J. PROTOZOOL.11(3), 317-344 (1964). 317 Speciation and Mating Behavior in Eudorina" 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 Chlorophyceae. 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.