Lack of variations in the salamander chytrid fungus, Batrachochytrium salamandrivorans, at its alleged origin: Updating its Japanese distribution with new evidence.

The chytrid fungus Batrachochytrium salamandrivorans [Bsal] is causing declines in the amphibian populations. After a decade of mapping the pathogen in Europe, where it is causing dramatic outbreaks, and North America, where its arrival would affect to the salamander's biodiversity hotspot, little is known about its current status in Asia, from presumably is native. Japan has several species considered as potential carriers, but no regulation is implemented against Bsal spreading. Previous Bsal known presence detected various cases on the Okinawa Island, southwestern Japan. Previous studies on its sister species, B. dendrobatidis presented a high genomic variation in this area and particularly on Cynops ensicauda. Here, we have done the largest monitoring to date in Japan on the Cynops genus, focusing on Okinawa Island and updating its distribution and providing more information to unravel the still unknown origin of Bsal. Interestingly, we have provided revealing facts about different detectability depending on the used molecular techniques and changes in its Japanese distribution. All in all, the Bsal presence in Japan, together with its low variability in the sequenced amplicons, and the lack of apparent mortalities, may indicate that this part of Asia has a high diversity of chytrids.

Discovery of ex situ individuals of Andrias sligoi, an extremely endangered species and one of the largest amphibians worldwide.

The South China giant salamander, Andrias sligoi, is one of the largest extant amphibian species worldwide. It was recently distinguished from another Chinese species, the Chinese giant salamander, Andrias davidianus, which is considered Critically Endangered according to the International Union for Conservation of Nature (IUCN) Red List. It appears too late to save this extremely rare and large amphibian in situ. Another extant species of the same genus, Andrias japonicus, inhabits Japan. However, the introduction of Chinese giant salamanders into some areas of Japan has resulted in hybridization between the Japanese and Chinese species. During our genetic screening of giant salamanders in Japan, we unexpectedly discovered four individuals of the South China giant salamander: two were adult males in captivity, and one had recently died. The last individual was a preserved specimen. In this study, we report these extremely rare individuals of A. sligoi in Japan and discuss the taxonomic and conservational implications of these introduced individuals.

Genetic and morphological variation analyses of Glandirana rugosa with description of a new species (Anura, Ranidae).

Glandirana rugosa is known to include several geographic groups differing in sex chromosomes, and has been proven to be paraphyletic in mitochondrial phylogeny with respect to G. susurra. By analyzing genetic and morphological variation in a large number of individuals of Glandirana, we studied their taxonomic relationships. A mitochondrial DNA phylogeny, with the G. tientaiensis as outgroup, revealed two major lineages containing respectively (1) the East group of G. rugosa, G. susurra, and the Central and Southeast-Kyushu groups of G. rugosa; and (2) G. emeljanovi, and the North and West groups of G. rugosa. In contrast, in a nuclear DNA phylogeny based on SNP data, lineages of (1) G. susurra and East group, and (2) the remaining groups of G. rugosa and G. emeljanovi, were split, indicating a distinct status of the East group among G. rugosa. In adult morphology, there were only minor differences between the East group and the remaining groups of G. rugosa, but in larvae, the East group had significantly more sparse skin glands than the others. The exact type locality of G. rugosa is most probably in western Japan, not including the range of the East group. From these results, we describe the East group as a new species, G. reliquia, distinct from the remaining groups of G. rugosa. The new species with sexually homomorphic chromosomes is thought to represent a basic stock of Japanese Glandirana, which existed far before G. rugosa originated.

Competition between the tadpoles of Japanese toads versus frogs.

