Masayuki Sumida

• Professor Emeritus at Hiroshima University

The endangered crocodile newt, Echinotriton andersoni, is a relatively large species of the family Salamandridae and is distributed on six islands in the central part of the Ryukyu Archipelago, Japan. Because of an originally small distribution range and recent habitat loss, this species has been steadily declining in number. To elucidate fine-scal...

For more than a century, Hylarana tytleri (Theobald, 1868) has been confused with two congeneric species, H. taipehensis and H. erythraea, in Bangladesh and neighboring countries, due to phenetic similarities as well as a lack of sufficient molecular and morphometric data. To resolve these problems, we conducted molecular and morphological surveys...

In light of reproductive isolation being a fundamental aspect of the biological species concept, we performed crossing experiments using six species from three genera (Hoplobatrachus, Euphlyctis and Fejervarya) of family Dicroglossidae to explore postmating isolation in dicroglossid frogs. Our results revealed gametic isolation among these genera,...

Aim Amphibians are considered poor transoceanic dispersers because of their permeable skin. However, overseas dispersal of amphibian species has been revealed by recent phylogeographical studies and the role of overseas colonization of amphibians on their evolution and diversification has also been highlighted. However, no studies have investigated...

The five frog species of the genus Hoplobatrachus are widely distributed in Asia and Africa, with Asia being considered the genus\' origin. However, the evolutionary relationships of Asian Hoplobatrachus species remain ambiguous. Additionally, genetic diversity and fundamental differentiation processes within species have not been studied. We condu...

Physiological tolerance to variable environmental conditions is essential for species to disperse over habitat boundaries and sustain populations in new habitats. In particular, salinity and temperature are one of the major factors determining species’ distributions. The tree frog Buergeria japonica is the most widely distributed amphibian species...

We have succeeded in creating see-through frogs from natural color mutants of the Japanese brown frog Rana japonica, which usually possesses an ochre or brown back; this coloration enables the organs, blood vessels, and eggs to be observed through the skin without performing dissection. We crossed two kinds of recessive color mutant (black-eyed and...

The five frog species of the genus Hoplobatrachus are widely distributed in Asia and Africa, with Asia being considered the genus' origin. However, the evolutionary relationships of Asian Hoplobatrachus species remain ambiguous. Additionally , genetic diversity and fundamental differentiation processes within species have not been studied. We condu...

Sex determination in frogs (anurans) is genetic and includes both male and female heterogamety. However, the origins of the sex chromosomes and their differentiation processes are poorly known. To investigate diversity in the origins of anuran sex chromosomes, we compared the chromosomal locations of sex-linked genes in 4 species: the African clawe...

Babina holsti is a ranid frog that occurs on northern Okinawa (Yanbaru) and Tokashiki Islands of southwest Japan. Because of its narrow distribution and recent habitat destruction, its population size is believed to have decreased, and the species has been listed as an endangered species on the IUCN Red List. To promote effective conservation manag...

Aim Pelophylax frogs in East Asia provide an opportunity to explore the impact of glacial cycling on demographic and genetic dynamics, because it has been suggested that they experienced distribution shifts and subsequent mt DNA introgression from Pelophylax plancyi to Pelophylax nigromaculatus in association with climatic oscillations. However, th...

The Western clawed frog, Xenopus tropicalis, is a highly promising model amphibian, especially in developmental and physiological research, and as a tool for understanding disease. It was originally found in the West African rainforest belt, and was introduced to the research community in the 1990s. The major strains thus far known include the Nige...

The rich biodiversity of frogs and toads in Bangladesh is unexpectedconsidering the rather simple topographic features of the country. Indeed, anew species, Microhyla nilphamariensis, has recently been described fromNilphamari District (northern region), Bangladesh. However, additionalgenetic, acoustic, and morphometric data are necessary to precis...

The dorsal blastopore lip (known as the Spemann organizer) is important for making the body plan in amphibian gastrulation. The organizer is believed to involute inward and migrate animally to make physical contact with the prospective head neuroectoderm at the blastocoel roof of mid- to late-gastrula. However, we found that this physical contact w...

