Postmating isolation and evolutionary relationships among Fejervarya species from Lesser Sunda, Indonesia and other Asian countries revealed by crossing experiments and mtDNA Cytb sequence analyses.

To evaluate the degree of postmating isolation and the evolutionary relationships among frog species in the genus Fejervarya from Indonesia (Lesser Sunda), Bangladesh, China, and Japan, crossing experiments and molecular phylogenetic analyses were carried out. Crossing experiments revealed that reciprocal hybrids among F. iskandari, F. verruculosa, and F. sp. large type and between F. multistriata and F. kawamurai are viable through metamorphosis, while those between the F. iskandari group and F. limnocharis group were completely or partially inviable at the tadpole stage and those between Southeast Asian and South Asian Fejervarya groups were completely inviable at the embryonic stage. The mature reciprocal hybrids between F. iskandari and F. verruculosa from Lesser Sunda, Indonesia, showed some degree of abnormality in spermatogenesis. In phylogenetic analyses based on mtDNA Cytb sequences, F. iskandari formed a sister clade with F. verruculosa from Lesser Sunda, Indonesia, with 8.1% sequence divergence. F. multistriata from China formed a clade with populations of F. limnocharis in Thailand, Malaysia, and Indonesia (topotype), and these taxa showed sister relationships to F. kawamurai from Japan with 8.9% sequence divergence. Fejervarya sp. small type from Bangladesh formed a clade with the other South Asian members of the Fejervarya group and formed a sister clade with the Southeast Asian Fejervarya group, with 23.1% sequence divergence in the Cytb gene. These results showed that the degree of postmating isolation reflects molecular phylogenetic relationships and that F. iskandari and F. verruculosa from Indonesia (Lesser Sunda) are reproductively isolated by abnormalities in spermatogenesis and show genetic differentiation.

Effect of region-wide use of prehospital stroke triage scale on management of patients with acute stroke.

BACKGROUND: Prehospital stroke triage scales help with the decision to transport patients with suspected stroke to suitable hospitals. OBJECTIVE: To explore the effect of the region-wide use of the Japan Urgent Stroke Triage (JUST) score, which can predict several types of stroke: large vessel occlusion (LVO), intracranial hemorrhage (ICH), subarachnoid hemorrhage (SAH), and cerebral infarction other than LVO (CI). METHODS: We implemented the JUST score and conducted a retrospective and prospective multicenter cohort study at 13 centers in Hiroshima from April 1, 2018, to March 31, 2020. We investigated the success rate of the first request to the hospital, on-scene time, and transport time to hospital. We evaluated the door-to-puncture time, puncture-to-reperfusion time, and 90-day outcome among patients with final diagnoses of LVO. RESULTS: The cohort included 5141 patients (2735 before and 2406 after JUST score implementation). Before JUST score implementation, 1269 strokes (46.4%) occurred, including 140 LVO (5.1%), 394 ICH (14.4%), 120 SAH (4.4%), and 615 CI (22.5%). The JUST score was used in 1484 (61.7%) of the 2406 patients after implementation, which included 1267 (52.7%) cases of stroke (186 LVO (7.7%), 405 ICH (16.8%), 109 SAH (4.5%), and 567 CI (23.6%)). Success rate of the first request to the hospital significantly increased after JUST score implementation (76.3% vs 79.7%, p=0.004). JUST score implementation significantly shortened the door-to-puncture time (84 vs 73 min, p=0.03), but the prognosis remained unaltered among patients with acute LVO. CONCLUSIONS: Use of prehospital stroke triage scales improved prehospital management and preparation time of intervention among patients with acute stroke.

Inter- and intra-specific genetic divergence of Asian tiger frogs (genus Hoplobatrachus), with special reference to the population structure of H. tigerinus in Bangladesh.

