Microsatellites: A tool for evolutionary genetic studies of western Palearctic water frogs

Genotypic variation at six of 67 microsatellite loci we developed from Rana ridibunda, containing di‐ or trinucleotide simplesequence repeats, confirms the value of microsatellites as an evolutionary genetic tool for studying western Palearctic water frogs, a model system characterized by clonal reproduction in natural hybrid lineages. R. ridibunda, Rana lessonae. and their hemiclonal hybrid Rana esculenta, which transmits only its ridibunda genome to gametes, are highly variable in central Poland and northern Switzerland: with one exception (a locus that is part of a coding region), each locus was polymorphic in each taxon when scorable, and the number of alleles was high (in Swiss lessonae‐esculenta populations, 1‐6 per locus, total 13, for ridibunda genomes; 1‐9 per locus, total 23, for lessonae genomes; 3 shared by both genomes). Estimated repeat numbers ranged from 3 to 14 for trinucleotide‐ and from 6 to 27 for dinucleotide‐repeats. Nonamplifying (null) alleles, problematic because they underestimate genotypic variability if undetected, occur in ridibunda genomes of Swiss R. esculenta at one locus: apparent lessonae null alleles at another locus were subsequently resolved for several segregating alleles evidenced by bands of much lower intensity. Two loci had multiple ridibunda alleles in Swiss R. esculenta and thus discriminate between hemiclones. with a resolution superior to that of allozymes. Adding these two loci to our protein electrophoretic data (7 loci) more than doubled the number of hemiclones detected, showed that several allozyme‐defined hemiclones are composites of multiple microsatellite hemiclones, and revealed clonal diversity in populations that are uniclonal by allozyme data. The extent to which microsatellite‐defined hemiclones result from primary hybridizations or from subsequent mutations in ridibunda genomes of R. esculenta lineages remains to be determined. A sample of newly metamorphosed R. ridibunda from hybrid × hybrid matings collected at a Swiss population in the fall had a significant heterozygote excess at the two loci that varied in ridibunda genomes; this shows that most, probably all, of them originated from inter‐ rather than intrahemiclonal matings, in accord with the idea that the usual hybrid × hybrid inviability in lessonae‐esculenta systems is caused by deleterious recessive alleles on the clonally inherited ridibunda genomes. Three of four microsatellite loci tested amplified in most species of western Palearc