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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 .

An integrative taxonomic analysis reveals a new species of loticHynobiussalamander from Japan