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 conducted molecular phylogenetic analysis on Asian Hoplobatrachus frogs and population genetic analysis on H. tigerinus in Bangladesh using the mitochondrial CYTB gene and 21 microsatellite markers. The resultant phylogenetic tree revealed monophyly in each species, notwithstanding the involvement of cryptic species in H. chinensis and H. tigerinus, which are evident from the higher genetic divergence between populations. Bayesian inference of population structure revealed genetic divergence between western and eastern H. tigerinus populations in Bangladesh, suggesting restricted gene flow caused by barriers posed by major rivers. However, genetic distances among populations were generally low. A discrete population is located in the low riverine delta region, which likely reflects long-distance dispersal. These results strongly suggest that the environment specific to this river system has maintained the population structure of H. tigerinus in this region.

The first see-through frog created by breeding: description, inheritance patterns, and dermal chromatophore structure.

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 gray-eyed) frogs through artificial insemination, and F2 offspring produced frogs whose skin is translucent throughout the life cycle. Three kinds of dermal chromatophores--xanthophores, iridophores, and melanophores--are observed in a layered arrangement in the skin of wild-type frogs, but few chromatophores were present in the skin of the see-through frogs. The translucent skin enables observation of organ growth and cancer formation and progression in the animal, which can be monitored over its entire life without the need for dissection. See-through frogs thus provide a useful animal model for environmental, medical, and biological research.

Inbreeding Ratio and Genetic Relationships among Strains of the Western Clawed Frog, Xenopus tropicalis.

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 Nigerian and Ivory Coast strains. However, due to its short history as an experimental animal, the genetic relationship among the various strains has not yet been clarified, and establishment of inbred strains has not yet been achieved. Since 2003 the Institute for Amphibian Biology (IAB), Hiroshima University has maintained stocks of multiple X. tropicalis strains and conducted consecutive breeding as part of the National BioResource Project. In the present study we investigated the inbreeding ratio and genetic relationship of four inbred strains at IAB, as well as stocks from other institutions, using highly polymorphic microsatellite markers and mitochondrial haplotypes. Our results show successive reduction of heterozygosity in the genome of the IAB inbred strains. The Ivory Coast strains clearly differed from the Nigerian strains genetically, and three subgroups were identified within both the Nigerian and Ivory Coast strains. It is noteworthy that the Ivory Coast strains have an evolutionary divergent genetic background. Our results serve as a guide for the most effective use of X. tropicalis strains, and the long-term maintenance of multiple strains will contribute to further research efforts.

Extraordinary Diversity in the Origins of Sex Chromosomes in Anurans Inferred from Comparative Gene Mapping.

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 clawed frog (Xenopus laevis), the Western clawed frog (Silurana/X. tropicalis), the Japanese bell-ring frog (Buergeria buergeri), and the Japanese wrinkled frog (Rana rugosa). Comparative mapping data revealed that the sex chromosomes of X. laevis, X. tropicalis and R. rugosa are different chromosome pairs; however, the sex chromosomes of X. tropicalis and B. buergeri are homologous, although this may represent distinct evolutionary origins. We also examined the status of sex chromosomal differentiation in B. buergeri, which possesses heteromorphic ZW sex chromosomes, using comparative genomic hybridization and chromosome painting with DNA probes from the microdissected W chromosome. At least 3 rearrangement events have occurred in the proto-W chromosome: deletion of the nucleolus organizer region and a paracentric inversion followed by amplification of non-W-specific repetitive sequences.

The Spemann organizer meets the anterior-most neuroectoderm at the equator of early gastrulae in amphibian species.

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 was already established at the equatorial region of very early gastrula in a wide variety of amphibian species. Here we propose a unified model of amphibian gastrulation movement. In the model, the organizer is present at the blastocoel roof of blastulae, moves vegetally to locate at the region that lies from the blastocoel floor to the dorsal lip at the onset of gastrulation. The organizer located at the blastocoel floor contributes to the anterior axial mesoderm including the prechordal plate, and the organizer at the dorsal lip ends up as the posterior axial mesoderm. During the early step of gastrulation, the anterior organizer moves to establish the physical contact with the prospective neuroectoderm through the "subduction and zippering" movements. Subduction makes a trench between the anterior organizer and the prospective neuroectoderm, and the tissues face each other via the trench. Zippering movement, with forming Brachet's cleft, gradually closes the gap to establish the contact between them. The contact is completed at the equator of early gastrulae and it continues throughout the gastrulation. After the contact is established, the dorsal axis is formed posteriorly, but not anteriorly. The model also implies the possibility of constructing a common model of gastrulation among chordate species.