Competition within and among species can play a key role in structuring the assemblages of anuran tadpoles. Previous studies have reported that tadpoles of the invasive cane toad (Rhinella marina) are more strongly disadvantaged by the presence of native frog tadpoles than by the same number of conspecific toad tadpoles. That effect might arise from a lack of coevolution of the invasive toad with its competitors; and/or from a generalized superiority of frog tadpoles over toad tadpoles. To clarify those possibilities, we conducted experimental trials using the larvae of a native rather than invasive toad (Bufo japonicus formosus in Japan) exposed to larvae of native anurans (the sympatric frogs Rana japonica and Rana ornativentris and the parapatric toad Bufo japonicus japonicus). In intraspecific competition trials, higher densities of B. j. formosus prolonged the larval period and reduced size at metamorphosis, but did not affect survival. In interspecific competition trials, the effects of the other anuran species on B. j. formosus were similar to the effects of the same number of conspecific larvae. This similarity in impact of interspecific versus intraspecific competition argues against any overall competitive superiority of frog larvae over toad larvae. Instead, the vulnerability of larval cane toads to frog tadpoles may result from a lack of coevolutionary history.

Description of a new subterranean breeding brown frog (Ranidae: Rana) from Japan.

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.

Unique Evolution of Hyla hallowellii Among Amphibians of the Central Ryukyus, Japan (Anura: Hylidae).

Fauna of the Central Ryukyus includes a high percentage of endemic species, and Hallowell's tree frog Hyla hallowellii Thompson, 1912 is one of such elements, occurring in a total of eight islands in the Amami and Okinawa Island groups. Using samples representing all of these eight island populations, we studied variations in morphology, karyotype, allozyme, and mtDNA, to clarify the pattern of geographic differentiation of H. hallowellii and consider factors for its formation. We could not clearly discriminate one population from another in morphology, nor could we find any interpopulation difference in karyotype. From genetic analyses, using allozymes and cyt b, we found low overall differentiations among populations. However, the southern populations from Okinawajima and Yoronjima were genetically nearly identical with the northern Amamioshima population. From that group the geographically intermediate Tokunoshima and Kakeromajima populations showed prominent differentiations. These patterns of geographical differentiation greatly differ from those known in other amphibian species of the Central Ryukyus, and suggest that H. hallowellii has evolutionary history unique to the species.

A new Occidozyga from Sarawak, Malaysian Borneo (Anura: Dicroglossidae).

We found a uniquely colored dicroglossid frog of the genus Occidozyga from western Sarawak, East Malaysia. It is divergent from other congeners in morphology and mtDNA sequences. In a molecular phylogeny, this species is the sister lineage to the continental species O. lima and O. martensii with weak support. The species is small with SVL 16-18 mm in males and 18-19 mm in females, without dorsolateral fold but with transverse wrinkles on dorsum, tips of fingers lacking disks but of toes with disks, only first and second toes webbed to disks, and orange-brown dorsum with dark brown band. We thus describe it as a new species.

Systematics of Calliophis intestinalis with the Resurrection of Calliophis nigrotaeniatus (Elapidae, Serpentes).

The red-bellied form of Calliophis intestinalis (Laurenti, 1768) sensu lato was originally reported from Pahang, west Malaysia. To determine the taxonomic status of this form, we examined the type specimens of Elaps sumatranus Lidth De Jeude, 1890, Calliophis intestinalis everetti (Boulenger, 1896), and Callophis furcatus var. nigrotaeniatus Peters, 1863. The results indicated that the red-bellied form of C. intestinalis should be named as Calliophis nigrotaeniatus comb. nov., whose valid species status was based on morphological and molecular analyses. We designate a lectotype and redescribe the species, which is genetically close to Calliophis bilineatus (Peters, 1881) from the Philippines, and is clearly distinguishable from other congeners by possessing a pair of gray or dark blue lateral stripes and by being bright red on the ventrum. Elaps sumatranus and C. i. everetti are relegated to subjective junior synonyms of C. nigrotaeniatus.

Two New Ansonia from Mountains of Borneo (Anura, Bufonidae).

