Reassessment of species delimitation using nuclear markers in three lentic-breeding salamanders from the Chugoku District of Japan (Amphibia: Caudata: Hynobiidae).

Hynobius akiensis sensu lato has recently been split into three species based on short sequence analyses of cyt-b gene of mtDNA and without data of nuclear DNA, and strange sympatric distribution in some areas has been indicated in two species. We analyzed nuclear DNA marker (SNPs) and complete sequence of cyt-b in H. akiensis sensu lato to reassess species delimitation and genetic introgression among species. As a result, we found two lineages with discordant mitochondrial and nuclear DNA in some areas. Of H. akiensis sensu lato, each of the two contains the type locality of two species recently reported (H. sumidai and H. geiyoensis), and the use of these names has been previously advocated. However, their sympatric distribution was rejected based on nuclear DNA data, which we consider is more reliable than mtDNA. We thus clarify geographic boundary of these two species and revise the species delimitations.

A new species of lotic breeding salamander (Amphibia, Caudata, Hynobiidae) from Shikoku, Japan.

Background: Hynobius hirosei is a lotic-breeding salamander endemic to Shikoku Island in western Japan. Significant allozymic and morphological differences have been found among the populations of this species; however, the degree and pattern of intraspecific variation have not been surveyed using a sufficient number of samples. Methods: For the taxonomic revision of H. hirosei, we conducted genetic and morphological surveys using samples collected throughout the distribution. Phylogenetic analysis using the cytochrome b region of mitochondrial DNA and population structure analysis using single nucleotide polymorphisms were conducted to evaluate the population structure within the species and the degree of genetic differentiation. Subsequently, a morphological survey based on multivariate and univariate analyses was performed to assess the morphological variation. Results: Genetic analyses revealed three genetic groups (Tsurugi, Central, and Nanyo) within H. hirosei, with the Nanyo group distributed allopatrically from the others, and the Tsurugi and Central groups distributed parapatrically with the formation of a hybrid zone between them. The Nanyo group was morphologically distinguishable from the remaining samples, including the topotype of H. hirosei, based on a smaller body size and several ratio values of characters to snout-vent length, longer axilla-groin distance, shorter tail length, shorter internarial distance, longer upper eyelid length, and larger medial tail width. These results support the notion that the Nanyo group is an undescribed species. However, the remaining genetically differentiated groups could not be divided in the present study. Herein, we described the Nanyo group as a new species.

Wide Distribution but Low Differentiation: Phylogenetic Relationships and Phylogeography of Hynobius nigrescens (Amphibia: Caudata).

We surveyed the genetic structure of Hynobius nigrescens Stejneger, 1907, a lentic breeding salamander widespread throughout montane and lowland regions of northeastern Japan. We performed a mitochondrial DNA analysis to explore intraspecific genetic variation and infer the evolutionary population history of H. nigrescens. Complete 1141 bp sequences of the mitochondrial cytochrome b gene were studied for 134 adult and larval individuals collected from 62 localities, encompassing the known range of the species. Hynobius nigrescens proved to be monophyletic, including two major clades (Clade II from southwestern Hokuriku and Clade I from all other localities). The latter clade comprises four well-supported and geographically structured subclades, which show genetic distances smaller than those seen in the widely sympatric species Hynobius lichenatus. Results of population statistical analyses indicated that Clade II of the westernmost range of H. nigrescens seems to have maintained a constant population size, while Clade I from most of the northeastern species range shows a tendency of recent population expansion, which is evident in Subclades I-A from the northernmost range and I-B from southern Tohoku to northern Kanto and eastern Chubu. In contrast, Subclades I-C from northeastern Chubu and Sado Is. and I-D from northwestern Chubu to Hokuriku seem to have been relatively stable in population size. Hynobius nigrescens differs greatly from other salamander species from northeastern Japan in its much more recent periods of genetic differentiation and its pattern thereof, and is suggested to be a young faunal element in this region.

A revision of Hynobius stejnegeri, a lotic breeding salamander from western Japan, with a description of three new species (Amphibia, Caudata, Hynobiidae).

