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.

Composition of the Cutaneous Bacterial Community in Japanese Amphibians: Effects of Captivity, Host Species, and Body Region.

The cutaneous microbiota plays a significant role in the biology of their vertebrate hosts, and its composition is known to be influenced both by host and environment, with captive conditions often altering alpha diversity. Here, we compare the cutaneous bacterial communities of 61 amphibians (both wild and captive) from Hiroshima, Japan, using high-throughput amplicon sequencing of a segment of the 16S rRNA gene. The majority of these samples came from a captive breeding facility at Hiroshima University where specimens from six species are maintained under highly standardized conditions for several generations. This allowed to identify host effects on the bacterial communities under near identical environmental conditions in captivity. We found the structure of the cutaneous bacterial community significantly differing between wild and captive individuals of newts, Cynops pyrrhogaster, with a higher alpha diversity found in the wild individuals. Community structure also showed distinct patterns when comparing different species of amphibians kept under highly similar conditions, revealing an intrinsic host effect. Bacterial communities of dorsal vs. ventral skin surfaces did not significantly differ in most species, but a trend of higher alpha diversity on the ventral surface was found in Oriental fire-bellied toads, Bombina orientalis. This study confirms the cutaneous microbiota of amphibians as a highly dynamic system influenced by a complex interplay of numerous factors.

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.

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. 

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.

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.

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.

Taxonomic status of three types of Fejervarya cancrivora from Indonesia and other Asian countries based on morphological observations and crossing experiments.

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 Selangor (Malaysia), Cianjur (Java, Indonesia), Trat (Thailand), Khulna (Bangladesh), and Makassar (Sulawesi, Indonesia) were morphologically observed and subjected to crossing experiments. Principal component and clustering analyses revealed that these five populations could be organized into three groups corresponding to three observed morphological types: a Selangor and Cianjur group (large-type), a Trat and Khulna group (mangrove-type), and a Makassar group (Sulawesi-type). The limited crossing experiments revealed that hybrids between Selangor females and Cianjur and Trat males developed normally, whereas hybrids between Selangor females and Khulna males showed incomplete gametic isolation. Histological observations of the testes of mature males revealed the presence of pycnotic nuclei in the hybrids between Selangor females and Khulna males in addition to normal bundles of spermatozoa. In contrast, no pycnotic nuclei were observed in the Selangor controls. Although meiotic metaphases in the controls were normal, those in hybrids showed several abnormalities, such as the appearance of univalents and an increase in rod-shaped bivalents. Based on our findings from the morphological observations and crossing experiments, we conclude that each of three identified types represents a distinct species. We propose that the large-type is F. cancrivora, the mangrove-type is F. moodiei, and the Sulawesi-type represents an undescribed species.

Genetic divergence and evolutionary relationship in Fejervarya cancrivora from Indonesia and other Asian countries inferred from allozyme and MtDNA sequence analyses.

To elucidate genetic divergence and evolutionary relationship in Fejervarya cancrivora from Indonesia and other Asian countries, allozyme and molecular analyses were carried out using 131 frogs collected from 24 populations in Indonesia, Thailand, Bangladesh, Malaysia, and the Philippines. In the allozymic survey, seventeen enzymatic loci were examined for 92 frogs from eight representative localities. The results showed that F. cancrivora is subdivided into two main groups, the mangrove type and the large- plus Pelabuhan ratu types. The average Nel's genetic distance between the two groups was 0.535. Molecular phylogenetic trees based on nucleotide sequences of the 16S rRNA and Cyt b genes and constructed with the ML, MP, NJ, and BI methods also showed that the individuals of F. cancrivora analyzed comprised two clades, the mangrove type and the large plus Pelabuhan ratu / Sulawesi types, the latter further split into two subclades, the large type and the Pelabuhan ratu / Sulawesi type. The geographical distribution of individuals of the three F. cancrivora types was examined. Ten Individuals from Bangladesh, Thailand, and the Philippines represented the mangrove type; 34 Individuals from Malaysia and Indonesia represented the large type; and 11 individuals from Indonesia represented the Pelabuhan ratu / Sulawesi type. Average sequence divergences among the three types were 5.78-10.22% for the 16S and 12.88-16.38% for Cyt b. Our results suggest that each of the three types can be regarded as a distinct species.

Genetic divergence and evolutionary relationships in six species of genera Hoplobatrachus and Euphlyctis (Amphibia: Anura) from Bangladesh and other Asian countries revealed by mitochondrial gene sequences.

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 clades corresponding to several cryptic species as well as to nominal species in the two genera. The results suggested monophyly of Asian Hoplobatrachus species, but the position of African Hoplobatrachus occipitalis was not clarified. Nucleotide divergence and phylogenetic data suggested the presence of allopatric cryptic species allied to Euphlyctis hexadactylus in Sundarban, Bangladesh and several parapatric cryptic species in the Western Ghats, India. The presence of at least two allopatric cryptic species among diverged Euphlyctis cyanophlyctis in Bangladesh, India, and Sri Lanka was also suggested. In some cases, our estimated divergence times matched the paleogeological events of South and Southeast Asian regions that may have led to the divergence of Hoplobatrachus and Euphlyctis taxa. Especially, land formation at Bangladesh (15-10Ma) may have allowed the spread of these frog taxa to Southeast Asian areas, and the aridification of central India (5.1-1.6Ma) might have affected the gene flow of widely distributed species. The present study revealed prior underestimation of the richness of the amphibian fauna in this region, indicating the possible occurrence of many cryptic species among these groups.

