Genotypic Diversity in Cypridopsis Vidua (Ostracoda: Cyprididae)

Heredity 62 (1989) 383—392 The Genetical Society of Great Britain Received 19 October 1988

Apomicticparthenogenesis and genotypic diversity in Cypridopsis vidua (Ostracoda: Cyprididae)

John E. Havel and Department of Biological Sciences, University of Paul D. N. Hebert Windsor, Windsor, Ontario, N9B 3P4, Canada. Based on sex ratios, the crustacean subclass Ostracoda appears to have made frequent transitions from sexual to asexual reproduction. However few genetic data are available verifying their mode of reproduction. The current report provides such evidence for apomictic pathenogenesis in the cosmopolitan cypridid ostracode Cypridopsis vidua. Sex ratio analysis indicated that natural populations are exclusively female. Allozyme phenotypes in 18 of 20 natural populations showed strong departures from Hardy—Weinberg expectations at single loci, and three of four populations had non-random associations of phenotypes at MPI and PGM. Allozyme phenotypes at these loci suggested that polyploids were present in half these populations and were more common in the low arctic site than in the temperate sites. Most natural populations of C. vidua displayed high levels of genotypic diversity, with a total of 77 unique clones detected and up to 24 clones per pond. Breeding studies on 64 isofemale lines established that parthenogenesis is apomictic and that mutation rates are less than i0.

INTRODUCTION in this group. Since ostracodes are cosmopolitan and live in a wide variety of habitats, this group Althoughsexual reproduction is the dominant should provide useful insight into the evolution of breeding mode, asexual reproduction is wide- sex and factors maintaining genotypic diversity. spread in both plant and animal kingdoms (Bell, Previous genetic work on ostracodes has been 1982; Ellstrand and Roose, 1987). Since genetic limited. Cytogenetic work of Bauer (1940) estab- recombination is absent, early theorists assumed lished differences in chromosomal behaviour that genetic variation would be low in asexuals between parthenogenetic and sexual "races" of (Williams, 1975). However, a variety of studies Heterocypris incongruens, and Tetart's (1978) study have now shown that this view is incorrect. Studies of 24 freshwater ostracode species from France of morphological, chromosomal, allozymic, and confirmed that the majority of species reproduced histocompatibility diversity have revealed abun- asexually, with several asexuals exhibiting cyto- dant variation on both macro- (Lyman and logical variation. Sywula and Lorenc (1982) Ellstrand, 1984; Hebert et a!., 1988) and micro- studied esterase polymorphisms in Cyprinotus geographic scales (Hebert and Crease, 1980; incongruens, but the genetic basis of the variation Hoffmann, 1986; Ellstrand and Roose, 1987). they found was uncertain, Allozyme studies on Species of the crustacean subclass Ostracoda other ostracode species were unsuccessful due to apparently employ both breeding modes. Based their small size (Sywula et a!., 1985). Recently, on the presence or absence of males, both asexual cellulose acetate electrophoresis has provided an and sexual species are common in freshwater habi- improved method for assaying allozyme diversity tats (Tressler, 1959; Bell, 1982). Four genera in small organisms (Easteal and Boussy, 1987), include exclusively asexual species, three exclus- and has, in the present study, permitted the resol- ively sexual species, while 14 include species ution of multiple enzymes from very small employing both breeding systems. In contrast, ostracodes. most marine species are believed to be sexual (Bell, The current study examines the extent of clonal 1982). Its broad taxonomic distribution suggests diversity in natural populations of the freshwater that asexuality has evolved many different times cypridid Cypridopsis vidua (0. F. Muller 1776) 384 J. E. HAVEL AND P. D. N. HEBERT

