SIMOCEPHALUS SERRULATUS

Transcription

1 POLYMORPHISM I A CYCLIC PARTHEOGEETIC SPECIES: SIMOCEPHALUS SERRULATUS M. YAO SMITH AD ALEX FFtASER Department of Biological Sciences, University of Cincinnati, Cincinnati, Ohio 5 Manuscript received September, 975 Revised copy received June 5, 976 ABSTRACT A survey of sixteen isozyme loci using electrophoretic techniques was conducted for three isolated natural populations and one laboratory population of the cyclic parthenogenetic species, Simocephalus serrulatus. The proportion of polymorphic loci (33%6%) and the average number of heterozygous loci per individual (6%3%) in the three natural populations were found to be comparable to those found in most sexually reproducing organisms. Detailed analyses were made for one of these populations using five polymorphic loci. The results indicated that () seasonal changes in genotypic frequencies took ple, () apomictic parthenogenesis does not lead to genetic homogeneity, and (3) marked gametic disequilibrium at these five loci was present in the population, indicating that selection ted on coadapted groups of genes. ATURAL populations have long been known to be genetically highly polymorphic. The degree of polymorphism in natural populations was first estimated by LEWOTI and HUBBY ( 966) using electrophoretic techniques. Since then, the degree of genetic variability in a number of natural populations of sexually reproducing organisms have been determined, including mice (SELADER, HUT and YAG 969) and Drosophila (AYALA, POWELL and TRACEY 97). These populations were found to be polymorphic for 57% of their loci. Close inbreeding and parthenogenesis are modes of reproduction which were considered to lead to genetic homogeneity within a population ( SUOMALAIE 95; WHITE 95). However, a species of predominantly selfpollinating plants has been shown to be highly polymorphic (MARSHALL and ALLARD 97) and to have a proportion of heterozygous loci per individual that is higher than predicted on the basis of their mating system alone (JAI and ALLARD 96; ALLARD and WORKMA 963) ; and diploid and polyploid species of parthenogenetic weevils have also been shown to be highly polymorphic. The mechanisms for the maintenance of large amounts of polymorphism in natural populations have been a central problem in population genetics. The large percentage of polymorphic loci in natural populations generates an infinite array of allelic combinations. This leads one to wonder how the Combinations which confer high fitness to a population are sorted out from this effectively Genetics 8: ovember, 976.

2 63 M. Y. SMITH AD A. FRASER infinite array of genotypes. The electrophoretic survey of isozymes was extended to the cyclic Parthenogenetic species, Simocephalus serrulatus. This species was folund to be genetically as variable as most sexually reproducing organisms, showing that apomictic parthenogenesis does not lead to monomorphism (WHITE 95). Strong gametic disequilibrium was found in one of the populations studied. Reasons are given to support the thesis that natural selection is responsible for the maintenance of polymorphism in these populations. MATERIALS AD METHODS Simocephalus serrulatus, like most species of the order Cladocera, has a cyclic parthenogenesis, in which several parthenogenetic generations alternate more or less regularly with a sexual reproduction is revealed by the ourrence of ephippial females. Such females have a large, yellowish egg in contrast to parthenogenetic females which have several small greenish eggs. Sexual (ephippial) females are rare except during Fall. The following three populations of S. serrulatus were collected and surveyed. () The Rustic Ranch Lake (RR), in Yankeetown, Ohio, was sampled on three oasions (early June, mid August and eprly October). These three samples will be referred to as RRSP, RRS and RRF, respectively, in future discussions. () The Gromley Farm Pond (GF) located in the orth East area of Cincinnati was sampled once in the Fall (973). (3) The ature Center Pond (C) located in the Cincinnati s ature Center, was also sampled once in the Fall of 973. The laboratory population (RRL) consisted of clones derived from single parthenogenetic females collected from the RR lake between May and December, 97. The isozyme survey was made using polyrylamide gel electrophoresis (E. C. apparatus). Individual eggcarrying parthenogenetic females were homogenized in tris borate solution (.M ph 8.9), centrifuged and the whole supernatant applied to the gel pocket. The same procedure was used for the survey of the laboratory population, except that two eggcarrying parthenogenetic females were used per gel pocket. The isozyme systems assayed were alkaline phosphatase (APH), esterases (EST), AD dependent malate dehydrogenase (MDH), ltic dehydrogenase (LDH), xanthine dehydrogenase (XDH) and idophenol oxidase (IPO). The assays used were those developed by MURTHY (97). In August, 973, over a hundred eggcarrying parthenogenetic females were isolated in individual plastic ice cube dishes and allowed to release their young. These young were allowed to mature and eh clone was assayed for eh of the six isozymes systems mentioned above. RESULTS The genetic interpretations of the isozyme phenotypes have been described elsewhere (SMITH 97). Sixteen loci were postulated to control the expression of the six isozyme systems. The prolportion of polymorphic loci, the proportion of heterozygous loci per individual and average number of alleles per locus are given in Table. The proportions of heterozygous loci per individual showed a slight tendency to increase as parthenogenetic reproduction progressed. This increase, holwever, was not statistically significant. The genotypic frequency distributions for five loci (MDH, XDH, APH, ESTI and EST) were determined for all populations studied. The allelic frequencies for eh of these loci and the deviation of heterozygous frequencies from the observed frequencies assuming HardyWeinberg equilibrium are given in Table. All genotypic frequencies at these loci were significantly different from HardyWeinberg proprtion (P <.5), except for the fixed loci and the APH locus of the C ppulation. In order to test whether the gene frequencies and genotypic frequencies of