Microsatellite marker development by multiplex ion torrent PGM sequencing: a case study of the endangered Odorrana narina complex of frogs.

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. Because of varying distribution patterns, this species complex is an intrinsic model for speciation and adaptation. For effective conservation and molecular ecological studies, further genetic information is needed. For rapid, cost-effective development of several microsatellite markers for these and 2 other species, we used next-generation sequencing technology of Ion Torrent PGM™. Distribution patterns of repeat motifs of microsatellite loci in these modern frog species (Neobatrachia) were similarly skewed. We isolated and characterized 20 new microsatellite loci of O. narina and validated cross-amplification in the three-related species. Seventeen, 16, and 13 loci were cross-amplified in O. amamiensis, O. supranarina, and O. utsunomiyaorum, respectively, reflecting close genetic relationships between them. Mean number of alleles and expected heterozygosity of newly isolated loci varied depending on the size of each inhabited island. Our findings suggested the suitability of Ion Torrent PGM™ for microsatellite marker development. The new markers developed for the O. narina complex will be applicable in conservation genetics and molecular ecological studies.

Development and characterization of 27 new microsatellite markers for the Indian bullfrog Hoplobatrachus tigerinus and its congeneric species.

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 characterized microsatellite loci for these frogs. We obtained genomic data using an Ion Torrent PGM sequencer and designed 54 primer sets for candidate loci. By screening for polymorphic loci in individuals of H. tigerinus and its congeneric species, we isolated 27 loci as highly polymorphic microsatellite loci. Eight of these loci were commonly applicable for all species except H. chinensis. Within two populations of H. tigerinus, the total number of alleles per locus and expected heterozygosity ranged from 2 to 18 and 0.271 to 0.938, respectively. No significant linkage disequilibrium was observed across all loci, and five showed a significant deviation from Hardy-Weinberg equilibrium in some populations after Bonferroni correction. Consequently, our findings suggest that these novel markers will be applicable for conservation genetic studies across varying scales from inter-population to inter-individual.

Description of two new species of Microhyla (Anura: Microhylidae) from Bangladesh.

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 cytochrome b gene from these new species are substantially diverged from M. fissipes (8.9 and 10.2% [3.6 and 4.2% for 16S ribosomal RNA gene] uncorrected pairwise divergence, respectively), and the estimated phylogenetic splits from their closest relative is in the Pliocene (3.4 Mya) and middle Miocene (10.5 Mya). The first new species (Microhyla mukhlesuri sp. nov.) can be diagnosed from its nearest congener (M. fissipes) by the following characteristics: SVL: 16.5-21.0 mm, finger length 1 < 4 < 2 < 3, tips of finger and toes not swollen, subarticular tubercles distinct, an inverse U-shaped mark on the anus, and a distinct X-shaped marking on the dorsum. Although the second new species (M. mymensinghensis sp. nov.) shares some morphological characteristics with the first new species, it can be readily diagnosed from its close congeners by its longer hindlimbs (HLL/SVL), tibia (TIL/SVL) and forearm width (FAW/SVL), in addition to a combination of the following characteristics: SVL: 14.2-21.3 mm, snout truncate, a crescent-shaped marking on the anus, and an X-shaped marking on the dorsum. The tibio-tarsal articulation extends to the eye in M. fissipes but ranges from the eye to the tip of the snout in the two new species. 

Development and characterization of 14 microsatellite markers for Buergeria japonica (Amphibia, Anura, Rhacophoridae).