Brady 1867, and supplements this with laboratory gel. Gels were run for 15 minutes at 200 V (2 mA studies to confirm its mode of reproduction. per gel plate), then stained using standard methods Although early workers suggested that this species (Harris and Hopkinson, 1976). was a cyclic parthenogen (Weismann, 1880, cited Two populations (Baseline-2, near Windsor, in Kesling, 1951),subsequentlaboratory studies Ontario, and T78, near Churchill, Manitoba) were and numerous collections from natural popula- screened for variation at 15 enzymes. Four enzymes tions have failed to detect males, leading recent were monomorphic, five were polymorphic but workers to assume the species is asexual. C. vidua unreliable, and four did not stain. Two enzymes is nearly cosmopolitan in distribution, being repor- were polymorphic and readily scored for allozyme ted from North America, Europe, Africa, and variation: mannose phosphate isomerase (MPI) South America (Kesling, 1951). The other 15 and phosphoglucomutase (PGM). As a result, species of Cypridopsis in North American are less other populations were scored only for MPI and widely distributed, with most being reported from PGM. a single state or province. Seven of these species Alleles were designated by their relative migra- are sexual and are presently restricted to localities tion rates: 0 fastest, 7 slowest. Although the genetic in the gulf states and Mexico (Tressler, 1959;Fer- basis of allozyme variation could not be verified guson, 1964), although Delorme (1970) reported with breeding studies because of C. vidua's asexual fossil records of one sexual species (C. okeechobei) mode of reproduction, banding patterns for puta- from the Canadian prairies. The eight other tive heterozygotes were consistent with their asexual species are endemic to either northern or known quaternary structure (both MPI and PGM southern locales. monomeric; Harris and Hopkinson, 1976). After establishing laboratory cultures, all subsequent gel plates included 2-4 individuals from clones of MATERIALS AND METHODS known genotype to act as standards. Allozyme data were collected on 23 popula- Collections tions and genotypic frequencies at single poly- Ostracodes were collected from ponds and lakes morphic loci were tested for fit to Hardy-Weinberg between May 1987 and August 1988 by sweeping (HW) expectations using x2tests.Loci were con- a 200 pm-mesh hand net through macrophytes and sidered polymorphic if the frequency of the most along the bottom. Lake collections were restricted common allele was less than 090 and sample size to the littoral zone. Samples were sorted on a light exceeded 20. Rare classes were pooled so that box and C. vidua identified using Tressler (1959). minimum expected values exceeded five. Three- Collections were made in Manitoba, Ontario, banded and unbalanced phenotypes were pooled Quebec, New Brunswick, Wisconsin, Michigan, with the closest two-banded phenotype for HW and Texas. C. vidua was collected during March analysis. Two-locus genotypes were examined for in Texas, July-August in Manitoba, and May-June deviation from random association by G-tests of in the remainder of the sites. C. vidua were held independence (Sokal and Rohlf, 1981). alive at 4°C for one week or less prior to electro- Genotypic diversity was estimated by two phoresis, or otherwise frozen at —70°C. Samples different statistics. The first was the total number from half the populations were examined for males of multilocus genotypes (clones, k) identified in at lOOx by crushing 20 individuals under a cover each sample. The second statistic was observed slip. Males of other species can be recognized by genotypic diversity (G0, Hoffmann, 1986), which the presence of sperm tubes, ejaculatory ducts, or combines estimates of clonal abundance with hemipenes, mature females by ovaries and eggs evenness. (Tressler, 1959; personal observation). G0 g Allozymevariation Electrophoresiswas carried out on Titan III whereg is the frequency of the ith of k genotypes. cellulose acetate gels (Helena Laboratories, The range of G0 is 1 to k, with G0 reaching its Beaumont, Texas) and Tris Glycine buffer (3 g maximum value when the clonal frequencies are Trizma base, 144g glycine, 1 litre water; pH 85). all equal. Gels were presoaked and run in the same butler Clonal composition and distribution were com- solution. Cypridopsis were crushed individually in pared among 19 populations for which standard sample wells and 12-24 samples applied to each markers were included on gels. CLONAL DIVERSITY IN CYPRIDOPSIS 385

