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.

Molecular phylogeny of the diversified frogs of genus Fejervarya (Anura: Dicroglossidae).

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 countries and sequenced part of the mitochondrial (mt) Cytb gene. We also sequenced the mt 12S and 16S rRNA genes and seven nuclear genes (BDNF, CXCR4, NCX1, RAG-1, RAG-2, Rhod, and Tyr) for 25 Fejervarya taxa. These molecular markers appear to be adequate for the identification of species. We subjected the molecular data molecular to phylogenetic analyses. In the resulting trees, topotypic F. limnocharis and "F. multistriata" (from China) formed a clade. On the other hand, neither "F. limnocharis" from the Japan mainland nor "F. limnocharis" from eastern Taiwan formed a clade with the real F. limnocharis, and the genetic divergences were larger than the species threshold for frog taxa proposed in previous studies (> 3% for 16S). These results may suggest that "F. multistriata" is a junior synonym of F. limnocharis, or that only some of the populations now recognized as "F. multistriata" correspond to F. limnocharis. Our results also suggest that several cryptic species may be included among the widely distributed Fejervarya species. Finally, our datasets support paraphyly for the genus Fejervarya, although alternative phylogenetic topologies, including Fejervarya monophyly, were not rejected by KH and SH tests.

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 divergences and phylogenetic relationships among the Fejervarya limnocharis complex in Thailand and neighboring countries revealed by mitochondrial and nuclear genes.

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 distinct haplotypes for 12S and 16S rRNA genes. Nucleotide similarities and the phylogenetic relationships indicated that the haplotype 1 group corresponded to the real "F. limnocharis", the haplotype 2 group was F. orissaensis or closely related to it, and the haplotype 3 group was possibly an undescribed species. Mitochondrial gene data also showed two major clades of the genus Fejervarya, the Southeastern and South Asian groups. Although F. orissaensis is so far known only from Orissa in India, the haplotype 2 group was observed in Thailand. This distribution pattern and the phylogeny suggested that the origin of F. orissaensis and the haplotype 2 group might lie in Southeast Asia. There was also evidence suggesting that the haplotype 3 group originated in the South Asian area and has spread to northern Thailand. The nuclear gene data did not support the monophyly of the haplotypes recognized by mitochondrial genes. This incongruence between the mitochondrial and nuclear data seems to be caused by ancestral polymorphic sites contained in nuclear genes. Although neither the mitochondrial nor the nuclear data clarified intergeneric relationships, the nuclear data rejected the monophyly of the genus Fejervarya.

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.

Morphological divergence, reproductive isolating mechanism, and molecular phylogenetic relationships among Indonesia, Malaysia, and Japan populations of the Fejervarya limnocharis complex (Anura, Ranidae).

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% (xbar=0.2%) and 0.3-1.5% (xbar=1.0%), respectively, between the Malaysia and Indonesia populations, and 2.4-2.6% (xbar=2.5%) and 11.0-12.0% (xbar=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.