A lotic-breeding salamander Hynobius stejnegeri, formerly called H. yatsui, from western Japan is revised based on genetic and morphological evidence, and three species are described: H. guttatus sp. nov. from Chubu-Kinki districts of Honshu Island, H. tsurugiensis sp. nov. from east highland of Shikoku Island, and H. kuishiensis sp. nov. from other parts of Shikoku Island. Thus, H. stejnegeri sensu stricto is restricted to Kyushu Island. Of these four species, H. kuishiensis sp. nov. contains two distinct mitochondrial lineages, but this split is not reflected in differentiation of allozyme (nuclear genome) markers. These species are morphologically similar to each other but can be differentiated by several characteristics, especially in combination of dorsal coloration, the number of vomerine, upper, and lower jaw teeth, and depth of vomerine teeth series. In coloration, H. guttatus sp. nov. is brown or dark brown mostly with tiny white to brownish white marking, while H. tsurugiensis sp. nov. is dark brown with bright yellow continuous markings. Hynobius kuishiensis sp. nov. is reddish purple or dark brown with small to continuous brownish white markings, in contrast to reddish purple or dark brown with small to large brownish white markings in H. stejnegeri.

Phylogeny and historical demography of Cynops pyrrhogaster (Amphibia: Urodela): taxonomic relationships and distributional changes associated with climatic oscillations.

We investigated the phylogenetic relationships and estimated the historical demography of the Japanese fire-bellied newt, Cynops pyrrhogaster, from Japanese mainlands using 1407-bp sequences of the mitochondrial DNA (NADH6, tRNAglu, cyt b) and 1208-bp sequences of nuclear DNA (Rag-1) genes. Phylogenetic trees based on mitochondrial DNA revealed four major haplotype clades (NORTHERN, CENTRAL, WESTERN, and SOUTHERN clades) within this species. Degree of genetic differentiation among major haplotype clades was very large for intraspecific variation, suggesting this species to be composed of four species lineages that replace each other geographically. Nuclear genetic variation presented no obvious patterns of geographic structure except for the distinctness of populations diagnosed by NORTHERN clade of mitochondrial haplotypes, suggesting results of incomplete lineage sorting. Current distribution and estimated divergence times for the genus Cynops suggest that the common ancestor of two Japanese species (C. pyrrhogaster and C. ensicauda from the Ryukyu Islands) had diverged at the edge of the continent corresponding to the present East China Sea and Central Ryukyus. Subsequent range expansion to Japanese mainland seems to have occurred in the middle Miocene. Population-genetic analyses indicated that all species lineages, except for the SOUTHERN one, experienced geographic population reductions and expansions associated with glacial and postglacial climatic oscillations.

Description of a New Salamander of the Genus Onychodactylus from Shikoku and Western Honshu, Japan (Amphibia, Caudata, Hynobiidae).

Recent phylogenetic studies using mtDNA and allozymes clarified the presence of multiple distinct genetic lineages in the Japanese clawed salamander, Onychodactylus japonicus, of which two from northern regions of the country have already been described as new species. Based on morphological analyses of the remaining genetic lineages, we describe the lineage from Shikoku Island and Chugoku Mountains of western Honshu, in western Japan, as a new species, Onychodactylus kinneburi sp. nov. It belongs to the O. japonicus complex and is morphologically similar to O. japonicus (sensu stricto), but is distinguishable from all the other members of the complex by sharply defined yellowish-orange dorsal stripe on black ground color, lack of dark marking on chest, whitish ventrum, comparatively large body size, and relatively narrow head, usually with 19 presacral vertebrae, 13 costal grooves, and relatively smaller number of vomerine tooth series. The new species occurs exclusively in Shikoku Island, but is sympatric with O. japonicus in Chugoku Mountains.

Allozymic variation in the Japanese clawed salamander, Onychodactylus japonicus (Amphibia: Caudata: Hynobiidae), with special reference to the presence of two sympatric genetic types.