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. Microsatellite marker is ideal genetic marker for such studies at inter- and intra-population level. We therefore developed microsatellite markers of B. japonica utilizing Ion PGM™ sequencing. As a result of the screening, we developed a total of 14 polymorphic markers. To test availabilities of these markers, we genotyped four island populations. The total number of alleles and expected hetelozygosities per locus ranged from 4 to 21 and 0.00 to 0.864, respectively. The phylogenetic relationship among the four populations based on the genetic distances of these markers was congruent with general divergence pattern of amphibians and reptiles in Ryukyu area. These markers developed in this study are considered to be useful for future studies about phylogeography and demography of this species.

Afrobatrachian mitochondrial genomes: genome reorganization, gene rearrangement mechanisms, and evolutionary trends of duplicated and rearranged genes.

BACKGROUND: 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 been available. Several molecular models for mitochondrial (mt) gene rearrangements have been proposed, but observational evidence has been insufficient to evaluate them. Furthermore, evolutionary trends in rearranged mt genes have not been well understood. To gain molecular and phylogenetic insights into these issues, we analyzed the mt genomes of four afrobatrachian species (Breviceps adspersus, Hemisus marmoratus, Hyperolius marmoratus, and Trichobatrachus robustus) and performed molecular phylogenetic analyses. Furthermore we searched for two evolutionary patterns expected in the rearranged mt genes of ranoids. RESULTS: Extensively reorganized mt genomes having many duplicated and rearranged genes were found in three of the four afrobatrachians analyzed. In fact, Breviceps has the largest known mt genome among vertebrates. Although the kinds of duplicated and rearranged genes differed among these species, a remarkable gene rearrangement pattern of non-tandemly copied genes situated within tandemly-copied regions was commonly found. Furthermore, the existence of concerted evolution was observed between non-neighboring copies of triplicated 12S and 16S ribosomal RNA regions. CONCLUSIONS: Phylogenetic analyses based on mitogenomic data support a close relationship between Afrobatrachia and Microhylidae, with their estimated divergence 100 million years ago consistent with present-day endemism of afrobatrachians on the African continent. The afrobatrachian mt data supported the first tandem and second non-tandem duplication model for mt gene rearrangements and the recombination-based model for concerted evolution of duplicated mt regions. We also showed that specific nucleotide substitution and compositional patterns expected in duplicated and rearranged mt genes did not occur, suggesting no disadvantage in employing these genes for phylogenetic inference.

Mitochondrial genomes of Japanese Babina frogs (Ranidae, Anura): unique gene arrangements and the phylogenetic position of genus Babina.

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 frogs, B. holsti, B. okinavana, and B. subaspera and 1 ranid frog Lithobates catesbeianus. The genes of NADH dehydrogenase subunit 5 (nad5) and the control region (CR) were found to have high sequence divergences and to be usable for population genetics studies. At present, no consensus on the phylogenetic position of genus Babina has been reached. To resolve this problem, we performed molecular phylogenetic analyses with the largest dataset used to date (11,345 bp from 2 ribosomal RNA- and 13 protein-encoding genes) in studies dealing with Babina phylogeny. These analyses revealed monophyly of Babina and Odorrana. It is well known that mt gene rearrangements of animals can provide usable phylogenetic information. Thus, we also compared the mt gene arrangements among Babina species and other related genera. Of the surveyed species, only L. catesbeianus manifested typical neobatrachian-type mt gene organization. In the B. okinavana, an additional pseudogene of tRNA-His (trnH) was observed in the CR downstream region. Furthermore, in the B. holsti and B. subaspera, the trnH/nad5 block was translocated from its typical position to the CR downstream region, and the translocated trnH became a pseudogene. The position of the trnH pseudogene is consistent with the translocated trnH position reported in Odorrana. Consequently, the trnH rearrangement seems to be a common ancestry characteristic (synapomorphy) of Babina and Odorrana. Based on the "duplication and deletion" gene rearrangement model, a single genomic duplication event can explain the order of derived mt genes found in Babina and Odorrana.