Two lineages of stream toads in the genus Ansonia from Malaysian Borneo have long been suspected to be specifically distinct on the basis of molecular data. We assessed the taxonomic status of these lineages using morphological and additional genetic data. In mtDNA phylogeny, each lineage-one from Bario, Kelabit Highlands of Sarawak, the other from Mt. Mulu of Sarawak and the Crocker Range of Sabah-is separated from other congeners by large genetic distances, comparable with those observed between heterospecific species in the genus. These lineages are also morphologically distinguishable from other species, and are considered to represent valid, independently evolving species. We therefore describe them as A. kelabitensis sp. nov. and A. kanak sp. nov.

Large-scale phylogenetic analyses provide insights into unrecognized diversity and historical biogeography of Asian leaf-litter frogs, genus Leptolalax (Anura: Megophryidae).

Southeast Asia and southern China (SEA-SC) harbor a highly diverse and endemic flora and fauna that is under increasing threat. An understanding of the biogeographical history and drivers of this diversity is lacking, especially in some of the most diverse and threatened groups. The Asian leaf-litter frog genus Leptolalax Dubois 1980 is a forest-dependent genus distributed throughout SEA-SC, making it an ideal study group to examine specific biogeographic hypotheses. In addition, the diversity of this genus remains poorly understood, and the phylogenetic relationships among species of Leptolalax and closely related Leptobrachella Smith 1928 remain unclear. Herein, we evaluate species-level diversity based on 48 of the 53 described species from throughout the distribution of Leptolalax. Molecular analyses reveal many undescribed species, mostly in southern China and Indochina. Our well-resolved phylogeny based on multiple nuclear DNA markers shows that Leptolalax is not monophyletic with respect to Leptobrachella and, thus, we assign the former to being a junior synonym of the latter. Similarly, analyses reject monophyly of the two subgenera of Leptolalax. The diversification pattern of the group is complex, involving a high degree of sympatry and prevalence of microendemic species. Northern Sundaland (Borneo) and eastern Indochina (Vietnam) appear to have played pivotal roles as geographical centers of diversification, and paleoclimatic changes and tectonic movements seem to have driven the major divergence of clades. Analyses fail to reject an "upstream" colonization hypothesis, and, thus, the genus appears to have originated in Sundaland and then colonized mainland Asia. Our results reveal that both vicariance and dispersal are responsible for current distribution patterns in the genus.

Genomic Displacement and Shift of the Hybrid Zone in the Japanese Fire-Bellied Newt.

Hybridizations on a secondary contact zone between 2 diverged lineages can have various evolutionary consequences, including the genetic replacement of one lineage by another. We detected such a case between 2 lineages (the Central and Western lineages) of the Japanese fire-bellied newt, Cynops pyrrhogaster in the Chugoku district of western Japan. We genotyped 269 individuals from 30 localities using the mitochondrial cytochrome b gene and 11 microsatellite loci. The mitochondrial DNA (mtDNA) analysis revealed that the 2 lineages were mostly distributed parapatrically to each other but co-occurred around the contact zone, whereas the microsatellite analyses indicated the presence of a hybrid zone. Geographic cline analysis revealed that the cline width of mtDNA is wider than the width of the microsatellite loci. The migration rate estimation and the NewHybrids analysis revealed that the Central lineage has expanded their range into the range of the hybrid zone, suggesting the possibility of range displacement of the 2 lineages as a consequence of the shift of their hybrid zone. We explored the process of asymmetric gene flow associated with the invasion of the Central lineage to explain this pattern.

A New Species of Pelophryne from Western Sarawak, Malaysian Borneo (Anura, Bufonidae).

A new small, semi-arboreal toad of the genus Pelophryne is described from western Sarawak, Malaysian Borneo, on the basis of molecular and morphological evidence. Of the two morphotypes recognized in the genus, the new species belongs to the one in which the tips of the fingers are expanded into truncate discs. Among the species in the morphotype, the new species is most similar to P. murudensis, but differs from it by body size, relative hindlimb length, and dorsal coloration. The new species is currently known only from a limited area on Gunung (= Mt.) Penrissen, and future measures of its habitat conservation are necessary.

