An electrophoretic survey of allozyme variation was conducted in four, highly polymorphic loci on nine populations of ostracod Candona neglecta Sars from three different environments: the profundal of post-glacial lakes, deep muddy bottom of the Baltic Sea and small astatic water bodies. The results suggest lack of genetic isolation between populations from lake profundal and the Baltic Sea. On the other hand a very distinct founder effect can be noted in the case of young, isolated populations from small astatic basins. It is suggested that a population inhabiting a large lake may be genetically subdivided due to differentiated eutrophication.
Candona neglecta G.O. Sars, 1887, is a common eurytopic and amphigonic ostracod. It was described by Sars on the basis of individuals found in the Italian lake Garda and individuals raised in his aquaria out of dried mud from Algeria. It was later reported from several locations in Europe. Unfortunately, some data concern allied species, which only became obvious when the structural details of the penis are taken as a diagnostic feature (Petkovski, 1959; Sywula, 1974). C. neglecta is a peculiar species as it inhabits three extremely different types of environment: primarily, the profundal and, to a lesser extent, the littoral of post-glacial lakes; secondly, small permanent and temporary water bodies, limnocrene and helocrene swamps in particular; and finally the deep muddy and muddy-sandy bottom of the Baltic Sea (Sywula, 1974). Adult specimens, as well as eggs and larvae can not swim.
Each of the three types of environment imposes extremely different living conditions, which demand specific adaptations. One may thus put forward the hypothesis that populations inhabiting particular types of environment boast some genetic specificity or even make up groups of sibling species. The aim of this paper is to verify the above hypothesis.
MATERIALS AND METHODS
Samples were obtained from nine sites (Fig. 1, Table 1), the site code also being the population code. The animals were collected using a fine mesh (100 Lμ) hand net (shallow waters) or a dredge (deep waters). Samples were taken to the laboratory, specimens being picked out immediately and frozen at −80°C.
Randomly selected adult specimens were sexed, homogenized and subjected to electrophoresis. Cellulose acetate electrophoresis was applied to study glucose phosphate isomerases (GPI, EC 184.108.40.206), leucine aminopeptidases (LAP, EC 220.127.116.11) and esterases (EST, EC 3.1.1). These procedures and staining methods were after Hebert and Beaton (1989). Each specimen was examined in respect of all three enzyme systems, alleles being designated according to the decreasing electrophoretic mobilities of the corresponding proteins. Esterases were evidently coded by several loci but only two of them could be genetically interpreted without special breeding data. None of the examined loci proved to be sex-linked.
Observed (direct count) and expected (based on Hardy-Weinberg equilibrium) heterozygosities were calculated and averaged across all loci for each population.
Representative samples of C. neglecta were tested for conformity with Hardy-Weinberg proportions using the χ2 test for quality of fit. The fixation index FST was used to estimate the amount of inbreeding due to population subdivision: FST =(HT −HS)/ HT (where HS, the expected heterozygosity of an individual in an equivalent random mating subpopulation; HT, the expected heterozygosity of an individual in an equivalent random mating total population).
FST-statistics were calculated according to Hartl and Clark (1989). The statistical significance of differences in the allele frequency between populations was tested by means of a likelihood-ratio test G2 (Adam, 1987). Genetic identity (I) and genetic distance (D) between local populations were estimated after Nei (1972). The dendrogram was constructed by Unweighted Pair Group Method using Arithmetic Averages (UPGMA) (Sneath and Sokal, 1973).
The obtained electrophoretic patterns were in accordance with the model predicted for dimeric proteins coded by one locus with four alleles in the case of Gpi, monomeric proteins coded by two loci with three alleles for two loci of Est and monomeric protein coded by one locus with two alleles in the case of Lap. Of 12 alleles of four loci only one (Gpid) was rare and found in only two populations. The others were common and found in at least six populations.