An Attempt at Captive Breeding of the Endangered Newt Echinotriton andersoni, from the Central Ryukyus in Japan.

Anderson's crocodile newt (Echinotriton andersoni) is distributed in the Central Ryukyu Islands of southern Japan, but environmental degradation and illegal collection over the last several decades have devastated the local populations. It has therefore been listed as a class B1 endangered species in the IUCN Red List, indicating that it is at high risk of extinction in the wild. The species is also protected by law in both Okinawa and Kagoshima prefectures. An artificial insemination technique using hormonal injections could not be applied to the breeding of this species in the laboratory. In this study we naturally bred the species, and tested a laboratory farming technique using several male and female E. andersoni pairs collected from Okinawa, Amami, and Tokunoshima Islands and subsequently maintained in near-biotopic breeding cages. Among 378 eggs derived from 17 females, 319 (84.4%) became normal tailbud embryos, 274 (72.5%) hatched normally, 213 (56.3%) metamorphosed normally, and 141 (37.3%) became normal two-month-old newts; in addition, 77 one- to three-year-old Tokunoshima newts and 32 Amami larvae are currently still growing normally. Over the last five breeding seasons, eggs were laid in-cage on slopes near the waterfront. Larvae were raised in nets maintained in a temperature-controlled water bath at 20 °C and fed live Tubifex. Metamorphosed newts were transferred to plastic containers containing wet sponges kept in a temperature-controlled incubator at 22.5 °C and fed a cricket diet to promote healthy growth. This is the first published report of successfully propagating an endangered species by using breeding cages in a laboratory setting for captive breeding. Our findings on the natural breeding and raising of larvae and adults are useful in breeding this endangered species and can be applied to the preservation of other similarly wild and endangered species such as E. chinhaiensis.

Postmating isolation in six species of three genera (Hoplobatrachus, Euphlyctis and Fejervarya) from family Dicroglossidae (anura), with special reference to spontaneous production of allotriploids.

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, although the intergeneric hybrids between female E. cyanophlyctis and male H. chinensis were not viable at the tadpole stage, while the hybrids between female E. cyanophlyctis and male H. tigerinus were inviable at the hatching stage. These results showed complete hybrid inviability between the two genera. Almost all interspecific hybrids between female H. tigerinus and male H. chinensis died of underdevelopment at the tadpole stage, whereas several hybrids developed normally and survived to maturity. Chromosomal observations and mtDNA and allozyme analyses confirmed that these mature hybrids were allotriploid, with two maternal genomes and one paternal genome. The present results suggest that the allotriploids were produced spontaneously, and histological observations confirmed their sex as sterile males. We also investigated the molecular relationships between H. tigerinus, H. chinensis, and the interspecific allotriploids by mitochondrial Cytb, 12S and 16S rRNA gene analyses. The maternal inheritance mode of mitochondrial genomes was retained in the hybrids. Finally, the present results suggest that the degree of postmating isolation reflects phylogenetic relationship. In addition, we speculate that allotriploids may be produced via hybridization among cryptic species.

Distributional change and epidemic introgression in overlapping areas of Japanese pond frog species over 30 years.

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 areas. To clarify the distribution areas and hybrid zones of the frogs, and to understand the mode of introgressive hybridization and its impact on the frog populations, we conducted exhaustive sampling at each basin and performed allozyme and mtDNA analyses of 233 individuals. Analysis using genetic markers clearly detected nine F1 hybrids and 94 hybrid descendants of P. nigromaculatus and P. porosus from the overlapping areas of both basins. Allozyme and mtDNA data suggest directional hybridization between female P. p. porosus and male P. nigromaculatus in the Matsumoto basin. Over the past 30 years, the distribution of P. p. porosus has been narrowed and fragmented by the invasion of P. nigromaculatus, seemingly because of directional hybridization in the Matsumoto basin. In the Ina basin, the "pure" P. p. brevipoda (n = 8) population was extremely reduced by gene introgression from P. nigromaculatus, yet its distribution was barely changed compared to the Matsumoto basin. Consequently, this study shows that P. porosus populations are threatened by interspecific hybridization with P. nigromaculatus, and that introgressive hybridization damaged P. porosus populations by different means in each basin.