We conducted an electrophoretic survey to examine geographic genetic variation in samples from 17 localities of the Japanese clawed salamander, Onychodactylus japonicus. This species was divided into six genetic groups (N-Tohoku, S-Tohoku, Tsukuba, SW-Honshu, Kinki, and Shikoku) that were largely concordant with clades or subclades recognized in our previous mtDNA study. Although the relationships among these six groups were not clarified, genetic distances between them were not small (mean Nel's D=0. 146-0.471). Among these groups, the geographically isolated Tsukuba group was genetically distinct, possibly as a result of population isolation. In a locality of western Honshu, two groups, SW-Honshu and Shikoku, were found to occur sympatrically. Although several presumable hybrid individuals were found, hybridization between these two groups seemed to occur very rarely. These results indicate that the Shikoku group is specifically distinct from the SW-Honshu group, whose range includes the type locality of O. japonicus.

Phylogenetic relationships of two Salamandrella species as revealed by mitochondrial DNA and allozyme variation (Amphibia: Caudata: Hynobiidae).

We conducted molecular phylogenetic analyses to confirm taxonomic relationships and to delimit distributional ranges of Siberian salamanders, Salamandrella keyserlingii and Salamandrella schrenckii, and to elucidate the origin of the isolated population of this species complex on Hokkaido, Japan. Phylogenetic trees constructed by MP, NJ, ML, and Bayesian methods, using complete sequences of mitochondrial cytochrome b genes, all indicated monophyly of Salamandrella and of each of the two species. Identical relationships were found on UPGMA, NJ, and CONTML trees derived from electrophoretic analysis of variation in 18 inferred allozyme loci. Populations from Hokkaido and northeastern China proved to be S. keyserlingii, while populations from Khabarovsk and Lazovsky are S. schrenckii. Genetic differentiations of S. keyserlingii within Sakhalin, and between Sakhalin and Hokkaido, are substantial. The Hokkaido population is hypothesized to have been isolated on the island since early Pleistocene, much earlier than isolation of sympatric anuran populations from their Sakhalin relatives. In contrast, the continental populations of S. keyserlingii are only slightly differentiated from some Sakhalin populations, and are thought to have expanded their ranges in the late Pleistocene.

Systematic Relationships of Hynobius okiensis among Japanese Salamanders (Amphibia: Caudata).

We conducted an electrophoretic survey to examine systematic relationships of a lotic-breeding salamander Hynobius okiensis endemic to Dogo Island of the Oki Islands, Japan, with several lentic and lotic-breeding Japanese species. Genetically H. okiensis with 2n=56 chromosomes was closer to the lentic-breeding H. nebulosus group (H. nebulosus and H. dunni) with the same chromosome number than to the lotic-breeding H. naevius group (H. naevius and H. kimurae) and H. boulengeri with 58 chromosomes. Chromosome number reduction from 58 to 56, possibly accompanied with a change in breeding environment from streams to still waters, is estimated to have first occurred in the nebulosus group of Hynobius. A reversal only in breeding habits then seems to have followed in steep, montane environments of the small island of Dogo, resulting in the speciation of H. okiensis.

Morphological and allozymic variation in Hynobius boulengeri and H. stejnegeri (Amphibia: Urodela: Hynobiidae).

We studied morphological and allozymic variation in populations of Japanese salamanders, Hynobius boulengeri and H. stejnegeri. Adult H. boulengeri showed sexual dimorphism, and juveniles differed greatly from adults in many morphological characters. From the results of multivariate analyses of morphological characters, the populations were divided into four groups: (I) H. boulengeri from Honshu, (II) H. boulengeri from Shikoku, (III) H. boulengeri from the Sobo-Katamuki Mountains of Kyushu and H. stejnegeri, and (IV) H. boulengeri from the Amakusa Islands and the Osumi Peninsula. Phenotypic relationships among the four groups were identical to relationships clarified by allozymic analyses, except for group IV, which was included in group III in the allozyme tree. Some morphometric characters were significantly correlated with environmental variables. We consider H. stejnegeri to be a valid species based on its unique color pattern, morphometric characters, and allelic composition, even though it was nested within group III of H. boulengeri by both morphological and allozymic analyses. We propose that group I from Honshu and group II from Shikoku should be treated as H. boulengeri sensu stricto and H. hirosei, respectively. Resolving the taxonomic status of the remaining populations of groups III and IV from Kyushu requires further study.