Breeding study There was no evidence of consistent heterozygote excess or deficiency, as F', values included similar Fora direct test of apomictic parthenogenesis, 126 numbers of positive and negative values (15 and cultures were initiated with adult C. vidua collec- 11) and were not significantly different from zero ted from nine locations. These were placed (Wilcoxin Signed Rank Test, T=037, P>050). individually in 120 ml cups containing synthetic In four populations both loci were sufficiently pond water (Hebert and Crease, 1980) and mass- polymorphic so that it was possible to test for cultured green algae (primarily Ankisrodesmus sp. deviations from random association of genotypes and Chiorella sp.). After 3 months, 64 cultures (following pooling of rare classes). These tests contained abundant C. vidua, and were likely mix- indicated that genotypes at the two loci were non- tures of several generations (Kesling, 1951). Ten individuals from each of the 64 cups were randomly associated in three of the four popula- examined at lOOx for males. MPI and PGM tions and in the 19 populations pooled (table 3). phenotypes were determined for eight additional individuals from each cup to ascertain whether Clonaldiversity isofemale lines were composed of genetically iden- Atotal of 77 different clones were detected among tical individuals. the 842 animals analysed, with a mean of 83 clones per pond (table 2). Although many ponds were multiclonal, four ponds were uniclonal. Two of RESULTS these ponds shared the same clone and were from eastern Canada. Since clonal frequencies were Collections uneven within populations, the genotypic diversity Sufficient numbers of C. vidua for genetic study measure (G0) was usually much less than the were collected from 23 populations over a wide observed number of clones (table 2). The effective geographic range, extending from southern Texas number of clones in each population ranged from to northern Manitoba (fig. 1, table 1). Microscopic 1 to 107, with an average of 38. examination of adults from 12 different collections Comparison of clonal genotypes among popu- revealed only females, as did repeated observations lations was carried out on 19 of the 23 populations. of cultured animals. The studies showed that most clones were rare, with 48 of the 77 clones found in only a single variation location (fig. 2). However, some clones were Allozyme apparently widely distributed. For instance, clone Allozymestudies revealed abundant variation in 23-33 (MPI-PGM) was found in 12 of the 19 C. vidua. Seven alleles were detected at MPI, with populations and distributed from northern and a mean of 44 alleles per population, and 5 alleles eastern Canada to southern United States (table 2). at PGM, with a mean of 23 alleles (table 2). In Although most populations were sampled only most cases, heterozygotes had two bands of equal once, the Warner Road population was sampled staining intensity. However, in ten populations twice in 1988, and these samples had significantly some MPI and PGM phenotypes were either three- different clonal arrays (G=22.6, 4df, P<0005). banded or had unbalanced activity, with one zone The 6 April sample contained only juveniles, with staining at least twice as intensely as the other 6 clones detected in 20 animals surveyed, while in (tables 1 and 2). The incidence of these phenotypes the 10 May sample 14 clones were detected in 47 appeared to be higher in arctic than temperate animals surveyed. In addition to additional rare populations, with values ranging from 006 to 015 clones detected in the larger sample, two new near Windsor and 019 to 051 near Churchill genotypes were in high abundance (frequencies (Wilcoxin Rank Sum Test, W=40, P=0004). 017 and 0.23). None of the four lake populations included any of these phenotypes, though these populations Breedingstudy were polyclonal (table 1). At single polymorphic loci, 18 of 20 popula- Microscopicexamination of samples from each of tions showed significant departures from HWE at the 64 laboratory cultures showed only females, 1 or more loci (table 1). Direction of departure with most of the adults carrying eggs. Allozyme was estimated by the inbreeding coefficient, F, = studies indicated no genotypic variation within 1—H0/He, where H0 and He are the number of these cultures. Most cultures contained heterozy- heterozygotes observed and expected under HWE. gotes for at least one locus, so the absence of 386 J. E. HAVEL AND P. D. N. HEBERT

