Midori Nishioka

Aspects of the Reproductive Biology of Japanese Anurans

Seventeen anurans occur on the four main islands of Japan and on the Islands of Tsushima situated between Japan and Korea. Two of these, Rana nigromaculata and R. brevipoda, are pond frogs allied to Rana esculenta.1 Six other species, Rana japonica, R. ornativentris, R. tagoi, R. chensinensis, R. tsushimensis and R. dyboswkii, are brown frogs allied to Rana temporaria, although Rana tagoi differs from the others in many respects. In addition to these species, two small, dark-colored frogs, Rana rugosa and R. limnocharis, and a large American frog, Rana catesbeiana, are widely distributed in Japan. In other genera Hyla arborea japonica, Bufo bufo japonicus, Bufo torrenticola and three species of Rhacophorus occur. Although it is generally believed that Bombina orientalis occurs on Tsushima, no one has recently found it there. During the last ten years, we have made a number of studies of reproductive biology using most of the Japanese anurans and some foreign ones.

Inter- and intraspecific evolutionary relationships of the rice frog Rana limnocharis and the allied species R. cancrivora inferred from crossing experiments and mitochondrial DNA sequences of the 12S and 16S rRNA genes

The rice frog Rana limnocharis is widely distributed in Southeast Asia and the rest of the Asian region extending from India to Japan. In Japan, the Sakishima-island populations of this species were regarded as a distinct species based on morphological and genetic divergences. The main purposes of this study were to confirm the presence of intraspecific reproductively isolating mechanisms in the Sakishima-island populations of R. limnocharis, and to clarify molecular inter- and intraspecific relationships of R. limnocharis and an allied species, Rana cancrivora. The hybridization experiments revealed that there were no reproductively isolating mechanisms between the Sakishima-island populations and other populations of R. limnocharis. The molecular evolutionary relationships were investigated by analyzing nucleotide sequences of the mitochondrial 12S and 16S rRNA genes using 12 populations of R. limnocharis from Japan and Taiwan, and two populations of R. cancrivora from Thailand and the Philippines. The phylogenetic trees constructed by the NJ method showed that the two populations of R. cancrivora were clearly separated from the 12 populations of R. limnocharis, and that the 12 populations of R. limnocharis were broadly divided into three clades; the first comprising eight populations from the main islands of Japan, the second comprising the Sakishima-island populations, and the third comprising the Okinawa-island and Taiwan populations. Interestingly, the Okinawa-island and Taiwan populations of R. limnocharis showed a close relationship that possibly reflected a secondary contact between the two populations. Based on the present crossing experiments and molecular data, it seems reasonable to regard the Sakishima-island populations as a single subspecies of R. limnocharis.

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

Abstract 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.

Sex-linked genes and linkage maps in amphibians.

This paper reviews sex-linked genes and linkage maps in amphibians. It appears that there is no common ancestral or conserved sex-linkage group in amphibians, whereas an important proportion of other linkage groups has been conserved in amphibians. Comparisons of amphibian linkage maps with those of fishes and mammals reveal several syntenic associations apparently conserved over a very long period of vertebrate divergence.

Reproductive isolating mechanisms and molecular phylogenetic relationships among Palearctic and Oriental brown frogs.

Abstract Crossing experiments were made among various brown frog species and populations collected from Japan, China, Russia and Taiwan. The main purpose of these experiments was to confirm the existence of reproductive isolating mechanisms among Rana pirica from Japan, R. chensinensis from China and R. chensinensis from Russia, and between these three taxa and the other brown frogs distributed in the Palearctic and Oriental regions. It was found that there was no or a slight gametic isolation among the three taxa. While there was a nearly equal number of male and female offspring in the control groups, the hybrid frogs were all males, and completely sterile upon attaining sexual maturity. Thus, each of the Japanese R. pirica and the Russian R. chensinensis is a valid species, distinct from the Chinese R. chensinensis. The phylogenetic tree based on nucleotide sequence data from the mitochondrial 12S and 16S rRNA genes of the Palearctic and Oriental brown frogs showed that the three taxa are included in a cluster together with the other species with 2n=24 chromosomes. The present crossing experiments and molecular data support the hypothesis that each of them is a separate but closely related species.

Genetic Relationships and Reproductive-isolation Mechanisms among the Fejervarya limnocharis Complex from Indonesia (Java) and Other Asian Countries

Abstract 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.

Genetic Divergence and Reproductive Isolation in the Genus Fejervarya (Amphibia: Anura) from Bangladesh Inferred from Morphological Observations, Crossing Experiments, and Molecular Analyses

Abstract 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 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 Nels 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.

Morphological Divergence, Reproductive Isolating Mechanism, and Molecular Phylogenetic Relationships Among Indonesia, Malaysia, and Japan Populations of the Fejervarya limnocharis Complex (Anura, Ranidae)

Abstract 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 observations using hybrids among these populations. Principal component and cluster analyses demonstrated that these populations could be clearly separated from one another. Abnormal testes were found in the hybrids between the Japan and Indonesia populations and between the Japan and Malaysia populations, but testes of the controls and hybrids between the Malaysia and Indonesia populations were quite normal. The mean number of univalents per cell was 5.42, 4.58, and 0.20 in hybrids between the Indonesia and Japan populations, Malaysia and Japan populations, and Indonesia and Malaysia populations, respectively. Sequence divergences in 16S rRNA and Cyt b genes were 0–0.4% (x̄=0.2%) and 0.3–1.5% (x̄=1.0%), respectively, between the Malaysia and Indonesia populations, and 2.4–2.6% (x̄=2.5%) and 11.0–12.0% (x̄=11.5%) between the Japan population and F. limnocharis complex, including the Malaysia and Indonesia populations and F. multistriata from China. This study indicated that the Malaysia population and F. multistriata from China should be designated as a subspecies of topotypic F. limnocharis, and that the Japan population should be regarded as a distinct species.

Genetic Variation and Population Divergence in the Mountain Brown Frog Rana ornativentris

Abstract 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. japonica. The UPGMA dendrogram constructed from Neis genetic distances showed that R. ornativentris from Honshu was divided into two distinct groups, western and eastern, and that the latter split into three subgroups, southern, central and northern. Genetic divergence was distinct between western and eastern populations of R. ornativentris at three loci, PEP-A, SOD-1 and Hb-1, with the Fst values of Wright of 0.624, 0.635 and 0.876, respectively. The average value of Fst (Fst), excluding the five invariant loci, was 0.306. Neis genetic distances among the four western populations of R. ornativentris were 0.015∼0.061, 0.043 on average. Those among the 12 eastern populations were 0.011∼0.179, 0.063 on average, whereas those between the four western and 12 eastern populations were 0.128∼0.313, 0.225 on average. The genetic distances between the 16 populations of R. ornativentris and one population of R. japonica were 0.579∼0.956, 0.793 on average. The UPGMA dendrogram showed that R. ornativentris was distinctly separated from R. japonica.