The Japanese clouded salamander (Hynobius nebulosus) is a lentic-breeding species distributed throughout western Japan. Threats, such as habitat loss, have led to it being categorized as a vulnerable species. To explore the phylogeographic features and population differentiation among clouded salamanders in Shiga prefecture, we analyzed sequences of the mitochondrial cytochrome b gene. DNA samples were collected from 29 distinct breeding sites, and 53 cytochrome b haplotypes were identified. On the basis of comparison of the composition and frequency of haplotypes in each breeding site, salamanders in each habitat appeared to have distinct characteristics. Significant genetic differentiation was observed in 93.3% of possible pairs of habitats in Shiga prefecture, and 67.7% of habitat pairs within the same locality were found to be significantly different. These results suggest that the salamanders' poor locomotion combined with topographic effects may have contributed to the diversity of locally distributed salamanders in Shiga. Phylogenetic analysis showed that haplotypes of H. nebulosus in Shiga can be divided into five groups (the Nagahama-Maibara group, Otsu group, Konan group 1, Konan group 2, and Takashima group), each with a distinct geographical distribution. Haplotypes of the Otsu group, however, were exceptionally widely distributed. The results of the present study will contribute to the future of H. nebulosus conservation management in Shiga.
Keywords: Lake Biwa; amphibians; cytochrome b; molecular phylogeny; salamander.