Figure 1 Locations sampled. A =AnnArbor, Michigan; C Churchill, Manitoba; H =Houston,Texas; I =Isabella,Michigan; M =Madison,Wisconsin; N Moncton, New Brunswick; 0 =Crosby,Ontario; Q =St.Rose, Quebec; W =Windsorand Rondeau Park, Ontario. recombination was convincing (table 4). Since genesis. In congruence with prior studies (Kesling, eight individuals were electrophoresed from each 1951), no males were observed in natural or labora- culture, the chance of detecting no recombinants tory populations. Genetic data confirm the asexual in individual cultures if segregation was occurring mode of reproduction. Genotypic frequencies in would be small ((0.5)=0.004). The absence of 18 of 20 natural populations were out of HWE and recombination among all the cultures carries an linkage disequilibrium between genotypes at MPI extremely small joint probability (table 4). and PGM was frequent. Similar genotypic characteristics have been reported in studies of other DISCUSSION asexual taxa (Hoffmann, 1986; Innes et al., 1986). The laboratory rearing studies indicated that single Thecurrent study confirms that C. vidua from C. vidua females could initiate cultures and that North America breeds by apomictic partheno- their offspring were genetically identical. The lack CLONAL DIVERSITY IN CYPRIDOPSIS 387

Table 1 Summary of allozyme results for populations of Cypridopsis vidua. Populations examined for sex ratios (f). Proportion of individuals with 3-banded or unbalanced phenotypes: prop. polypl. x2valueswere tested for fit to HWE at indicated degrees of freedom (df); probability values (P): ns P> 005, *P<005, **P<001; —notsufficiently polymorphic or insufficient data to test

MPI PGM Locale No. No. prop. population md. clones polypl. x2 (df) P x2 (df) P Churchill, Man. 1. Goose-3 (f) 45 9 051 227 (1) — — — ** 2. Goose Cr. (f) 42 13 045 15 (1) ns 163 (1) 3. T14 9 4 067 — — — — — — ** 4. T49 43 7 019 359 (1) — — 5. T78 85 10 022 1359 (3) 850 (1) ** 6. T204 (f) 44 1 0 440 (1) — — — 7. T205-B 58 1 0 370 (1) — — — 8. T208 45 4 0 — — — — — — Moncton, N.B. 9. NB-2 22 1 0 220 (1) — — — St. Rose, Que. ** 10. Qu-20 22 1 0 220 (1) — — — Crosby, Ont. 11. Rideau L. 10 5 0 — — — — — — Rondeau Pk., Ont. 12. Rondeau-I (f) 35 9 0 07 (1) ns — — — ** 13. Rondeau-2 (f) 46 15 015 56 (3) ns 220 (1) Windsor, Ont. 14. Ojibway-2 (f) 46 12 013 325 (2) not done 15. Ojibway-5 (f) 36 14 011 78 (1) 17 (1) ns 16. Ojibway-6 (f) 30 8 007 01 (1) ns — — — 17. Baseline-2 (f) 48 24 0 125 04 ns (2) ** (1) 18. L. St. Clair 42 10 0 122 (2) — — Big Bay de Noc, Mi. ** 19. L. Michigan 47 8 0 120 (2) — Ann Arbor, Mi. 20. Warner (f) 67 16 006 622 (6) — — — * 21. Textile-B 24 15 0 353 (7) 78 (2) Madison, Wis. 22. L. Monona (f) 78 10 0 243 (1) — — — Houston, Tex. 23. FM-2090 (f) 69 11 0 383 (2) k — —

of segregation at heterozygous loci confirms that ostracode species he examined. The allozyme data eggs are produced apomictically in this taxon. for C. vidua suggest there may be a latitudinal These data are the first genetic confirmation of pattern in polyploidy, with the lowest incidence in asexual reproduction in an ostracode, and suggest the south and highest in the north. In temperature that other species in which males have not been latitudes, it appears that polyploids are more com- found are also apomicts. mon in ponds than in lakes. Eleven populations of C. vidua included The current study revealed surprisingly high individualswith 3-banded or unbalanced levels of genotypic diversity in C. vidua. Based on phenotypes at the monomeric loci MPI and PGM. only two polymorphic loci, up to 24 clones were Such phenotypes are ordinarily indicative of poly- detected in single ponds (average 8.3). Such high ploidy (Weider and Hebert, 1987; Beaton and clonal diversity is unprecedented for asexuals liv- Hebert, 1988), but DNA quantification and cyto- ing in discrete habitats. For example, in a survey logical studies are necessary to verify the polyploid of obligately asexual Daphnia pulex from ponds status of the clones in C. vidua. There is little prior near Churchill, Manitoba, one to four clones were evidence of polyploidy in ostracodes, although detected per pond (average 1.5) and only 16 in the Tetart's (1978) cytological studies revealed a low entire region (Weider and Hebert, 1987). Surveys proportion of triploids in one of 17 asexual of the same species throughout the Great Lakes * Table 2 Clonalfrequencies in populations of Cypridopsis vidua. Allele designations: 0 = fastest. = unbalanced hererozygote,with greater activity in marked allele. Baseline-2 frequencies are based on laboratory cultures. G0=observed genotypic diversity (Hotlmann, 1986). Population numbers are identified in table 1. A total of 77 clones were detected

