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.

Addressing the taxonomic confusion of Mesocoelium Odhner, 1910 (Trematoda: Plagiorchioidea: Mesocoeliidae) in Japanese urodelan and anuran amphibians.

Trematodes of the genus Mesocoelium Odhner, 1910 (Digenea: Plagiorchioidea: Mesocoeliidae) are globally distributed and parasitize amphibians, reptiles, or occasionally fishes. This genus is one of the most confusing taxa in trematodes because of its poor morphological features. In this study, we examined species of Mesocoelium collected from Japanese amphibians and found that they can be morphologically assigned to two species of Mesocoelium. Mesocoelium brevicaecum Ochi in Goto and Ozaki, 1929 parasitizes various both urodelan and anuran amphibians and occurred widely in Japan, while M. japonicum Goto and Ozaki, 1930 parasitizes a few hynobiid species in a limited part of Japan. We proposed ceca length as a valid key characteristic for species identification in this genus. M. elongatum Goto and Ozaki, 1929, M. lanceatum Goto and Ozaki, 1929, M. minutum Park, 1939, M. ovatum Goto and Ozaki, 1930, and M. pearsei Goto and Ozaki, 1930 are junior synonyms of M. brevicaecum, while M. japonicum can be distinguishable from them by morphologically and molecularly. Our molecular study supported the validity of both species and showed intraspecific divergence associated with geographic distance. Molecular identification suggests that the land snail Euhadra quaesita can serve as the first intermediate host for M. japonicum in Japan. This study also indicates the extremely low specificity of this genus for vertebrate hosts. Finally, we conclude that at least three species of Mesocoelium (M. brevicaecum, M. japonicum, and Mesocoelium sp. 1) are distributed in Japan. Further studies in other regions are undoubtedly required for a better understanding of the taxonomy and ecology of the genus Mesocoelium.

Larval Body Shape and Eye Size Variation in Japanese Toads (Anura: Bufonidae: Bufo): An Ecological Implication for Lentic and Lotic Habitats.

Variation in morphological traits of anurans has evolved due to the pressures imposed by inhabiting different environments. The Japanese stream toad, Bufo torrenticola, breeds in running water, such as mountain streams, where the larvae grow. This lotic-breeding habit of B. torrenticola was suggested to have evolved from a lentic-breeding ancestor. Evolutionary shift of breeding habit from lentic- to lotic-breeding caused larval morphological changes to adapt to the stream habitat. However, morphological adaptation associated with the larval habitat of these three types of Japanese toads has not been explored well. In this study, we applied geometric morphometrics and distance measurements to compare body and eye characters among the tadpoles of three Japanese toads. The results showed that B. torrenticola has more dorsally and posteriorly positioned eyes, and a longer and wider rostrum because of having a larger mouth and more-developed oral muscles than its close relatives. These characters might be related to lotic lifestyle. Meanwhile, tadpoles of B. torrenticola and its sister taxon B. japonicus japonicus showed similar eye growth pattern, supporting their close phylogenetic relationship. Some of the lotic-adapted characters have also been reported in other lotic tadpoles, which is indicative of convergent evolution among stream-adapted tadpoles.

Population genetic structure and hybrid zone analyses for species delimitation in the Japanese toad (Bufo japonicus).

Hybridization following secondary contact may produce different outcomes depending on the extent to which genetic diversity and reproductive barriers have accumulated during isolation. The Japanese toad, Bufo japonicus, is distributed on the main islands of Japan. In the present study, we applied multiplexed inter-simple sequence repeat genotyping by sequencing to achieve the fine-scale resolution of the genetic cluster in B. j. japonicus and B. j. formosus. We also elucidated hybridization patterns and gene flow degrees across contact zones between the clusters identified. Using SNP data, we found four genetic clusters in B. j. japonicus and B. j. formosus and three contact zones of the cluster pairs among these four clusters. The two oldest diverged lineages, B. j. japonicus and B. j. formosus, formed a narrow contact zone consistent with species distinctiveness. Therefore, we recommend that these two subspecies be elevated to the species level. In contrast, the less diverged pairs of two clusters in B. j. japonicus and B. j. formosus, respectively, admixed over a hundred kilometers, suggesting that they have not yet developed strong reproductive isolation and need to be treated as conspecifics. These results will contribute to resolving taxonomic confusion in Japanese toads.