Fig. S1.Rate of each type in the experiment of neutral red injection into blastocoel roof. Fig. S2. Not dorsal lip but dorsal blastocoel floor acts as anterior organizer in normal development. Fig. S3. Anterior axial tissue is prevented from labeling surface cells at blastopore appearance. Fig. S4. Proposed model of amphibian gastrulation is compar...

Movie S1.Time-lapse movie of vegetal view of Rana nigromaculata embryo. Movie S2.Time-lapse movie of vegetal view of Xenopus laevis embryo.

Two species of the true frog genus Rana (R. japonica and R. ornativentris) are widely distributed on the main islands of Japan with some degree of habitat overlap. These species could be key indicators for environmental conditions and accurate identification is important. However, morphological characters in early stages of development are often in...

The endangered Ryukyu tip-nosed frog Odorrana narina and its related species, Odorrana amamiensis, Odorrana supranarina, and Odorrana utsunomiyaorum, belong to the family Ranidae and are endemically distributed in Okinawa (O. narina), Amami and Tokunoshima (O. amamiensis), and Ishigaki and Iriomote (O. supranarina and O. utsunomiyaorum) Islands. Be...

The Indian bullfrog Hoplobatrachus tigerinus and its four congeneric species are common frog species and distributed throughout South Asia. Due to recent human activity, they are facing a changing environment and reduction in natural population size. For effective conservation and molecular ecological studies, we therefore isolated and characterize...

The Indonesian archipelago is an ideal setting for the study of speciation and biogeography. This archipelago is divided into three island groups based on zoogeography: Sundaland, Wallacea and the Australian region. In this paper we used frogs in genus Fejervarya (Bolkay) to study biogeography and examine patterns of gene flow across proposed zooge...

To elucidate the genetic divergences of several Asian frog taxa, the mitochondrial 16S rRNA gene (16S) sequences of 81 populations across 6 Asian countries were analyzed. In total, 109 haplotypes were found, and the concept of a 3% difference in 16S sequence corresponding to species threshold was applied to define candidate amphibian species, for w...

Xenopus tropicalis has many advantages as a next-generation model animal for biological research. However, the temperature tolerance range of this frog is narrow, making transportation of live specimens difficult under extreme air temperatures in the summer and winter. This seasonal constraint diminishes the usefulness of X. tropicalis as an experi...

Two new frog species belonging to genus Microhyla from the southeast, central and northeast regions of Bangladesh are described. Based on a molecular phylogeny derived from mitochondrial DNA sequences, one of the new species forms a clade with M. fissipes, while the second new species is sister to this clade. The DNA sequences of the mitochondrial...

Buergeria japonica is a common frog species distributed throughout almost all islands in Ryukyu Archipelago. Because of their exceptionally wide distribution and higher physiological tolerance comparing to the other anurans, their demographic history and formation of distribution are intrinsic topics in the herpetological fauna of Ryukyu. Microsate...

The Indian bullfrog Hoplobatrachus tigerinus and its four congeneric species are common frog species and distributed throughout South Asia. Due to recent human activity, they are facing a changing environment and reduction in natural population size. For effective conservation and molecular ecological studies, we therefore isolated and characterize...

Two new frog species belonging to genus Microhyla from the southeast, central and northeast regions of Bangladesh are described. Based on a molecular phylogeny derived from mitochondrial DNA sequences, one of the new species forms a clade with M. fissipes, while the second new species is sister to this clade. The DNA sequences of the mitochondrial...

Mitochondrial genomic (mitogenomic) reorganizations are rarely found in closely-related animals, yet drastic reorganizations have been found in the Ranoides frogs. The phylogenetic relationships of the three major ranoid taxa (Natatanura, Microhylidae, and Afrobatrachia) have been problematic, and mitogenomic information for afrobatrachians has not...

Simple Summary We naturally bred the endangered Anderson’s crocodile newt (Echinotriton andersoni) and tested a laboratory farming technique using near-biotopic breeding cages with several male and female pairs collected from Okinawa, Amami, and Tokunoshima Islands. This is the first published report of successfully propagating an endangered specie...