Manitoba New Ontario Michigan Genotype Bruns. Quebec — Wisconsin Texas MPI PGM 1 2 4 5 6 7 8 9 10 11 15 16 17 18 19 20 21 22 23 Total

014 33 02 33 2 2 11 23 1 1 2 11 33 11 13 1*2 33 1 2 12 23 1 1 12 33 3 3 12* 11 1 1 12* 23 1 1 13 23 1 1 13 33 1 2 3 6 134 33 1 1 14 33 1 1 15 33 1 6 7

22 1 1 22 23 3 1 1 5

22 33 27 4 1 1 6 3 14 8 5 33 1 103 22 3*4 7 8 15 23 23 5 1 9

23 33 1 1 22 22 2 1 2 3 10 1 27 38 130 2*3 33 4 9 2 15 23 34 3 1 1 5 234 33 3 3 24 1 1 24 33 1 3 13 1 5 3 9 7 42 24 34 1 1 2 2*4 33 30 30

245* 33 1 1 25 33 1 1 4 2*5 33 2 2 26 23 12 1 13

26 33 1 1

33 22 1 1

33 23 3 1 1 5 0 33 33 1 1 1 10 16 5 9 43 - 34 1 1 34 13 1 1 P 34 22 58 58 z 34 23 2 1 2 5 34 33 44 1 11 3 7 2 1 71 34 34 1 1 W

3*4 23* 1 1

347 33 1 2 3 CLONAL DIVERSITY IN CYPRIDOPSIS 389

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N '('.n 'f0O'.O* N*N d en en en en ., , o '.0 '.0 '.0 '.0 390 J. E. HAVEL AND R D. N. HEBERT

Table 3 G-tests of independence between MPI and PGM phenotypes following pooling of rare classes. Pooled data are from all 19 populations in table 2 60 Cypridopsis vidua Population G df P-value N 78 Goose Cr. 223 4 <0005 T78 1163 4 <0005 50 Ojibway-5 55 2 <010 Baseline-2 102 3 <005 <0005 Pooled 196 5 40 0 0 0 watershed revealed an average of only 28 clones 0 per pond (Hebert et al., 1988). One to nine clones z 30 per population were detected in the brine shrimp Anemia parthenogenetica (Abreu..Grobois and Beardmore, 1982), while a total of three hemi- 20 clones were detected in 13 populations of the hybridogenetic triploid fish Poeciliopsis monacha lucida (Vrijenhoek, 1978). Seventy-seven clones of C. vidua were detected 10 in the 23 ponds surveyed. Several clones were apparently widespread, with one ranging from Churchill, Manitoba, to Houston, Texas. However with clonal discrimination based on only two loci, 1 2 3 45 6 7 89 10 11 12 it is likely that this "clone" represents a clonal complex whose members happen to share a com- No. Populations mon genotype at these loci, while differing at other Figure2 Histogram showing the number of populations in loci. The number of C. vidua clones detected is which each clone was detected. Raw data are presented in almost certainly a small fraction of the total present table 2. (Hebert et a!., 1988). The proximate cause of the high clonal diversity in C. eidua is unclear. Two problems need to current breeding study suggests that C. vidua does be considered: the origin of diversity in single not have exceptionally high mutation or recombi- habitats and the origin of diversity in the entire nation rates during subitaneous egg formation. The gene pool. Potential sources of diversity in single lack of variation within the lab cultures indicated habitats include mutational or recombinational that recombination rates were no higher than 19 x diversification of single colonists and multiple iO- (67 total heterozygous loci x8 individuals) colonizations of the habitat. The results from the and mutation rates no higher than 49 x 10 (64