3 POLYMORPHISM AD PARTHEOGEESIS 633 TABLE The percentage of polymorphism, the percentage of heterozygous loci per individual and the average number of alleles per locus per population in all populations surveyed Populations RRL RRSP RRS RRF GF C Percentage polymorphic loci 9% 6% 6% 6% 53% 33% Average heterozygous loci per individual*.om6,66.83,38. Average number of alleles per locus ~ _ ~ ~ _ ~~ _ * Based on the five polymorphic loci scored. TABLE Allelic frequencies distribution at five polymorphic loci of Simocephalus serrulatus populrctions and deviation of observed from expected frequencies of heterozygotes as a proportion of expected for eh polymorphic locus studied Alleles RRSP RRS RRF GF C Mdh a Mdh b Mdh c Mdh d Xdh a Xdh b Xdh c Aph a Aph b Est la Est Ib Est IC Est a Malate dehydrogenase _ go8 Xanthine dehydrogenase.77, Alkaline phosphatase (APH ) Esterase I (EST ) ,.a Esterase (EST ) Est b Est c Est d Est e SO

4 ~ 63 M. Y. SMITH AD A. FRASER TABLE 3 Seasonal comparison of gene and genotypic frequencies of the Rustic Ranch population of Simocephalus serrulatus Populations Contingency Variance Enzyme compared x test value df P heterogeneity df P MDH XDH APH EST EST 3.8***.9**.so 6.3* *** * **.8***.3* * * *** 9. * **.6 3 <,5 5. >.5 <.5 >.lo >.5 <.5 >.@ <,5 <.5 <,5 >.5 <.5 <.OM >.5 7.8**.37*. 9.73***.8* ***.67* ** *** 8.*** 7. I <.Ol <.5 >.5 <.O5 <.5 >.5 <.Ol >.5 >.lo <,5 <.5 >.5 <.5 <.5 >.lo the RR population remained constant during its parthenogenetic cycle, pairwise comparisolns of the seasonal genotypic and gene frequencies were made using the variance heterogeneity test and the contingency Chisquare test, respectively (Table 3). The joint fivelocus distributions observed in single parthenogenetic females are given in Table. Expected numbers in eh class were computed by taking the products of the relevant singlelolcus genotypic frequencies. Of the 7 classes of joint fivelocus genotypes expected, only were observed. The deviation ot TABLE umber of genoiypes observed and their expected number computed by taking the product of the indiuidual genotypic frequency in the populations sampled in 973 Alleles umber umber MDH XDH APH EST EST observed exuected Deviation ab ab dd ee be bc ee 8 9 e , g ,999.o

5 POLYMORPHISM AD PARTHEOGEESIS 635 TABLE 5 umber of obserued gametic types, their expected number obtained by using the product of their allelic frequencies and their deviation from the expected value Gametic umber umber types observed expected Deviation umber observed 6 umber expected Deviation l.l ** * ** ** ** *Deviation computed by combining sum of expected in category ** as expected value in gametic type. x = In this table eh number or represents one of the alternate alleles at eh locus in the following order: Mdh a is allele, Mdh c is allele ; Xdh b is allele and Xdh c is allele ; Apha is allele and Aphb is allele ; Estla is allele and Estlb is allele ; Estla, Estb and Estc are combined to form synthetic allele and Estd and Este are combined to form synthetic allele. the observed frequencies from the expected frequencies were consequently all negative except for eight classes in which definite excesses were observed. There were two alleles present at all loci except at EST locus for which 5 alleles were detected in the population. The presence of these 5 alleles made the number of joint fivelocus gametic types very large. In order to reduce the data to a more manageable size the alleles at the EST locus were combined to create two allelic classes (Table 5). This method was shown not to affect the picture of gametic phase disequilibrium (CLEGG, ALLARD and KAHLER 97). In the case of the double heterozygote the gametic types were chosen as the most probable types found in the population. DISCUSSIO The proportions of polymorphic loci, proportions of heterozygous loci per individual and the average number of alleles per lolcus (Table ) in natural populations of S. serrulatus are comparable to those found in most sexually reproducing organisms. The constancy of these proportions throughout the parthenogenetic reproductive cycle indicates that apomictic parthenogenesis does not lead to genetic homogeneity.