Demographic and ecological niche dynamics of the Vietnam warty newt, Paramesotriton deloustali: Historical climate influences.

Quaternary climatic cycles strongly affected the genetic diversification and ranges of organisms, shaping current genetic structures and distribution patterns. Urodeles provide ideal examples for exploring these dynamics over time and across space. In this study, we integrated a phylogeographic approach and ensemble species distribution modeling (eSDM) to infer the historical demography and distribution patterns of the Vietnam warty newt, Paramesotriton deloustali. Mitochondrial data revealed two groups, West and East, which diverged approximately 1.92 million years ago (Mya). Diversification was likely driven by change in the climate during early stages of the Pleistocene, with increasing monsoon and drought intensities. Biogeographic analysis indicated that the newt's current distribution formed as a result of vicariance events. In addition, the two groups occupy distinct ecological niches. Demographic reconstruction showed signs of expansion in the effective population sizes of the two major groups beginning around 0.11 and 0.15 Mya, respectively. However, eSDM showed fluctuating predicted distributions during the last interglacial, last glacial maximum, mid-Holocene, and present. Mountain systems in northern Vietnam are likely to have served as climatic refuges and to have played a crucial role in safeguarding species from the effects of climate change.

Molecular and Morphological Assessment of Juvenile and Adult Forms in the Giant Worm Eunice Cf. Aphroditois (Annelida: Eunicidae) and Its Phylogenetic Position in the Family.

Eunice aphroditois (Pallas, 1788) is a large polychaete worm (up to 3 m in length) and the type species of the genus. In Japan, a similar but potentially different species, Eunice cf. aphroditois, is distributed mainly in the rocky shores of the temperate and warm Pacific coasts. Juveniles and adults were suggested to be distinguished by their body color. The juvenile form was previously regarded as distinct species, Eunice flavopicta Izuka, 1912 and Eunice ovalifera Fauvel, 1936, although they are now considered synonymous with E. aphroditois. In this study, we revisited the validity of the present taxonomy based on morphological observations including SEM and microCT, and three molecular markers (cytochrome c oxidase subunit I [COI], 16S rRNA, and histone H3 genes) and investigated the phylogenetic position of E. cf. aphroditois in the family Eunicidae using the combined dataset of three genes (COI + 16S rRNA + 18S rRNA). The adult and juvenile forms were different in body size, color, the distribution of the branchiae and subacicular hooks, and maxillae shape, but not in other characteristics. One individual showed an intermediate body color between the two forms. The adult and juvenile forms shared major haplotypes and the maximum K2P genetic distance of COI was 1.7%, which can be considered within intraspecific variation. In the phylogenetic tree based on the combined gene dataset, E. cf. aphroditois was closely related to Eunice roussaei Quatrefages, 1866 and Eunice cf. violaceomaculata Ehlers, 1887, which are large species from the Mediterranean Sea and the Caribbean Sea, respectively.

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.

Two new kathlaniid species (Nematoda: Cosmocercoidea) parasitic in salamanders of the genus Andrias (Amphibia: Caudata: Cryptobranchidae).