Table 4 Allozyme results from C. vidua cultures. Eight individuals from each culture were electrophoresed at MPI and PGM

Total no. Initial no. No. of heterozygous loci No. of Location of isolates cultures detected recombinants prob.

Baseline-2 72 33 43 0 26x iO° Ojibway-l 3 2 2 0 16x 10 Ojibway-2 5 1 2 0 16x i0 Ojibway-5 5 1 0 — — Ojibway-6 21 20 15 0 75 Xi0< Warner 5 3 2 0 l-6x10< NB-2 5 2 2 0 16x10 L. Monona 7 1 1 0 3.9 x 10< L. Michigan 3 1 0 —--

Total 126 64 67 0 CLONAL DIVERSITY IN CYPRIDOPSIS 391

cultures x 2 loci x 8 x 2 alleles) (table 4). There is BAyER, i. 1940. Uber die Chromosomen der bisexuellen und a clear need to extend such studies to investigate der parthenogenetischen Rasse des Ostracoden mutation and recombination rates among the pro- Heterocypris incongruens Ramd. Chromosoma, 1, 620—637. BEATON, M. J. AND1-IEBERT, P.D. N. 1988. Geographic geny of resting eggs. Rare endomeiosis during rest- parthenogenesis and polyploidy in Daphnia pulex (Ley- ing egg oogenesis could, for example, account for dig). Am. Nat., 132, 837-845. the large number of genotypic combinations BELL, G. 1982. The Masterpiece of Nature: The Evolution and observed in the present study. Rare production of Genetics of Sexuality. University of California Press, Berkeley. males would have a similar eflect, but extensive DELORME, L. D. 1970. Freshwater ostracodes of Canada. Part field surveys have never detected males in this II. Subfamily Cypridopsinae and Herpetocypridinae, and species (Furtos, 1933; Kesling, 1951; Delorme, family Cyclocyprididae. Can. J. Zool., 48, 253-266. 1970). Multiple colonizations of each habitat could EASTEAL, S. AND BOUSSY, 1. A. 1987. A sensitive and efficient isoenzyme technique for small arthropods and other also account for the high clonal diversity in local invertebrates. Bull. ent. Res., 77, 407—415. habitats. Two vectors for gene flow have been ELLSTRAND,N. C.ANDROOSE, M. L. 1987. Patterns of identified for C vidua: adults 'can remain alive genotypic diversity in clonal plant species. Amer. J. Bot., following passage through the guts of fish (Vin- 74,123—131. FERGUSON, E. 1964. The ostracod (Crustacea) genus Cypridop- yard, 1979) and eggs hatch following passage sis in North America and a description of Cypridopsis through the guts of ducks (Proctor, 1964). howei, Sp. Nov. Trans. Amer. Micros. Soc., 83, 380—384. Polyphyletic origins of asexuality have been FURTOS, N. C.1933.The Ostracoda of Ohio. Ohio Biol. Survey, shown to be a cause of high clonal diversity in 5411-524. other asexual species (Harshman and Futuyma, HARRIS, H. AND HOPKINSON. D. A.1976. Handbook of Enzyme Electrophoresis in Human Genetics. North-Holland Pubi., 1985; Hebert 1987). This mechanism is probably Amsterdam. not currently important in C. vidua, as this study HARSI-IMAN, L. G. AND F(JTUYMA, D. J. 1985. The origin and examined populations over a wide geographic distribution of clonal diversity in Alsophila pometaria range and found no evidence of conspecific sexual (Lepidoptera: Geometridae). Evolution, 39, 315-324. populations. However, sexual species in the genus HEBERT, P. fl N. 1987. Genotypic characteristics of the Cladocera. Hydrobiologia, 145,183-193. Cypridopsis presently co-occur with C. vidua in HEBERT, P. D. N. AND CREASE, T. j.1980.Clonal coexistence the Gulf States (Tressler, 1959; Ferguson, 1964) in