Mohammad Shafiqul Alam

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 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.

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

Abstract 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 Neis (1972) 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.

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.