Phylogenetic relationships and phylogeography of Hynobius tokyoensis (Amphibia: Caudata) using complete sequences of cytochrome b and control region genes of mitochondrial DNA.

Using complete sequences of cytochrome b (cyt b) and control region (CR; D-loop) genes of mitochondrial DNA, we investigated the phylogenetic relationships and phylogeography among populations of the salamander Hynobius tokyoensis in northern Japan. Inclusion of populations from Aichi, Chubu region within Hynobius nebulosus by previous results was confirmed because they formed a clade with H. nebulosus. Monophyly of H. tokyoensis was supported in all trees analyzed, and two clades consistently emerged: clade A from northeastern Kanto (Ibaraki) to southern Tohoku (Fukushima), and clade B from the remaining area south of northern Kanto (Tochigi). These two clades are estimated to have an old history of divergence during the late Pliocene, when the present-day Kanto Plain was under the sea. Within clade B, three haplotypes groups (I-III) are hypothesized to have diverged more recently, i.e., between the early and mid-Pleistocene, probably in relation to glacial events. The ancestral groups I and II were separated at the present-day western Kanto and Boso Peninsula areas, respectively, and subsequently, group II invaded northward to the present-day northern Kanto area and gave rise to group III. Populations of the Miura Peninsula are thought to have multiple origins, one directly from Boso and another from a more recent southward invasion from the northern Kanto area. Cytochrome b appears to have evolved more quickly than CR genes in H. tokyoensis, and thus will be more useful for phylogenetic analyses and formulating conservation measures.

Phylogenetic relationships of Hynobius naevius (Amphibia: Caudata) as revealed by mitochondrial 12S and 16S rRNA genes.

Using mitochondrial 12S and 16S rRNA sequences, we investigated phylogenetic relationships among populations of the endemic Japanese salamander Hynobius naevius. Monophyly of this species was recovered only in the maximum parsimony tree and was unresolved in maximum likelihood and Bayesian trees. Instead the following four haplotype clades consistently emerged clearly: Clade 1 from northwestern Kyushu, Clade 2 from Chugoku and northeastern Kyushu, Clade 3 from western Shikoku and Kyushu, and Clade 4 from Chubu-Kinki and central-eastern Shikoku. Of these, Clades 1 and 2, and Clades 3 and 4, respectively, correspond to Groups A and B previously recognized from the analyses of allozyme data in this species, but monophyly of these groups was not strongly supported. Unlike the previous results, the western and eastern samples from Shikoku did not form a clade, and were grouped with Kyushu-B in Clade 3 and Chubu-Kinki in Clade 4, respectively. The reason for this conflict between mtDNA and allozyme results is unknown, but might be related to retention of ancestral mtDNA polymorphism in Shikoku populations. Nearly simultaneous divergence of as many as four lineages in wide-ranging H. naevius is inferred for the late Miocene-Pliocene history of this taxon.

Morphological discrimination of two genetic groups of a Japanese salamander, Hynobius naevius (Amphibia, Caudata).

Hynobius naevius, distributed on western Honshu, Shikoku, and Kyushu Islands of Japan, includes two genetically distinct groups (Groups A and B) that have never been delimited morphologically. Using specimens from the entire species range, we investigated the possibility of distinguishing these groups morphologically. Multivariate analyses of morphometric characters resulted in recognition of two groups that corresponded well to the two genetic groups. One (Group A) was characterized by larger body, compressed tail, shallower vomerine tooth series, bluish- or reddish-purple ground color, and pale-white lateral markings. In contrast, another (Group B) was characterized by smaller body, cylindrical tail, longer vomerine tooth series, reddish-brown ground color, and white lateral markings. Group A was composed of populations from the Chugoku District of Honshu and northern Kyushu, and could not be divided into subgroups, while Group B encompassed populations from the Chubu and Kinki Districts of Honshu, Shikoku and Kyushu, and was subdivided into three local subgroups that are geographically separated by marine straits. Morphometric differentiation in Group A is presumed to have been less affected by genetic factors than by other factors, such as ecological relationships with other, coexisting species. Differentiation in Group B is assumed to have been enhanced not only by genetic but also by climatological factors.