Genetic differentiation of the Fejervarya limnocharis complex from Bangladesh and other Asian countries elucidated by allozyme analyses.

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 from the Philippines as an outgroup. Based on body size, the F. limnocharis complex from Bangladesh was divided into three distinct groups: large, medium and small types. Allozyme analyses were carried out with 28 loci encoding 20 enzymes and two blood proteins by horizontal starch-gel electrophoresis. When genetic distance was calculated, distinct divergence was found among the three types: mean genetic distance was 0.782 between the small and medium types, 1.458 between the large and medium types, and 1.520 between the large and small types. Phylogenetic trees based on genetic distance showed that all populations of Bangladesh small type strongly formed a cluster and were found to be most closely related to the Sri Lanka population; that all populations of Bangladesh large type formed a very strong cluster and were grouped with several populations from Thailand, Malaysia, Japan, and Taiwan; and that the medium type was segregated from all other groups. This may imply that each of the three types is a different species, and that the medium type is possibly an undescribed taxon.

Genetic divergence and reproductive isolation in the genus Fejervarya (Amphibia: Anura) from Bangladesh inferred from morphological observations, crossing experiments, and molecular analyses.

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 into four distinct groups: large, medium, small, and mangrove types. Crossing experiments indicated the involvement of three reproductive isolating mechanisms: gametic isolation between the large type and mangrove type, hybrid inviability between the large type and two other types, and hybrid sterility between the medium and small types. Experimental results also indicated that these four types of frogs merit the status of individual species of Fejervarya . Molecular analyses based on mtDNA gene sequences showed that the Bangladesh Fejervarya species were largely divided into three groups: the mangrove type, large type, and others, with the last further subdivided into the medium and small types. Comparison with other Asian Fejervarya species revealed that the Bangladesh mangrove type (which resembled F. cancrivora in morphology) was closely related to F. cancrivora from India, Thailand, and the Philippines; the large type belonged to the F. iskandari group and closely resembled F. orissaensis ; the small type was included in the South Asian or Indian group, and was closest to F. syhadrensis from India and Sri Lanka, whereas the medium type was most closely related to F. limnocharis from Myanmar among all described species of this genus.

Genetic relationships and reproductive-isolation mechanisms among the Fejervarya limnocharis complex from Indonesia (Java) and other Asian countries.

In order to elucidate the genetic relationships and reproductive-isolation mechanisms among the Fejervarya limnocharis complex from Indonesia and other Asian countries, allozyme analyses and crossing experiments were carried out using 208 individuals from 21 localities in eight Asian countries. The allozyme analyses revealed that 17 enzymes examined were controlled by genes at 27 loci, and that 7.9 phenotypes were produced by 5.2 alleles on average. The two species recognized in F. limnocharis sensu lato from Southeast Asia (i.e., F. limnocharis sensu stricto and F. iskandari) were found to occur sympatrically at three localities (Bogor, Cianjur and Malingping), all on Java, Indonesia. Fejervaya iskandari was dominant at each of these localities and showed substantial geographic genetic variation. Laboratory-produced hybrids between F. limnocharis and F. iskandari from Java became underdeveloped and died at the tadpole stage, suggesting that these species are completely isolated by hybrid inviability. Hybrids between topotypic F. limnocharis and the Malaysian and Japanese conspecific populations developed normally to metamorphosis. Likewise, hybrids between topotypic F. iskandari and the Thailand and Bangladesh conspecific populations also showed normal viability throughout larval development. The present allozyme analyses and crossing experiments strongly suggested the presence of two distinct forms, the large type and the small type, in the F. limnocharis complex from Asia, and further subdivision of the large type into the F. limnocharis assemblage and the F. iskandari assemblage. The small type was found in samples from India, Thailand, Bangladesh and Sri Lanka, and included at least three different species. The sample from Pilok, Thailand, was considered to represent an undescribed species.

Evolutionary relationships and reproductive isolating mechanisms in the rice frog (Fejervarya limnocharis) species complex from Sri Lanka, Thailand, Taiwan and Japan, inferred from mtDNA gene sequences, allozymes, and crossing experiments.

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 affinities within this species complex using eight populations (Sri Lanka; Bangkok and Ranong in Thailand; Taiwan; and Hiroshima, Okinawa, Ishigaki and Iriomote in Japan). We also conducted crossing experiments among four populations from Japan, Thailand, and Sri Lanka in order to find out more about the reproductive isolating mechanisms that might exist among the East, Southeast, and South Asian populations of this species complex. The crossing experiments revealed that the Sri Lanka population is reproductively isolated from the Hiroshima, Bangkok, and Ranong populations by complete hybrid inviability, and that the Bangkok population may be reproductively isolated from the Hiroshima population by partial hybrid inviability. Thus, it is not unreasonable to regard the Sri Lanka population as a species separated from F. limnocharis. The mtDNA and allozyme data showed that the Ranong population is most closely related to the Bangkok population in nuclear genome, but more similar to the Okinawa and Taiwan populations in mtDNA genome. The present, preliminary survey may raise questions about the species status of these particular populations and also about the nature of the biological species concept.