Unusually high genetic diversity in the Bornean Limnonectes kuhlii-like fanged frogs (Anura: Dicroglossidae).

A fanged frog Limnonectes kuhlii was once thought to be wide-ranging in Southeast Asia, but is now confined to its type locality Java through recent phylogenetic studies, which clarified heterospecific status of non-Javanese populations, and monophyly of Bornean populations. However, large genetic differences among Bornean populations suggest occurrence of cryptic species, which we test using dense geographic sampling. We estimated the phylogenetic relationships among samples of Bornean populations together with their putative relatives from the continental Southeast Asia, using 2517bp sequences of the 12S rRNA, tRNA(val), and 16S rRNA of mitochondrial DNA, and 2367bp sequences of the NCX1, POMC, and RAG1 of nuclear genes. In the mtDNA trees, Bornean L. kuhlii-like frogs formed a monophyletic group split into 18 species lineages including L. hikidai, with the deepest phylogenetic split separating L. cintalubang from the remaining species. Almost all of these lineages co-occur geographically, and two to three lineages were found syntopically in each locality. Co-occurrence of more than one lineage may be maintained by differential morphology and microhabitat selection. These syntopic lineages should be regarded as distinct species. Our results clearly indicate that taxonomic revision is urgent to clarify many evolutionary problems of Bornean L. kuhlii-like frogs.

A New Species of Meristogenys (Anura: Ranidae) from Sarawak, Borneo.

A cryptic Bornean torrent frog of the genus Meristogenys, which is divergent genetically and morphologically from all known congeners, is described from mountain streams of western Sarawak, East Malaysia (Borneo). The species occurs sympatrically with the type species of the genus, M. jerboa, but apparently differs from it in adult coloration and larval morphology, such as keratodont formulae and glands in tail fins. Females of the new species possess much larger and fewer eggs than in sympatric M. jerboa, suggesting significantly different reproductive traits between these species. A key to larvae of known species of the genus is provided.

Phylogeny and Differentiation of Wide-Ranging Ryukyu Kajika Frog Buergeria japonica (Amphibia: Rhacophoridae): Geographic Genetic Pattern Not Simply Explained by Vicariance Through Strait Formation.

To investigate geographic genetic structures and taxonomic relationships among isolated populations of Buergeria japonica, occurring very widely in various habitats of the Ryukyu Archipelago and Taiwan, we conducted phylogenetic and demographic analyses among individuals from various localities, representing their entire distributional ranges. Buergeria japonica is genetically greatly differentiated and comprises three major clades (the Southern Taiwan [ST] clade, the Northern Taiwan + Southern Ryukyu [NT/SR] clade, and the Central + Northern Ryukyu [CR/NR] clade), each of which seems to represent independent species. The first divergence in the species is estimated to have occurred in the middle to late Miocene in areas of current Taiwan, then eastern periphery of the Asian continent. Split of the ST and the remaining clades, and subsequent divergence between the NT/SR and the CR/NR clades in the latter, indicate consecutive south to north vicariant diversifications. However, these vicariances are not always associated with formation of significant barriers such as deep straits. Less but still prominently diverged subclades (the Amami + Tokara [AM/TK] and the Okinawa [ON] subclades) in the CR/NR clade were recognized in spite of the absence of an intervening deep strait. Contrariwise, individuals from Amami and Tokara Groups formed the AM/TK subclade in spite of the presence of the intervening Tokara Gap (a long-standing deep tectonic strait). Furthermore, in the AM/TK subclade, low but definite genetic divergence was found between the Northern Amami + Tokara (NAM/TK) lineage and the Southern Amami (SAM) lineage. Estimated divergence time and gene flow rate within the NAM/TK lineage indicate that this species reached northern Tokara from the south by overseas dispersal over the Tokara Gap long after its formation, but not by more recent artificial transportation. This overseas dispersal would have been facilitated by its more frequent occurrence around coastal habitats than other frogs.

