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Igawa T.,Hiroshima University | Nozawa M.,National Institute of Genetics | Nozawa M.,Graduate University for Advanced Studies | Nagaoka M.,Prefectural University of Hiroshima | And 4 more authors.
Journal of Heredity | Year: 2015

The endangered Ryukyu tip-nosed frog Odorrana narina and its related species, Odorrana amamiensis, Odorrana supranarina, and Odorrana utsunomiyaorum, belong to the family Ranidae and are endemically distributed in Okinawa (O. narina), Amami and Tokunoshima (O. amamiensis), and Ishigaki and Iriomote (O. supranarina and O. utsunomiyaorum) Islands. Because of varying distribution patterns, this species complex is an intrinsic model for speciation and adaptation. For effective conservation and molecular ecological studies, further genetic information is needed. For rapid, cost-effective development of several microsatellite markers for these and 2 other species, we used next-generation sequencing technology of Ion Torrent PGM". Distribution patterns of repeat motifs of microsatellite loci in these modern frog species (Neobatrachia) were similarly skewed. We isolated and characterized 20 new microsatellite loci of O. narina and validated cross-amplification in the three-related species. Seventeen, 16, and 13 loci were cross-amplified in O. amamiensis, O. supranarina, and O. utsunomiyaorum, respectively, reflecting close genetic relationships between them. Mean number of alleles and expected heterozygosity of newly isolated loci varied depending on the size of each inhabited island. Our findings suggested the suitability of Ion Torrent PGM" for microsatellite marker development. The new markers developed for the O. narina complex will be applicable in conservation genetics and molecular ecological studies. © The American Genetic Association 2014. All rights reserved.


Igawa T.,Hiroshima University | Oumi S.,Section of Agriculture and Forest | Katsuren S.,Okinawa Prefectural Institute of Health and Environment | Sumida M.,Hiroshima University
Heredity | Year: 2013

Isolation by distance and landscape connectivity are fundamental factors underlying speciation and evolution. To understand how landscapes affect gene flow and shape population structures, island species provide intrinsic study objects. We investigated the effects of landscapes on the population structure of the endangered frog species, Odorrana ishikawae and O. splendida, which each inhabit an island in southwest Japan. This was done by examining population structure, gene flow and demographic history of each species by analyzing 12 microsatellite loci and exploring causal environmental factors through ecological niche modeling (ENM) and the cost-distance approach. Our results revealed that the limited gene flow and multiple-population structure in O. splendida and the single-population structure in O. ishikawae were maintained after divergence of the species through ancient vicariance between islands. We found that genetic distance correlated with geographic distance between populations of both species. Our landscape genetic analysis revealed that the connectivity of suitable habitats influences gene flow and leads to the formation of specific population structures. In particular, different degrees of topographical complexity between islands are the major determining factor for shaping contrasting population structures of two species. In conclusion, our results illustrate the diversification mechanism of organisms through the interaction with space and environment. Our results also present an ENM approach for identifying the key factors affecting demographic history and population structures of target species, especially endangered species. © 2013 Macmillan Publishers Limited All rights reserved.


Kurabayashi A.,Hiroshima University | Nishitani T.,Hiroshima University | Katsuren S.,Okinawa Prefectural Institute of Health and Environment | Oumi S.,Section of Agriculture and Forest | Sumida M.,Hiroshima University
Genes and Genetic Systems | Year: 2012