The Otton frog (Babina subaspera) and Holst’s frog (Babina holsti) are both endangered sister species belonging to family Ranidae. For extensive genetic and ecological research of these species, we isolated and characterized 8 new microsatellite loci of the Otton frog and validated cross-amplification in Holst’s frog along with 8 previously reporte...

Genus Babina is a member of Ranidae, a large family of frogs, currently comprising 10 species. Three of them are listed as endangered species. To identify mitochondrial (mt) genes suitable for future population genetic analyses for endangered species, we determined the complete nucleotide sequences of the mt genomes of 3 endangered Japanese Babina...

Isolation by distance and landscape connectivity are fundamental factors underlying speciation and evolution. To understand how landscapes affect gene flow and shape population structures, island species provide intrinsic study objects. We investigated the effects of landscapes on the population structure of the endangered frog species, Odorrana is...

In light of reproductive isolation being a fundamental aspect of the biological species concept, we performed crossing experiments using six species from three genera (Hoplobatrachus, Euphlyctis and Fejervarya) of family Dicroglossidae to explore postmating isolation in dicroglossid frogs. Our results revealed gametic isolation among these genera,...

Pelophylax nigromaculatus, P. porosus porosus, and P. p. brevipoda are three pond frog species distributed in Japan. Their distributions overlap at two basins in central Japan (P. nigromaculatus and P. p. porosus in the Matsumoto basin, and P. nigromaculatus and P. p. brevipoda in the Ina basin), and hybrid descendants have been found in these area...

A new cryptic species of the genus Hoplobatrachus from Cox's Bazar district of Bangladesh is described and compared with its relevant congeners both in morphology and mitochondrial gene sequences. The new species differs from its close relative H. tigerinus in having a distinct broad black band from the eye, through the nostrils, to the anterior ed...

To survey the diversity of anuran species in Bangladesh, we compared mitochondrial 16S rRNA gene sequences (approximately 1.4 kbp) from 107 Bangladesh frog specimens. The results of genetic divergence and phylogenetic analyses incorporating data from related species revealed the occurrence of at least eight cryptic species. Hoplobatrachus tigerinus...

Crocodile newts, which constitute the genera Echinotriton and Tylototriton, are known as living fossils, and these genera comprise many endangered species. To identify mitochondrial (mt) genes suitable for future population genetic analyses for endangered taxa, we determined the complete nucleotide sequences of the mt genomes of the Japanese crocod...

Due to an originally small distribution range and recent habitat loss, Anderson’s crocodile newt (Echinotriton andersoni) has been steadily declining in number. For effective conservation of this species, a greater amount of genetic information is needed. Here, we isolated ten microsatellite loci of E. andersoni using three different methods, and p...

In order to investigate the morphological and allozyme variation of Indian bullfrog Hoplobatrachus tigerinus, 60 live individuals were collected from three different regions namely the Dinajpur, the Nilphamari and the Lalmonirhat the northern parts of Bangladesh. For morphological study, thirty one morphometric characters of frogs were measured. Th...

We describe a new species of dicroglossid frog of the Fejervarya limnocharis complex from western Honshu, Japan Mainland. The new species, Fejervarya kawamurai, is genetically closer to F. sakishimensis than to F. limnocharis. It differs from F. sakishimensis by smaller tympanum, head, forelimb, hindlimb, foot, and tibia lengths, all relative to sn...

Odorrana ishikawae is listed as a class IB endangered species in the IUCN Red List and is protected by law in both Okinawa and Kagoshima Prefectures, Japan. Here, in an effort to help effectively preserve the genetic diversity of this endangered species in the laboratory, we tested a farming technique involving the artificial breeding of frogs, and...

Recently, we identified nine novel antimicrobial peptides from the skin of the endangered anuran species, Odorrana ishikawae, to assess its innate immune system. In this study an additional antimicrobial peptide was initially isolated based on antimicrobial activity against Escherichia coli. The new antimicrobial peptide belonging to the palustrin-...

We recently reported the primary structures, antimicrobial activities and cDNA precursors of nine novel antimicrobial peptides from the skin of the endangered anuran species, Odorranaishikawae. Their cDNA clones revealed a highly conserved approximately 60 bp region upstream of the start codon. This conserved region was used in the "shotgun" cDNA c...

