Md. Mukhlesur Rahman Khan

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.

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.

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.

A new species of genus Hoplobatrachus (Anura, Dicroglossidae) from the coastal belt of Bangladesh

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 edge of the upper jaw, another black band along the lateral margin of the upper jaw, and a narrow inter-orbital distance relative to eyelid width and inter-nostril distance. Advertisement calls of the new species are similar to those of H. tigerinus but differ in dominant frequency and number of pulses. Based on mitochondrial DNA sequence data, this species was proved to genetically divergent from H. tigerinus at 3.2% for the 16S rRNA gene and 14.2% for the Cytb gene. The known distribution range of the new species is restricted to the southeastern corner of Bangladesh and it seems to be endemic in this coastal belt.

Genetic divergences of South and Southeast Asian frogs: a case study of several taxa based on 16S ribosomal RNA gene data with notes on the generic name Fejervarya

Mahmudul HASAN, Mohammed Mafizul ISLAM, Md. Mukhlesur Rahman KHAN, Takeshi IGAWA, Mohammad Shafiqul ALAM, Hon Tjong DJONG, Nia KURNIAWAN, Hareesh JOSHY, Yong Hoi SEN, Daicus M. BELABUT, Atsushi KURABAYASHI, Mitsuru KURAMOTO, Masayuki SUMIDA* Institute for Amphibian Biology, Graduate School of Science, Hiroshima University, Higashihiroshima, Japan Department of Fisheries Biology and Genetics, Faculty of Fisheries, Bangladesh Agricultural University, Mymensingh, Bangladesh Department of Genetics and Fish Breeding, Faculty of Fisheries, Bangabandhu Sheikh Mujibur Rahman Agricultural University, Gazipur 1706, Bangladesh Department of Biology, Faculty of Mathematics and Science, Andalas University, Padang, West Sumatra, Indonesia Department of Biology, Faculty of Mathematics and Science, Brawijawa University, Malang, East Java, Indonesia Laboratory of Applied Biology, St. Aloysius College, Mangalore, Karnataka, India Institute of Biological Sciences, Faculty of Science, University of Malaya, Kuala Lumpur, Malaysia 3-6-15 Hikarigaoka, Munakata, Fukuoka, Japan

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.