The endemic Japanese frog Rana tagoi is unique among Holarctic brown frogs in that it breeds in small subterranean streams. Using mitochondrial 16S ribosomal RNA and NADH dehydrogenase subunit 1 genes, we investigated genealogical relationships among geographic samples of this species together with its relative R. sakuraii, which is also a unique stream breeder. These two species together form a monophyletic group, within which both are reciprocally paraphyletic. Rana tagoi is divided into two major clades (Clade A and B) that are composed of 14 genetic groups. Rana sakuraii is included in Clade A and split into two genetic groups, one of which forms a clade (Subclade A-2) with sympatric R. tagoi. This species-level paraphyly appears to be caused by incomplete taxonomy, in addition to introgressive hybridization and/or incomplete lineage sorting. Rana tagoi strongly differs from other Japanese anurans in its geographic pattern of genetic differentiation, most probably in relation to its unique reproductive habits. Taxonomically, R. tagoi surely includes many cryptic species.

Taxonomic relationship of two forms of Rana tagoi, a brown frog endemic to Japan, is assessed. Based on morphological and bioacoustical comparisons, as well as molecular analysis, we consider the large-form-lineage as true R. tagoi, and describe the small-form-lineage as Rana kyoto sp. nov. The new species and R. tagoi occur sympatrically in many places around the Kinki District, although the former can be distinguished from the latter by smaller body size, more developed dark marking on ventral side of legs, less developed toe webs, and advertisement call with fewer notes and lower dominant frequency. Rana kyoto sp. nov. sympatric with R. tagoi show smaller body size than the allopatric ones, whereas R. tagoi sympatric with the new species show larger body size than the allopatric ones, suggesting that the character displacement of body size occurs between the two species around the type locality of Rana kyoto sp. nov.

We assessed the taxonomic relationships of an endangered Japanese small salamander, Hynobius hidamontanus Matsui, 1987, and its close relative H. tenuis Nambu, 1991 electrophoretically and found that they were not clearly distinguished from each other. This result, together with available morphological and ecological information, strongly indicates that H. tenuis Nambu, 1991 is a subjective junior synonym of H. hidamontanus Matsui, 1987. By this conclusion, the total distribution range of H. hidamontanus is greatly expanded, but its endangered status and the necessity of its conservation is not be changed since the habitats of this species are fragmented and not continuous. The distribution pattern of this species is interesting from the viewpoint of biogeography.

Two morphotypes, with a large and small body size, of a brown frog Rana t. tagoi occur sympatrically in the Kinki region, central Honshu of Japan. Previous mitochondrial (mt) DNA genealogical study recognized two main lineages (A and B) and several sublineages in R. tagoi, where the small type was placed in the group A-1b, and the large type in groups A-1a and B-2a. Using haplotype network and structure analysis of three nuclear genes, we examined the discrepancy between morphology and mitochondrial genealogy. The results showed that the small type is reproductively isolated from its co-occurring large type (A-1a or B-2a), and that unlimited gene flow occurred between parapatrically occurring two mtDNA lineages of large types (A-1a and B-2a). Discordant genetic relationships between mtDNA and nuclear DNA results may be caused by the past mitochondrial introgression, and possibly, the incomplete lineage sorting. These results also suggest a heterospecific relationship between the large (A-1a and B-2a) and small types (A-1b). The large type is identified as Rana t. tagoi as it is genetically very close to the topotypes of the nominal subspecies, while the small type remains unnamed.

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