A new tree frog of the genus Gracixalus from Thailand (Amphibia: Rhacophoridae).

We report a new tree frog of the genus Gracixalus from western Thailand and describe it as a new species Gracixalus seesom based on results of morphological and molecular analyses. The new species is a small-sized Gracixalus (male snout-vent length ca. 22 mm) and is morphologically similar to G. gracilipes, but is easily distinguished from it by its dorsal tan color in life, absence of white spot on lower lip, and black markings on its foot webbing. The new species also clearly differs from all the other members of the genus by the combination of small body size, triangular snout, and light yellowish brown dorsum without distinct tuberculations. Problems of phylogeny and taxonomy of the genus Gracixalus are briefly discussed.

Mitochondrial phylogeny of an Asian tree frog genus Theloderma (Anura: Rhacophoridae).

We assessed phylogenetic and systematic relationships among 17 out of 23 species of Theloderma and all three species of Nyctixalus from 2412bp sequences of the mitochondrial DNA genes of 12S rRNA, tRNA(val), and 16S rRNA using maximum likelihood and Bayesian inference methods. With the exception of T. moloch, Theloderma and Nyctixalus are confirmed to form a clade, in which each genus also forms a clade. Theloderma moloch is phylogenetically outside these clades and closer to samples from Chiromantis, Feihyla, Gracixalus, Kurixalus, Philautus, Polypedates, Raorchestes, and Rhacophorus. Within Theloderma, T. horridum and T. stellatum form the sister taxon to a clade comprising the remaining species. The basal split within the latter clade groups T. asperum, T. licin, T. petilum, and T. ryabovi as the sister to a clade comprising T. bicolor, T. chuyangsinense, T. corticale, T. gordoni, T. laeve, T. lateriticum, T. nebulosum, T. rhododiscus, and T. truongsonense. Our phylogenetic results indicate homoplastic evolution of four morphological characters: small vs. large body size, presence of vomerine teeth, presence of a vocal opening in males, and interdigital webbing on hands. The common ancestor of Theloderma and Nyctixalus is inferred to have arisen in the area including the current Sunda region.

Cytonuclear discordance and historical demography of two brown frogs, Rana tagoi and R. sakuraii (Amphibia: Ranidae).

Prior studies of mitochondrial genomic variation reveal that the Japanese brown frog Rana tagoi comprises a complex of cryptic species lineages, and that R. sakuraii arose from within this complex. Neither species forms a monophyletic group on the mitochondrial haplotype tree, precluding a simple explanation for the evolutionary origins of R. sakuraii. We present a more complete sampling of mitochondrial haplotypic variation (from the ND1 and 16S genes) plus DNA sequence variation for five nuclear loci (from the genes encoding NCX1, NFIA, POMC, SLC8A3, and TYR) to resolve the evolutionary histories of these species. We test hypotheses of population assignment (STRUCTURE) and isolation-with-migration (IM) using the more slowly evolving nuclear markers. These demographic analyses of nuclear genetic variation confirm species-level distinctness and integrity of R. sakuraii despite its apparent polyphyly on the mitochondrial haplotype tree. Divergence-time estimates from both the mitochondrial haplotypes and nuclear genomic markers suggest that R. sakuraii originated approximately one million years ago, and that incomplete sorting of mitochondrial haplotype lineages best explains non-monophyly of R. sakuraii mitochondrial haplotypes. Cytonuclear discordance elsewhere in R. tagoi reveals a case of mitochondrial introgression between two species lineages on Honshu. The earliest phylogenetic divergence within this species group occurred approximately four million years ago, followed by cladogenetic events in the Pliocene and early Pleistocene yielding 10-13 extant species lineages, including R. sakuraii as one of the youngest.

Discordance between mitochondrial DNA genealogy and nuclear DNA genetic structure in the two morphotypes of Rana tagoi tagoi (Amphibia: Anura: Ranidae) in the Kinki Region, Japan.

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.