Variation in Ventral Coloration Pattern and Aposematism in Tropical Warty Newts.

Aposematic coloration plays a crucial role in animal defense, and it is shaped by a complex interplay of factors such as physiological limitations and sexual and natural selection. Warty newts within the genus Paramesotriton exhibit significant variation in ventral coloration. In this study, we quantified the percentage of red ventral area to investigate aposematic ventral coloration in Paramesotriton deloustali and P. guangxiensis across eight populations in northern Vietnam. To assess the interaction between predators and the aposematic signals, we conducted experiments employing three types of clay replicas of newts: dorsal, red ventral, and black ventral models. Our findings revealed a significant variation in the red ventral area among different populations. Additionally, a significant correlation was detected between the red ventral area of the newt and the annual temperature range. In clay model experiments, a significant difference in predator attack rates was observed between dorsal and ventral clay models. Interestingly, there was no significant difference in attack rates between red and black ventral types. Our study suggested that the variation in the red ventral area of warty newts is probably influenced by multiple factors, including genetic constraints, sex, ambient environment, and diet. Furthermore, our results supported the effectiveness of displaying aposematic coloration as an antipredator defense mechanism in warty newts. However, variations in body size and the pressure of mammal predation might not play a significant role in determining aposematic coloration.

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.

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.

Discovery of Two Lineages of Hynobius tsuensis (Amphibia, Caudata) Endemic to Tsushima Island, Japan.

The Tsushima salamander, Hynobius tsuensis, is a lotic-breeding species endemic to Tsushima Island, Japan. We investigated genetic variation in this species using samples covering all known localities on the island. Phylogenetic analyses based on mitochondrial cytochrome b gene sequences divided H. tsuensis populations into two genetic groups: true H. tsuensis and an unknown species (Hynobius sp.) showing a closer relationship to Hynobius nebulosus than to H. tsuensis. The genetic distance between the two groups was greater than the genetic distances between different species of Hynobius. Both groups were found to breed in mountain streams, sometimes in the same streams syntopically. Population structure analysis based on several nuclear microsatellite loci strongly indicated reproductive isolation between the two groups in the syntopic localities. This is the first report confirming the syntopic occurrence of two lotic salamanders of Hynobius on a small island. We propose that the lotic breeding habits of the two salamanders were independently acquired.

Translucent in air and iridescent in water: structural analysis of a salamander egg sac.

Females of some Asian salamanders of the genus Hynobius deposit in streams their eggs embedded in a translucent envelope called an 'egg sac'. The edges of the envelope exhibit a spectacular blue-to-yellow iridescent glow, which instantaneously disappears when the sac is removed from water. First, our scanning electron microscopy analyses reveal that the inner surface of the 100 µm-thick envelope displays striations (length scale of about 3 µm), which are themselves covered by much smaller (190 ± 30 nm) and quasi-periodic corrugations. The latter could constitute a surface diffraction grating generating iridescence by light interference. Second, our transmission electron microscopy and focused-ion-beam scanning electron microscopy analyses show that the bulk of the egg sac wall is composed of meandering fibres with a quasi-periodic modulation of 190 ± 60 nm along the thickness of the envelope, generating a photonic crystal. Third, Fourier power analyses of 450 electron microscopy images with varying incident angles indicate that changing the surrounding medium from water to air shifts most of the backscattered power spectrum to the ultraviolet range, hence, explaining that the egg sac loses visible iridescence when removed out of the water. Fourth, the results of our photography and optical spectroscopy experiments of submerged and emerged egg sacs rule out the possibility that the iridescence is due to a thin film or a multilayer, whereas the observed non-specular response is compatible with the backscattering expected from surface diffraction gratings and volumetric photonic crystals with spatial 1D modulation. Finally, although we mention several potential biological functions of the egg sac structural colours and iridescence, we emphasise that these optical properties might be the by-products of the envelope material internal structure selected during evolution for its mechanical properties.

Intraspecific Variation in the External and Skeletal Morphology of Hynobius setouchi Matsui, Okawa, Tanabe et Misawa, 2019 (Amphibia: Urodela: Hynobiidae).

The hynobiid salamander genus Hynobius is a basal taxon in the urodeles that plays a key role in understanding the evolution of sexual dimorphism in urodeles. We examined the age (juveniles vs. adults) and sexual differences in the external and skeletal morphology of a Japanese lentic breeding salamander, Hynobius setouchi. We found that juveniles had larger heads than those of adults, and most characters were larger in adult males than in females, except for trunk size. This was also observed in skeletal morphology, although some tail characters differed only in the skeleton. We suggest that intra- and interspecific comparisons of the external and skeletal characters of salamanders should consider age, sexual, and seasonal differences.

A New Species of Micryletta (Amphibia: Anura) from Sumatra, Indonesia.