The allele frequencies and data on heterozygosity and χ2-values connected with Hardy-Weinberg expectations are presented in Table 2. Not all of four electrophoretic phenotypes could be interpreted genetically in some individuals and therefore in Table 2 different numbers of individuals analysed are given with reference to particular loci. Two samples S3 and S4 were taken from small populations and although not very numerous they were statistically representative. On the other hand, sample L4 should not be treated as representative due to its small size as it could only be analysed partially. There are no grounds for rejecting the hypothesis assuming that all local populations were in Hardy-Weinberg equilibrium: for Gpi p = 0.60 − 0.99; for Est-1 p = 0.50 − 0.95; for Est-2 p = 0.25 − 0.75; for Lap p= 0.25 − 0.99. Six of nine examined local populations (five from large water-bodies and one from a small one) were polymorphic in reference to all four loci and highly heterozygotic; the remaining ones (from small water-bodies) being polymorphic in only one locus.
Allelic frequencies observed at the Gpi, Est, Lap loci in nine local populations (N,sample size; H,observed heterozygosity; H̄, average heterozygosity over loci; χ2,values calculated for quality of fit to Hardy-Weinberg expectations of genotype frequencies).
The values of the FST indicator are presented separately for all populations and for populations from large water-bodies in Table 3. These values were in general quite low for populations from large water-bodies (B, L1-L4) and high for those from small ones. Genetic identity and genetic distance of local populations are presented in Table 4; Fig. 2 shows the populations clustering from the point of view of genetic identity. Our attention was drawn to the unexpected result that populations from large permanent water-bodies formed a single cluster, which also contained population S1 from a small water-body while other populations from small water-bodies were apart. Another unexpected result was that two samples from the same lake, one from mesotrophic basin L1 and the other from eutrophic basin L2, were relatively distant from each other. The statistical significance of differences in the allele frequency between these two samples was checked by a G2-test; 20.85 for locus Gpi, 2.23 for locus Est-1, 14.02 for locus Est-2 and 1.49 for Lap. The first and the third values were statistically important.
Estimates of genetic identity (I) (above diagonal) and genetic distance (D) (below diagonal) between eight local populations based on data for four loci Gpi, Est-1, Est-2, Lap.
Four of the results obtained are noteworthy. At first there is the resemblance in fairly old populations from large water-bodies despite very different environmental conditions, on a deep muddy Baltic Sea bottom and in the profundal of freshwater post-glacial lakes (stations B, L1–L4) (see Tables 2–4, Fig. 2). The genetic similarities within this set of populations (0.901–0.988) are not very high as compared with conspecific populations according to Ayala (1975). The low values of FST in this population set (Table 4) confirm the lack of genetic isolation between populations. In this context it is unlikely that the Central-European populations inhabiting the particular environment types are characterized by some genetic specificity or even make up a group of sibling species must be rejected. The examined ostracod can migrate only passively between lakes and Baltic Sea. The migration, probably mediated by birds and fish, might be an effective means of gene flow, which caused a fairly high level of genetic homogenization of quite old Central-European populations connected with large water bodies. A similar situation was found with a profundal ostracod Cytherissa lacustris Sars which is distantly related to Candona neglecta used in this study (Sywula, 1974; Sywula and Geiger,1990).
The second matter worth mentioning is the very distinct founder effect in the case of young, isolated populations from small astatic basins (stations S2 – S4). It can easily be traced by heterozygosity, genetic identity and FST.
Thirdly a different situation was noted at station S1. Although the basin is also young, small and astatic, the population had similar heterozygosity and allele frequencies to those of the old lake populations. (Tables 2,3, 4; Fig. 2). The difference between this population and the remaining young populations seems to be caused by frequent visit of wild-duck to station S1; according to Michno B (unpublished data) the mean number of wild-ducks from October to April was as high as 180–323 in a single counting at station S1 (countings were conducted weekly in 1983-1993). This could possibly indicate the role of the birds in the passive migration of ostracods, which was discussed previously in respect of the species Cytherissa lacustris Sars (Sywula et al.,1994) and Cyprideis torosa (Jones) (Sywula et al.,1995).
The fourth item worth discussing is the significant difference in the allele frequencies within Lake Drawsko (stations L1, L2). This finding indicate that two basins of the lake are inhabited by two separate subpopulations of C. neglecta. As both basins are eutrophic to different extents, one may assume that it is a sign of a selection process connected with progressive eutrophication. A similar situation was noted in respect of ostracod Cytherissa lacustris Sars in an Alpine lake (Attersee) (Sywula and Geiger, 1990).