross E. green flagellates Gonium pectorale( 65 ) and Pandorina elegans 56f x E. illinoisensis 808m expressed a sexual morum(l3), a 1:l segregation of the mating types potential which combined the potentialities of both was obtained indicating not only that inheritance of parents. apparently through the incorporation of both mating types is controlled by a single pair of alleles. mating type factors into the newly formed polyploid but that gene exchange occurs between the above nucleus. The male phenotype was dominant in these strains of E. elegans. Consequently this appears to be homothallic polyploids thus bearing a marked re- an excellent criterion to determine whether sexually semblance to the selfing males isolated from nature compatible strains of Eudorina share a common gene and to a lesser extent the homothallic strains of Eudo- pool. Before applying the concept of syngens within rim. It therefore appeared that natural hybridization the genus Eudorina a much more extensive genetic of heterothallic strains might account for the origin analysis is necessary, since preliminary data (Table 3) of selfing males and homothallic strains ; however, indicate possible significant differences in '/. survival chromosome counts revealed approximately 14 or 15 of gone colonies derived from crosses of strains within chromosomes for the 5 selfing male clones isolated and between natural populations. from nature (4sm, 47sm, Slsm, 67sm, and 72sm); 5, In light of the sexual compatibility exhibited by the 6. 7. and 14 chromosomes for certain of the homo- 3 species in Group 11' it appeared that a syngen in thallic monoecious strains of E. elegans; and 14 chro- Eudorina would have to include strains of different mosomes for the homothallic dioecious strain of E. species. However, when a genetic and cytological conradii. Therefore, it seems that the homothallic analysis was performed for the reciprocal interspecific condition observed in the polyploid hybrids is only crosses involving E. elegans 56mf and E. illinoisensis analogous to the naturally occurring homothallic mode 808mf. it was found that regardless of their sexual of sexuality. compatibility, gene flow was absent between the spe- A detailed cytological analysis of the meiotic events cies. This was indicated by the high rate of mortality in the hybrid zygotes is planned as well as a more of the F1 gones. the abnormal segregation of the detailed genetic analysis of interspecific hybridization SPECIATIONAND SEXUALITYIN Eudorina 3 43 with the use of other markers in addition to the mating vocineae (Rabenh.) . Nachrichten kaisl. Ges. Naturwiss. An- type locus. It is felt that herein lies an exceptional thropol. Ethnogr. 16, Heft 2. 27. Grove, W. B. 1915. Pleodorina illinoisensis Kofoid in system for the investigation not only of the genetic Britain. New Phytol. 14, 169-82. relationships between the species of Eudorina, but 28. Hartmann, M. 1921. Die dauernd agame Zuch von Eu- also a key to understanding the genetic control of dorina elegans, experimentelle Beitrage zum Befruchtungs-und Todproblem. Arch. Protistenk. 43, 223-86, sexuality in the genus. 29. Herndon, W. 1958. Studies on Chlorosphaeracean algae from soil. Am. J. Botany 45, 298-308. REFERENCES 30. Huber-Pestalozzi, G. 1961. Das Phytoplankton des Susswassers Systematik und Biologie. Chlorophyceae. Vol- 1. Akehurst, S. C. 1934. Eudorina elegans Ehrenberg forma ellipsoida sub. var. tubijera. J. Roy. Microscop. SOC.54, 99-103. vocales. Die Binnengewiisser, Band XVI, 5 Teil, 1-744. 31. Iyengar, M. 0. P. 1933. Contributions to our knowledge 2. Arce, G. & Bold, H. C. 1958. Some Chlorophyceae from Cuban soil. 492-503. of the colonial Volvocales of South India. J, Linn. SOC.Lon- Am. 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Pseudocohnilembidae n. fam., a Hymenostome Ciliate Family Containing One Genus, Pseudocohnilembus n. g., with Three New Species"

FREDERICK R. EVANS and JESSE C. THOMPSON, Jr.

Dppnrtnrent oi Zoology and Entoinology, Lvnivrrhi!p of 1-tah, Salt Lakp Citp, Utah, and Departnieirl of Biology, Hanipden-Sydney College, Hanipden-Sydney, Virginia

SY'SOPSIS. r\ new family (Pseudocohnilembidae) of hymeno- genus (Phetrdorohnileinbzi.,) with three species (one from stome ciliates is described based primarily upon the pattern oi Great Salt Lake, Utah, and two from the Atlantic coast) is the buccal infraciliature which consists of two parallel rows of described. These organisms bear superficial resemblance to kinetosomes, nearly equal in length, representing the bases of Cohnilenibicb ; an analysis of the buccal infraciliature of Pseudo- two large membranes. and of argentophilic fibrils arranged in rohni~rnthusis presented, including a discussion of and com- a marc or less uniform design in the buccal cavity. A new parison with that ot Cohizileinbus

NOSG protozoa that were found in water samples to warrant the establishment of the following species: A collected from the Great Salt Lake. Ctah, from Psmdocohnilenzbus persalinus n. g., n. sp.; Pseudo- the York River at Gloucester Point. I'irginia. and cohnilembus hargisi n. sp. ; Pseudocohnilembus longi- from Daytona Beach. Florida. there appeared hymeno- SPiZtS n. sp. stonie ciliates of a similar structural plan. It is becoming apparent that there is a great need The results of morphological studies on these ciliates for a new look at hymenostome taxonomy, and that indicated a general similarity to the family Cohnilem- the taxonomic history of the ciliate order Hymeno- bidae. but. due to differences primarily associated with stomatida needs to be rewritten in light of modern the buccal apparatus. a new family. Pseudocohnilem- techniques(3,6). Most past descriptions were written bidae. and a new genus. Pseudocohnilembus. are pro- primarily from microscopical observations of the living posed to receive these ciliates. Constant morphological organisms and they provide such superficial bases for variations among these organisms seemed peat enough ___ taxonomic determinations that it is virtually impos- * This investigaticn was supported by research grants to the sible. in most cases, to make specific assignment; even senior author from the University of Utah Research Fund and generic assignment is often difficult. from the Sational Science Foundation (#G22667). The junior \Ye feel that it is an error of omission, in dealing author acknowledges support from Sational Science Founda- tion grants #G?0851 and #GI5445 ; the latter (Research Par- with the hymenostome ciliates, to exclude modern ticipation for College Teachers) was awarded for the summer techniques in taxonomic publications. The silver im- of 1961 at the Virginia Institute of Marine Science. Gloucester Point, Virginia. The authors are indebted to Dr. John 0. pregnation techniques produce a "protozoan finger- Corliss for his counsel and critical evaluation of the manuscript. print"(20) which provides a precise basis for taxo-