The new nematode species, Falcaustra hanzaki n. sp. and Urodelnema takanoensis n. sp. (Cosmocercoidea: Kathlaniidae), were found from the intestine of giant salamanders in Kyoto Prefecture, Japan. The first species is featured by the number and arrangement of caudal papillae (3 pairs of precloacal papillae, 8 pairs of postcloacal papillae and a single ventral precloacal papilla), the presence of a single pseudosucker, spicules equal in size (520-638 µm long), and V-shaped gubernaculum in males; vulva situated about 3/5 of a body in females. The second species is characterized by the number and arrangement of caudal papillae (5 pairs of precloacal papillae, 6 pairs of postcloacal papillae and a single ventral precloacal papilla), spicules equal in size (403-593 µm long), V-shaped gubernaculum in males; vulva situated about 3/5 of a body in females. The molecular phylogenetic analysis was performed using the partial 18S and 28S ribosomal DNA and the internal transcribed spacers 1 region in the nuclear DNA. This phylogenetic study raised a question about the validity of Family Kathlaniidae and related families of Cosmocercoidea.

Morphological differentiation in giant salamanders, Andrias japonicus, A. davidianus, and their hybrids (Urodela, Cryptobranchidae), and its taxonomic implications.

For a long time, it has been debated whether the two giant salamanders, Andrias japonicus from Japan and A. davidianus from China, are conspecific or heterospecific. Morphological information about their diagnostic characteristics has been limited, without considering sexual dimorphism and/or body size variation. Recently, A. davidianus, which was introduced into Japan sometime in the past, has been found to hybridize with A. japonicus in situ. Taxonomic identification of individuals involved in this unusual breeding is made based on mitochondrial and nuclear DNA analyses. This identification method is time-consuming and costly. Thus, developing easier methods of identification, such as utilizing external morphological characteristics, is urgently needed. In this study, we verify previous descriptions showing that A. davidianus has a longer relative tail length than A. japonicus, and the tubercles on the lower jaw and throat were present in both sexes of A. davidianus. In addition, many head characteristics were found to be relatively larger in A. davidianus than in A. japonicus, which were new distinguishing characters. These morphological differences help support the idea that these are heterospecific lineages. In hybrids, relative values of head width and tail length were larger than those of A. japonicus, and the tubercles on their lower jaw and throat were present as in A. davidianus, suggesting that the hybrids and A. davidianus are distinguishable from A. japonicus.

Taxonomic reassessment of salamanders (genus Hynobius) from Tsushima Islands, Japan, with a resurrection of Hynobius tagoi Dunn, 1923 (Amphibia: Caudata).

To clarify the taxonomic status of salamanders from Tsushima Islands, Japan, we examined two species of salamanders, Hynobius tsuensis and Hynobius sp. from Tsushima Islands, and compared them with H. nebulosus from Kyushu. We found that the three taxa differ from each other in nuclear DNA, adult morphology, and egg-sac shape, and consider them to be independent species. Based on the investigation of type specimens of the synonyms, we identified Hynobius sp. as H. tagoi Dunn, 1923 and redescribe it herein. Also, we designate a neotype for H. tsuensis and redescribe H. tsuensis. The distribution areas of the two species partly overlap but they were distinguishable by their body colorations: H. tsuensis has uniformly blackish tail sides and a distinct yellow stripe on the upper tail edge; whereas H. tagoi has brown tail sides with numerous dark stippling and without a distinct yellow stripe on the upper tail edge.

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.

Taxonomic reappraisal of Hynobius tokyoensis, with description of a new species from northeastern Honshu, Japan (Amphibia: Caudata).

Previous phylogenetic studies based on mitochondrial DNA data have consistently suggested that Hynobius tokyoensis consists of two major clades, clade A (northern clade) and clade B (southern clade). In this study, we newly estimated their population genetic structure and phylogenetic relationships by nuclear SNPs, and the results suggested heterospecific relationships of the two mitochondrial clades, without present hybridization in between. They were also recognized as morphologically different. The type locality of H. tokyoensis is in Tokyo Prefecture, and therefore clade B corresponds to H. tokyoensis sensu stricto, leaving clade A without available scientific name. We, thus, describe the clade A from northeastern Kanto to southern Tohoku as a new species Hynobius sengokui. The new species is distinguished from H. tokyoensis by its relatively longer axilla-groin distance, shorter trunk, and deeper vomerine teeth series, and is estimated to have diverged from it during the late Pliocene.