Daphnia pulex: another planktonic paradox. Science, and such co-occurence may have been broader in 207, 1363—1365. the past (Delorme, 1970). Mating between congen- I-IEBERT, P. D. N., WARD, R. D. AND WElDER, L. j.1988.Clonal erics is a source of variation in some asexual fish diversity patterns and breeding system variation in Daphnia pulex, an asexual-sexual complex. Evolution, 42, 147-159. (Vrijenhoek, 1978), and remains a potential source I-IOFFMANN, R. .i. 1986. Variation in contributions of asexual of high genotypic diversity in C. vidua. reproduction to the genetic structure of populations of the In summary, the current study confirms sea anemone Metridium senile. Evolution, 40, 357—365. apomixis in C. vidua and suggests that polyploidy INNES, D. J., SCHWARTZ, S. S. AND FIEBERT, P. 0. N. 1986. is widespread, with a high incidence in arctic popu- Genotypic diversity and variation in mode of reproduction among populations in the Daphnia pulex group. Heredity, lations. The levels of genotypic diversity detected 57, 345—355. in this species are higher than that of other asexuals KESLING, R. V. 1951. The morphology of ostracod molt stages. living in discrete habitats. The broad geographic Ill. Biol. Monogr., 21, 1—324. distribution of C. vidua may be a consequence of LYMAN, J. C.ANDELLSTRAND, N. C. 1984. Clonal diversity in Taraxacum officinale (Compositae), an apomict. Heredity, this high diversity. 53, 1-10. PROCTOR, V. w. 1964. Viability of crustacean eggs recovered from ducks. Ecology, 45, 656-658. Acknowledgements We aregrateful toRachelle Masse and SOKAL, R. R. AND ROHLF, F J. 1981. Biometty. W. H. Freeman, Terrie Finston for maintaining the cultures, Denis Delorme for San Francisco. confirming identifications, and Marc Boileau for contributing SYWULA, T., AND LORENC, R. 1982. Esterase polymorphism the Houston sample. Larry Weider and an anonymous reviewer as a marker of interpopulation differentiation in Cyprinotus made helpful comments on the manuscript. Financial support incongruens (Ramd.) (Ostracoda, Crustacea). BulL Acad. was provided by a grant from the Natural Sciences and Polonaise Sciences, 29, 317-322. Engineering Research Council of Canada to PDNH. SYWULA, T., SUOMALAINEN, E., SAURA, A. AND LOKKI, J. 1985. Genetic structure in natural populations of Ostracoda. 1. Electrophoretic methods. Zeitschrift für zoologische Systematik und Evolutionforschung, 23, 194-198. REFERENCES TETART, i1978. Les garnitures chromosomiques des Ostracodes d'eau douce. Tray. Lab. Hydrobiol., 69-70, 113- ABREU-GROBOIS,F. A. AND BEARDMORE, J. A. 1982. Genetic 140. differentiation and speciation in the brine shrimp Anemia. TRESSLER, W. L. 1959. Ostracoda. In Edmondson, W. T. (ed.) In Berigozzi, C. (ed.) Mechanisms of Speciation, Alan R. 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VINYARD,G. 1979. An ostracod (Cypridopsis vidua) can reduce WEISMANN, A. 1880. Parthogenese bei den Ostracoden. predation from fish by resisting digestion. Amer. Mid. Nat., Zoologische Anzeiger, 3, 82-84. 102, 188-190. WILLIAMS .c.1975. Sex and Evolution. Princeton Univ. Press. vRIJENHOEK. IS. C. 1978. Coexistence of clones in a heterogeneous environment. Science, 199, 549-552. WElDER,L. J.AND FILBERT, P. D. N. 1987. Microgeographic genetic heterogeneity of melanie Daphnia pulex at a low arctic site. Heredity, 58, 391—399.