Due to an originally small distribution range and over-exploitation of primary forest, Ishikawa’s frog (Odorrana ishikawae) has been steadily declining in number. For effective conservation of this species, a greater amount of genetic information of this species is needed. Here, we isolated and characterized 12 microsatellite loci of O. ishikawae u...

Time trees from calibration A and D. Time trees from the calibration A and D are shown.

Estimated ages from calculations A-F. Detailed estimated ages from the calibrations A-F are written in tabular form.

Time trees from calibrations H and J. Time trees from the calibration H and J are shown.

Estimated ages from the calibrations G-J. Detailed estimated ages from the calibrations A-F are written in tabular form.

Information of sequence data and substitution models. Data partitions and fitted nucleotide substitution model for each partition in the alignment datasets are shown.

Phylogenetic trees from our data subsets and previous studies. Two ML trees from our data subsets (Aln-2 and 3) and three phylogenetic trees from previous studies.

Information of OTUs and sequence accession numbers. List of taxa used for this study and their sequence accession numbers.

Alignment datasets. All alignment datasets used (Aln-1, 2, 2 d, 3, and 3 d) are provided in nexus format.

Microhylidae is a geographically widespread family of anurans. Although several extensive molecular analyses have attempted to elucidate their subfamilial relationships, and correlate these with Mesozoic and Cenozoic continental drifts, consensus has not been reached. Further, generic level relationships have not been well investigated in some micr...

The endangered frog, Odorrana ishikawae (Anura, Ranidae), is a species endemic to the mami and Okinawa Islands ofthe Ryukyu Archipelago, Japan. Segmentation of these islands has een considered to occur middle or upper Pleistocene. Our morphometric analyses revealed bvious differences between the Amami and Okinawa populations. Two distinct morphotyp...

In this study we sought to clarify the evolutionary relationships and biogeographic history of the bell-ring frog, Buergeria buergeri (family Rhacophoridae), and two congeneric species Buergeria japonica and Buergeria robusta, by analyzing three mitochondrial (mt) genes: 12S rRNA, Cytb, and ND5. Phylogenetic analyses based on gene data showed the m...

Although the crab-eating frog Fejervarya cancrivora is one of the most widely distributed species in Asian region, taxonomic relationships among different populations remain unclarified. In this study, we attempted to elucidate the taxonomic status of F. cancrivora from Indonesian and other Asian populations. Five populations of F. cancrivora from...

We determined the complete nucleotide sequences of mitochondrial (mt) genomes from two dicroglossid frogs, Hoplobatrachus tigerinus (Indian Bullfrog) and Euphlyctis hexadactylus (Indian Green frog). The genome sizes are 20462 bp in H. tigerinus and 20280 bp in E. hexadactylus. Although both genomes encode the typical 37 mt genes, the following uniq...

The endangered anuran species, Odorrana ishikawae, is endemic to only two small Japanese Islands, Amami and Okinawa. To assess the innate immune system in this frog, we investigated antimicrobial peptides in the skin using artificially bred animals. Nine novel antimicrobial peptides containing the C-terminal cyclic heptapeptide domain were isolated...

We determined the complete nucleotide sequence of the mitochondrial (mt) genome of an endangered Japanese frog, Odorrana ishikawae (family Ranidae). We also sequenced partial mt genomes of three other Odorrana and six ranid species to survey the diversity of genomic organizations and elucidate the phylogenetic problems remaining in this frog family...

Consensus on the taxonomic system and phylogenetic relationships for the anuran genus Fejervarya has yet to be established. Morphological characters in this genus are generally unsuitable for species identification. To carry out molecular species identification and solve phylogenetic problems, we collected 67 Fejervarya specimens from 12 Asian coun...

Fifteen enzymes and two blood proteins encoded by 24 presumptive loci were analyzed using starch-gel electrophoresis in 136 frogs of 16 populations of Rana ornativentris and 21 frogs of a sympatric population of Rana japonica, in order to elucidate the degree of geographic divergence of R. ornativentris in Honshu and its genetic relationships to R....