Genetic diversity and demography of Bufo japonicus and B. torrenticola (Amphibia: Anura: Bufonidae) influenced by the Quaternary climate.

The Quaternary climate affected the present species richness and geographic distribution patterns of amphibians by limiting their activities during the glacial period. The present study examined the phylogenetic relationships of Japanese toads (Bufo japonicus and B. torrenticola) and the demography of each lineage from the past to the present based on mitochondrial sequences and ecological niche models. Japanese toads are a monophyletic group with two main clades (clades A and B). Clade A represents B. j. formosus, including three clades (clades A1, A2, and A3). Clade B contains three clades, two of which corresponded to B. j. japonicus (clades B1 and B2) and the other to B. torrenticola. Clade B2 and B. torrenticola made a sister group, and, thus, B. j. japonicus is paraphyletic. Clades A and B diverged in the late Miocene 5.7 million years ago (Mya) during the period when the Japanese archipelago was constructed. The earliest divergence between the three clades of clade A was estimated at 1.8 Mya. Clades A1 and A2 may have diverged at 0.8 Mya, resulting from the isolation in the multiple different refugia; however, the effects of the glacial climate on the divergence events of clade A3 are unclear. Divergences within clade B occurred from the late Pliocene to the early Pleistocene (3.2-2.2 Mya). Niche similarity between the parapatric clade in clade B (clades B1 and B2) indicated their allopatric divergence. It was suggested that niche segregation between B. japonicus and B. torrenticola contributed to a rapid adaptation of B. torrenticola for lotic breeding. All clade of Japanese toads retreated to each refugium at a low elevation in the glacial period, and effective population sizes increased to construct the current populations after the Last Glacial Maximum. Furthermore, we highlight the areas of climate stability from the last glacial maximum to the present that have served as the refugia of Japanese toads and, thus, affected their present distribution patterns.

Phylogeography of Rhabdias spp. (Nematoda: Rhabdiasidae) collected from Bufo species in Honshu, Shikoku, and Kyushu, Japan including possible cryptic species.

The genus Rhabdias Stiles & Hassall, 1905 comprises lung parasites of amphibians and reptiles worldwide. In Japan, 9 species have been recorded, including Rhabdias incerta Wilkie, 1930 which has been reported only in Bufo species. In this study, to assess the diversity of R. incerta, we performed molecular analyses of Rhabdias species sampled from three species/subspecies of Japanese toads namely Bufo japonicus, B. japonicus formosus, and B. torrenticola, collected in various regions of Honshu, Shikoku, and Kyushu, Japan. DNA sequence divergence was compared using mtDNA (COI) and nuclear DNA (28S) to identify possible cryptic species. Morphological analysis was performed through light microscopy and scanning electron microscopy (SEM). The results revealed that Bufo spp. serve as hosts for at least three Rhabdias species. Morphologically, most samples were identified as R. incerta but with a longer body and larger buccal cavity than originally described. Rhabdias incerta appears to be specific to the genus Bufo and is further subdivided into two or three phylogroups based on subspecies divisions and biogeography of their host. Some Rhabdias specimens collected in this study resemble R. tokyoensis Wilkie, 1930, parasitic in hosts from the order Caudata, which suggests host switching. Both molecular and morphological analyses suggested the presence of undescribed and cryptic Rhabdias species within toads collected in Japan. This study was the first to molecularly characterize Rhabdias species in Japan, including novel sequences of R. incerta and two undescribed species.

Differentiated historical demography and ecological niche forming present distribution and genetic structure in coexisting two salamanders (Amphibia, Urodela, Hynobiidae) in a small island, Japan.