6 636 M. Y. SMITH AD A. FRASER The initial deviation of the genotypic distribution of the RRSP population from HardyWeinberg proportions may indicate that intense selection operated m the gene pm during the first few parthenogenetic generations. Alternatively, only a small proportion of epphippia produced in the Fall may have suessfully metamorphosed into adult females or nonrandom mating of sexual forms ourred or the population of sexual forms was small in the Fall or any combination of all of the above. Thus natural selection and random drift could both be responsible for the initial deviation of the RRSP population s genotypic frequencies from HardyWeinberg proportions. Under apomictic parthenogenesis one would expect the genotypic frequency to remain invariant throughout parthenogenetic reproduction if no disturbing forces ted on the population. The pairwise comparisons of seasonal gene and genotypic frequencies of the RR population indicate, however, that these frequencies have changed significantly from Spring to Summer at all lolci. These frequencies then remained stable at four loci (MDH, ESTI, XDH and EST), and changed cyclically at the APH locus (Table 3). The deficiencies of heterozygotes increased at three loci, decreased at one and changed cyclically at yet another (Table ). There is a general deficiency of heterozygotes which could not be a constant or intrinsic feature of these loci since heterozygotes for the MDH and the EST# loci were present in large excess in the GF and the C populations. It is important that there was no evidence of sexual recruitment in this population (HERBERT 97) and since the population remained very large throughout the sampling period and migration into the population was probably very rare, the changes in genotypic and gene frequencies observed were most likely caused by natural selection. The laboratory clonal population, which was obtained with considerable difficulty (only 3% of clones survived and were sampled), was much less polymorphic than the natural population from which it was derived (Table ). This indicates that stringent selection probably operated on the population as it made the transition from its natural habitat to the laboratory environment. The very marked gametic disequilibrium found (Table 5 ) involves complementary gametic types which is improbable on a hypothesis of random drift, and is in strong support for a hypothesis of selection. Tight linkage between the five loci studied could not explain the marked gametic disequilibrium observed in this population, since daphnids have an estimated chromosomes per cell (HOSSEIIE 966), making it extremely unlikely that these five randomly chosen loci are linked. Another possible explanation is that the five isozymes are all part of a single organelle or pathway. This possibility is not valid since the five isozymes sampled do not have closely related functions. Selection ting on coadapted gene combinations ting at a more subtle level of relationship is the most likely explanation for this observation. This selection keeps the population genetically highly structured. The advantage of cyclic parthenogenesis would be to maximize the capity of the species to keep highly fit coadapted units in the populations through generation after generation of parthenogenetic reproduction. Its sexual cycle would

7 POLYMORPHISM AD PARTHEOGEESIS 637 confer flexibility to cope with a changing environment. As the environment becomes unsuitable sexual reproduction takes ple. ew arrays of genotypes are created which could then be ted upon by natural selectioa. Although all observations made in this study are consistent with the theory that natural selection is responsible for the genic structure in this cyclic parthenogenetic species of organism, there is a possibility that small population sizes during the Fall sexual phase could cause the gametic disequilibrium which is then maintained by parthenogenetic reproduction. LITERATURE CITED ALLARD, R. W. and P. L. WORKMA, 963 Population studies in predominantly self pollinated species IV. Seasonal fluctuation in estimated values of genetic parameters in lima bean populations. Evolution 7: 78. AYALA, J. F., J. R. POWELL and M. C. TRACEY, 97 Enzyme variability in Drosophila willistoni group V. Genic variation in natural populations of Drosophila Equinoxialis. Genetic Research Cambridge : 9. CLEGG, I. T., R. W. ALLARD and A. L. KAHLER, 97 Is the gene the unit of selection? Evidence from two experimental plant populations. PAS 69 : 778. HERBERT, P. D.. and R. D. WARD, 97 magna. Genetics 7 : Inheritance during parthenogenesis in Daphnia HERBERT, P. D.., 97 Enzyme variability in natural populations of Daphnia magna. Genotypic frequencies in permanent populations. Genetics 77: HOSSEIIE, F., 966 The ecology and reproductive cytology of Daphnia middendorfiana fischer (Cladocera) from the Arctic. Ph.D. thesis, Department of Zoology, Indiana University. JAI, S. K. and R. W. ALLARD, 96 Population studies in predominantly self pollinated species I. Evidence of heterozygote advantage in a close population barley. PAS : LEWOTI, R. C. and J. L. HUBBY, 966 A molecular approh to the study of genic heterozygosity in natural populations. Genetics a: MARSHALL, D. R. and R. W. ALLARD, 97 Isozyme polymorphism in natural populations of Avena fatus and Avenrr barbata. Heredity 5: MURTHY, R., 97 Survey of various enzymes and a study of the biochemistry of ltate dehydrogenase in Daphnia magna. Ph.D. thesis, University of Cincinnati (Biological Sciences). SELADER, R. K., W. G. HUT and S. Y. YAG, 969 Protein polymorphism and genetic heterozygosity in two European subspecies of the house mouse. Evolution 3: SMITH, M. Y., 97 A study of polymorphism in Simocephalus serrulatus. Ph.D. thesis, University of Cincinnati (Biological Sciences). SUOMALAIE, E., 95 Parthenogenesis in animals. Advances in Genetics 3: 9353 SUOMALAIE, E. and A. SAURA, 973 Genetic polymorphism and evolution in parthenogenetic animals I. Polyploid Curculionidae. Genetics 7: WHITE, M. J. D., 95 Animal Cytology and Evolution. Second edition, Cambridge University Press. Corresponding editor: R. W. ALLARD