Neobatrachia is a phylogenetically-nested anuran group and contains approximately 85% of amphibian species. It is difficult to analyze the neobatra-chian mitochondrial (mt) genome due to accelerated nucleotide substitution rates and occasional gene rearrangements. To overcome these problems, we designed 15 PCR primers. These primers, together with...

Two new frog species of the genus Euphtyctls, which were shown to be two distinct taxa by mitochondrial DNA analyses, are described from Karnataka State, southwestern India. On the molecular phylogenetic tree, the first new species appears as a sister group with respect to E hexadactylus. The second new species forms a group with E cyanophlyctis. T...

Intraspecific differentiation of the Japanese brown frog Rana japonica was investigated by analyzing nucleotide sequences of the mitochondrial cytochrome b (cyt b) gene in order to clarify phylogenetic relationships among three population groups known to exist in this species. The nucleotide sequences of 447 base pair (bp) segments were determined...

In order to elucidate the nucleotide sequence divergence of mitochondrial DNAs between two Japanese pond frog species Rana nigromaculata and R. brevipoda, and the mode of inheritance of cytoplasmic genomes in the female hybrid lineages of the two species, the cleavage patterns of mtDNAs digested with 10 restriction endonucleases were examined by ag...

In the present study, morphological examinations, crossing experiments and molecular analyses were performed to elucidate the degree of genetic divergence and phylogenetic relationships within the genus Fejervarya from Bangladesh and other Asian countries. Morphological characteristics revealed that Fejervarya species from Bangladesh were divided i...

In Malagasy frogs of the family Mantellidae, the genus Mantella is known to possess highly reorganized mitochondrial (mt) genomes with the following characteristics: 1) some rearranged gene positions, 2) 2 distinct genes and a pseudogene corresponding to the transfer RNA gene for methionine (trnM), and 3) 2 control regions (CRs) with almost identic...

To elucidate the species composition, genetic divergence, evolutionary relationships, and divergence time of Hoplobatrachus and Euphlyctis frogs (subfamily Dicroglossinae, family Ranidae) in Bangladesh and other Asian countries, we analyzed the mitochondrial Cyt b, 12S, and 16S rRNA genes of 252 specimens. Our phylogenetic analyses showed 13 major...

To clarify the genetic divergence in the F. limnocharis complex from Thailand and neighboring countries and to elucidate the phylogenetic problems of this taxon, we analyzed partial sequences of the mitochondrial 12S and 16S rRNA genes and the nuclear CXCR4, NCX1, RAG-1, and tyrosinase genes. The F. limnocharis complex from Thailand had three disti...

The present study was conducted to elucidate the genetic divergence and the phylogenetic relationships in the F. limnocharis complex from Bangladesh and other Asian countries such as Sri Lanka, Thailand, Malaysia, Taiwan and Japan by allozyme analyses. We used a total of 95 frogs of the F. limnocharis complex from these countries and F. cancrivora...

We sequenced the whole mitochondrial (mt) genomes of three neobatrachian species: Japanese tree frog Hyla japonica, Japanese common toad Bufo japonicus, and narrow-mouthed toad Microhyla okinavensis. The gene arrangements of these genomes diverged from that of basal anurans (suborder Archaeobatrachia), but are the same as that of the members of der...

In order to elucidate the taxonomic status of the Fejervarya limnocharis complex relative to Malaysia and Japan populations, morphological observations and molecular phylogenetic analysis were carried out using three populations from Indonesia (type locality), Malaysia, and Japan. In addition, we conducted histological and spermatogenic observation...

We compared six taxa of the genus Fejervarya from central Western Ghats, southwestern India, including F. rufescens, F. sahyadris, and four taxa that possess distinct mtDNA haplotypes as demonstrated by our previous studies. Morphological comparisons with F. brevipalmata, F. keralensis, F. nilagirica, and F. syhadrensis on the basis of literature d...

The rice frog (Fejervarya limnocharis) species complex is widely distributed, from India to Japan, and most prevalently in Southeast Asia. Conspicuous morphological variation has been reported for this species complex throughout its distribution range. In the present study, we used mtDNA gene sequence and allozyme analyses to infer evolutionary aff...