An integrative taxonomic analysis reveals a new species of lotic Hynobius salamander from Japan

We examine the phylogenetic structure and morphological differentiation within the Hynobius kimurae–H. boulengeri species complex—a widely-distributed group of stream-breeding hynobiid salamanders, inhabiting montane areas of western, central and eastern parts of Honshu Island, Japan. Phylogenetic relationships were assessed based on analyses of mitochondrial (mtDNA) and nuclear (nuDNA) gene fragments for a total of 51 samples representing 23 localities covering the entire range of the species complex. Morphological study included one-way analysis of variance (ANOVA) and principal components analysis (PCA) analyses of 26 morphometric and six meristic characters for 38 adult specimens of H. kimurae and three adult specimens of H. boulengeri. MtDNA genealogy supported monophyly of the H. kimurae–H. boulengeri complex, which is sister to all other Hynobius except H. retardatus. The complex is subdivided into three major clades, corresponding to the Eastern (Clade I) and Western (Clade II) populations of H. kimurae sensu lato, and to H. boulengeri (Clade III). Monophyly of H. kimurae sensu lato is only moderately supported by mtDNA, while nuDNA suggested that the Western form of H. kimurae is closer to H. boulengeri than to the eastern form. The time of the split of the H. kimurae–H. boulengeri complex is estimated as late Miocene and coincides with intensive crust movement in western Japan. Divergence between Clades I and II took place in early Pliocene and was likely influenced by the uplift of Central Japanese Highlands. All three clades were found to be different in a number of morphological characters, allowing us to describe the eastern form of H. kimurae as a new species, Hynobius fossigenus sp. nov.