Micryletta inornata is a complex species that is widely distributed from Sumatra to mainland Asia, including the Thai-Malay Peninsula and Indochina. Recently, this species was confirmed to be endemic to regions near the type locality in Sumatra, and the populations from other regions were suggested to be different species. We examined phenotypic and genotypic characters of the Sumatran populations and found an unnamed lineage in addition to the true M. inornata. The newly found lineage can be distinguished from M. inornata and other congeners by both molecular and morphological traits and has been named Micryletta sumatrana sp. nov. The new species is characterized by having a small body size, golden brown dorsum with scattered dark spots, dark brown ventrum with diffuse cream mottling, dark brown lateral head with cream spots on lips and the tympanum region extending to the axilla, and tibiotarsal articulation reaching to the front of the eye. We discuss the taxonomic status of so-called M. inornata occurring outside of its type locality, especially of M. inornata lineata.

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.

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.

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.

Adaptation of cone pigments found in green rods for scotopic vision through a single amino acid mutation.

Most vertebrate retinas contain a single type of rod for scotopic vision and multiple types of cones for photopic and color vision. The retinas of certain amphibian species uniquely contain two types of rods: red rods, which express rhodopsin, and green rods, which express a blue-sensitive cone pigment (M1/SWS2 group). Spontaneous activation of rhodopsin induced by thermal isomerization of the retinal chromophore has been suggested to contribute to the rod's background noise, which limits the visual threshold for scotopic vision. Therefore, rhodopsin must exhibit low thermal isomerization rate compared with cone visual pigments to adapt to scotopic condition. In this study, we determined whether amphibian blue-sensitive cone pigments in green rods exhibit low thermal isomerization rates to act as rhodopsin-like pigments for scotopic vision. Anura blue-sensitive cone pigments exhibit low thermal isomerization rates similar to rhodopsin, whereas Urodela pigments exhibit high rates like other vertebrate cone pigments present in cones. Furthermore, by mutational analysis, we identified a key amino acid residue, Thr47, that is responsible for the low thermal isomerization rates of Anura blue-sensitive cone pigments. These results strongly suggest that, through this mutation, anurans acquired special blue-sensitive cone pigments in their green rods, which could form the molecular basis for scotopic color vision with normal red rods containing green-sensitive rhodopsin.

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.

Taxonomic Relationships of Hynobius stejnegeri and H. yatsui, with Description of the Amber-Colored Salamander from Kyushu, Japan (Amphibia: Caudata).

Examination of the holotype of Hynobius stejnegeri Dunn, 1923 revealed that the species is identical with H. yatsui Oyama, 1947 , recently resurrected from H. naevius (Temminck and Schlegel, 1838). The name H. yatsui is therefore relegated to a subjective junior synonym of H. stejnegeri. The latter name, however, has been widely applied to an amber-colored or tortoise-shell salamander from Kyushu, southwestern Japan, which now requires a new name. We, thus, describe the ambercolored salamander as a new species Hynobius ikioi. The new species is phylogenetically very close to H. amakusaensis Nishikawa and Matsui, 2014 and H. osumiensis Nishikawa and Matsui, 2014 , both also from Kyushu, but is easily distinguished from them by its uniquely bi-colored dorsum.

Historical Demography of an Endangered Salamander, Ranodon sibiricus (Amphibia, Urodela, Hynobiidae): A Reassessment.

Chinese populations of the endangered Siberian salamander Ranodon sibiricus are reported to have diverged only about 120 years ago, and to have the lowest genetic diversity of any amphibian. However, these conclusions require verification, as the main range of the species is in Kazakhstan. Moreover, the generation time used for estimating divergence time has a weak ground. In order to clarify these problems, we investigated the molecular phylogenetic relationship and historical demography of the species covering its whole distribution range using the mitochondrial DNA region reported for Chinese population (1072 bp sequences of the control region), while conducting skeletochronological analysis to estimate accurate generation time. As a result, the range expansion was estimated at 88,000-50,000 YA, based on the generation time of 6-10 years. Degree of intraspecific genetic differentiation is actually very small, but, as a single species, is not so small as had been reported for Chinese population alone.

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.

Life History of a Unique Asian Plethodontid Salamander, Karsenia koreana.

Using preserved specimens, we studied the basic life history of the topotypic population of the unique Asian plethodontid salamander, Karsenia koreana. Of 51 individuals examined, 11 males and 13 females were judged as mature from the development of gonads. The ovarian eggs were large (diameter 3.7-4.8 mm) and yellow to orange in color, and the clutch size was about 8-10. These values approximate those of actually spawned eggs recently reported. Skeletochronological analyses revealed the average age of males (5.3 years) to be lower than females (7.3 years). The age at maturity and maximum observed longevity were four and nine years in males and five and 10 years in females, respectively. In the growth curves estimated by a von Bertalanffy growth model, the growth coefficient and asymptotic SVL did not differ between the sexes, although males (40.6 mm) were smaller than females (45.3 mm) in the average snout-vent length. The time and place of courtship behavior, oval development, hatching, and especially, whether the species shows aquatic larval stage or direct development, are important topics to be resolved in future.

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.