Lake Biwa is an ancient freshwater lake that was formed approximately 4 Mya and harbours many coastal plants that commonly inhabit the seashore. We used chloroplast DNA haplotype analysis using two spacer sequences and simple sequence repeat (SSR) analysis using eight nuclear microsatellite markers to detect genomic signatures indicating long-term isolation of inland populations of Calystegia soldanella in Lake Biwa from coastal populations. We used 348 samples from 63 populations for haplotype analysis and 478 samples from 27 populations for SSR analysis covering the inland and coastal distribution of the species. We detected seven haplotypes, and the distribution pattern of these haplotypes was geographically highly structured between Lake Biwa and the coast. Nuclear SSR analysis also supported genetic differentiation between Lake Biwa and coastal populations (analyses of molecular variance, 43%), and the grouping of Lake Biwa and coastal populations by a Neighbour-joining tree. In addition, genetic diversity of the inland populations (mean HE = 0.153) was significantly lower than that of coastal populations (mean HE = 0.328). These results suggested that inland populations at Lake Biwa have been isolated from coastal populations for a very long time. The inland populations most likely experienced a bottleneck effect, resulting in sufficient in situ genetic divergence to clearly distinguish them from coastal populations.

Eight microsatellite loci of Calystegia soldanella useful for comparisons of the genetic structure of isolated populations in the ancient Lake Biwa and coastal populations in Japan were isolated and characterised. The number of alleles ranged from 2 to 5. The expected (H E ) and observed (H O ) heterozygosities were 0.097-0.583 and 0.000-0.380, respectively, from 100 individuals from Lake Biwa and coastal populations. Seven of the eight loci exhibited significantly fewer heterozygotes than expected based on the Hardy-Weinberg equilibrium (P \ 0.05). These primers amplifying microsatellites in C. soldanella may provide a population genetics tool useful in the establishment of a conservation strategy.