Mitochondrial genomes and divergence times of crocodile newts: inter-islands distribution of Echinotriton andersoni and the origin of a unique repetitive sequence found in Tylototriton mt genomes.

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 crocodile newt Echinotriton andersoni and Himalayan crocodile newt Tylototriton verrucosus. Although the control region (CR) is known as the most variable mtDNA region in many animal taxa, the CRs of crocodile newts are highly conservative. Rather, the genes of NADH dehydrogenase subunits and ATPase subunit 6 were found to have high sequence divergences and to be usable for population genetics studies. To estimate the inter-population divergence ages of E. andersoni endemic to the Ryukyu Islands, we performed molecular dating analysis using whole and partial mt genomic data. The estimated divergence ages of the inter-island individuals are older than the paleogeographic segmentation ages of the islands, suggesting that the lineage splits of E. andersoni populations were not caused by vicariant events. Our phylogenetic analysis with partial mt sequence data also suggests the existence of at least two more undescribed species in the genus Tylototriton. We also found unusual repeat sequences containing the 3' region of cytochrome apoenzyme b gene, whole tRNA-Thr gene, and a noncoding region (the T-P noncoding region characteristic in caudate mtDNAs) from T. verrucosus mtDNA. Similar repeat sequences were found in two other Tylototriton species. The Tylototriton taxa with the repeats become a monophyletic group, indicating a single origin of the repeat sequences. The intra-and inter-specific comparisons of the repeat sequences suggest the occurrences of homologous recombination-based concerted evolution among the repeat sequences.

Cryptic anuran biodiversity in Bangladesh revealed by mitochondrial 16S rRNA gene sequences.

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 from two districts diverged considerably, indicating the involvement of a cryptic species. Two Fejervarya sp. (large and medium types) and Hylarana cf. taipehensis formed lineages distinct from related species and are probably new species. Microhyla cf. ornata differed from M. ornata with respect to type locality area and involved two distinct species. In addition, we found that Hylarana sp. and Microhyla sp. did not match congeners examined to date in either morphology or 16S rRNA sequence. The occurrence of M. fissipes was tentatively suggested. Consequently, at least, 19 species were found from Bangladesh in this study. These findings revealed a rich anuran biodiversity in Bangladesh, which is unexpected considering the rather simple topographic features of the country.

A new species of the Fejervarya limnocharis complex from Japan (Anura, Dicroglossidae).

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 snout-vent length, and from F. multistriata by relatively shorter forelimb, hindlimb, foot, and tibia. From F. limnocharis and F. iskandari, it is differentiated by relatively smaller forelimb, hindlimb, foot, and tibia lengths. Taxonomic problems of Fejervarya populations occurring in Central Ryukyus, continental China, and Taiwan are discussed.

Artificial production and natural breeding of the endangered frog species Odorrana ishikawae, with special reference to fauna conservation in the laboratory.

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 also promoted natural breeding in the laboratory. Field-caught male/female pairs of the Amami and Okinawa Island populations were artificially bred using an artificial insemination method in the 2004, 2006, and 2008 breeding seasons (March to April). Although fewer than 50% of the inseminated eggs achieved metamorphosis, approximately 500, 300, and 250 offspring from the three respective trials are currently being raised in the laboratory. During the 2009 and 2010 breeding seasons, second-generation offspring were produced by the natural mating activities of the first offspring derived from the two artificial matings in 2004. The findings and the methods presented here appear to be applicable to the temporary protection of genetic diversity of local populations in which the number of individuals has decreased or the environmental conditions have worsened to levels that frogs are unable to survive by themselves.