Background: The climatic oscillations in the Quaternary period considerably shaped the distribution and population genetic structure of organisms. Studies on the historical dynamics of distribution and demography not only reflect the current geographic distribution but also allow us to understand the adaption and genetic differentiation of species. However, the process and factors affecting the present distribution and genetic structure of many taxa are still poorly understood, especially for endemic organisms to small islands. Methods: Here, we integrated population genetic and ecological niche modelling approaches to investigate the historical distribution and demographic dynamics of two co-existing salamanders on Tsushima Island, Japan: the true H. tsuensis (Group A), and Hynobius sp. (Group B). We also examined the hypothesis on the equivalency and similarity of niches of these groups by identity and background tests for ecological niche space. Results: Our result showed that Group A is considered to have undergone a recent population expansion after the Last Glacial Maximum while it is unlikely to have occurred in Group B. The highest suitability was predicted for Group A in southern Tsushima Island, whereas the northern part of Tsushima Island was the potential distribution of Group B. The results also suggested a restricted range of both salamanders during the Last Interglacial and Last Glacial Maximum, and recent expansion in Mid-Holocene. The genetic landscape-shape interpolation analysis and historical suitable area of ecological niche modelling were consistent, and suggested refugia used during glacial ages in southern part for Group A, and in northern part of Tsushima Island for Group B. Additionally, we found evidence of nonequivalence for the ecological niche of the two groups of the salamanders, although our test could not show either niche divergence or conservatism based on the background tests. The environmental predictors affecting the potential distribution of each group also showed distinctiveness, leading to differences in selecting suitable areas. Finally, the combination of population genetics and ecological modeling has revealed the differential demographic/historical response between coexisting two salamanders on a small island.

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.

Development and evaluation of a loop-mediated isothermal amplification (LAMP) assay for quick identification of the Japanese salamander Hynobius tokyoensis.

Species identification using molecular techniques has recently become common for various taxa. Loop-mediated isothermal amplification (LAMP) is one of the easiest and least expensive molecular identification methods. Although few studies have developed LAMP assays for amphibians, we believe that LAMP is also useful for identifying endangered amphibians. Hynobius tokyoensis and H. lichenatus occur in Honshu, Japan, and have parapatric distributions. They are similar morphologically, especially at early developmental stages, including eggs and larvae. Hynobius tokyoensis has been listed as a national endangered species in Japan since 2020, and unambiguous identification of these species is therefore important for their conservation and management. In this study, we developed a LAMP primer set for the mitochondrial cytochrome b region to detect H. tokyoensis, and we evaluated the LAMP assay using total genomic DNA from four H. tokyoensis and three H. lichenatus individuals from across most of their ranges. Our LAMP primer set could distinguish these two species. This study should help to establish LAMP assays for other endangered species and morphologically similar species.

Osteological characteristics of the Setouchi salamander Hynobius setouchi (Urodela, Hynobiidae).

This study described the detailed osteological features of the Setouchi salamander Hynobius setouchi. H. setouchi, which is endemic to central Japan, was recently delineated from the wide range of H. nebulosus species based on molecular and external morphological characteristics. However, the osteological features of the species belonging to the genus Hynobius have not been examined, which has hindered the detailed understanding of morphological diversity in the genus. To address this problem, this study elucidated the osteological features of the salamander using micro-computed tomography scanning and whole-mount double staining methods. The articular, a part of the hypobranchial I, a part of coracoid, pubis, carpus, and tarsus were cartilaginous in small-sized individuals and were partially or completely ossified in large-sized individuals. The degree of ossification varied in the operculum, basibranchial II, and ascending process of the palatoquadrate. However, ossification was not associated with body size. The lacrimal, ossified manus, and ossified pes exhibited varying degrees of articulation or fusion irrespective of body size. Moreover, the cranium and tarsus of H. setouchi exhibited several unique characteristics. These osteological characteristics will aid in revising the taxonomy and phylogeny of the Hynobius species.