Crocodile newts, which constitute the genera Echinotriton and Tylototriton, are known as living fossils, and these genera comprise many endangered species. To identify mitochondrial (mt) genes suitable for future population genetic analyses for endangered taxa, we determined the complete nucleotide sequences of the mt genomes of the Japanese crocodile newt Echinotriton andersoni and Himalayan crocodile newt Tylototriton verrucosus. Although the control region (CR) is known as the most variable mtDNA region in many animal taxa, the CRs of crocodile newts are highly conservative. Rather, the genes of NADH dehydrogenase subunits and ATPase subunit 6 were found to have high sequence divergences and to be usable for population genetics studies. To estimate the inter-population divergence ages of E. andersoni endemic to the Ryukyu Islands, we performed molecular dating analysis using whole and partial mt genomic data. The estimated divergence ages of the inter-island individuals are older than the paleogeographic segmentation ages of the islands, suggesting that the lineage splits of E. andersoni populations were not caused by vicariant events. Our phylogenetic analysis with partial mt sequence data also suggests the existence of at least two more undescribed species in the genus Tylototriton. We also found unusual repeat sequences containing the 3′ region of cytochrome apoenzyme b gene, whole tRNA-Thr gene, and a noncoding region (the T-P noncoding region characteristic in caudate mtDNAs) from T. verrucosus mtDNA. Similar repeat sequences were found in two other Tylototriton species. The Tylototriton taxa with the repeats become a monophyletic group, indicating a single origin of the repeat sequences. The intra-and inter-specific comparisons of the repeat sequences suggest the occurrences of homologous recombination-based concerted evolution among the repeat sequences.


Komaki S.,Hiroshima University | Igawa T.,Hiroshima University | Nozawa M.,National Institute of Genetics | Lin S.-M.,National Taiwan Normal University | And 2 more authors.
Genes and Genetic Systems | Year: 2014

Buergeria japonica is a common frog species distributed throughout almost all islands in Ryukyu Archipelago. Because of their exceptionally wide distribution and higher physiological tolerance comparing to the other anurans, their demographic history and formation of distribution are intrinsic topics in the herpetological fauna of Ryukyu. Microsatellite marker is ideal genetic marker for such studies at inter- and intra-population level. We therefore developed microsatellite markers of B. japonica utilizing Ion PGM™ sequencing. As a result of the screening, we developed a total of 14 polymorphic markers. To test availabilities of these markers, we genotyped four island populations. The total number of alleles and expected hetelozygosities per locus ranged from 4 to 21 and 0.00 to 0.864, respectively. The phylogenetic relationship among the four populations based on the genetic distances of these markers was congruent with general divergence pattern of amphibians and reptiles in Ryukyu area. These markers developed in this study are considered to be useful for future studies about phylogeography and demography of this species.


Kuramoto M.,3 6 15 Hikarigaoka | Satou N.,Hiroshima University | Oumi S.,Section of Agriculture and Forest | Kurabayashi A.,Hiroshima University | Sumida M.,Hiroshima University
Zootaxa | Year: 2011

The endangered frog, Odorrana ishikawae (Anura, Ranidae), is a species endemic to the mami and Okinawa Islands ofthe Ryukyu Archipelago, Japan. Segmentation of these islands has een considered to occur middle or upper Pleistocene. Our morphometric analyses revealed bvious differences between the Amami and Okinawa populations. Two distinct morphotypes were also recognized from the Amami Island (Amami common and Amami large types). Furthermore, the Amami and Okinawa populations could be distinguished clearly by coloration and dorsal uberculation. Based on 16SrRNA gene data, the Okinawa and Amami populations were phylogenetically separated but the genetic divergence (1.44-2.16%) was lower than the value suggested as species threshold in anurans (> 3% in 16S). Individuals of the Amami common and large types were nested within a single clade. Artificial hybridization experiments revealed normal hybrid viability between the two Amami types, with one exception. By contrast, between Okinawa females and two Amami type males, complete hybrid inviability was observed at early embryonic stages in the hybrids contrary to expectations from their low divergence in 16S. The reciprocal hybrids between two Amami type females and Okinawa males were viable, but spermatogenesis in the hybrid males showed some degree of abnormality. These results strongly indicate specific separation of the Amami population from the Okinawa population of O. ishikawae. Thus, we describe the Amami population as a new species, which is readily distinguishable from O. ishikawae by smaller ruggedly edged dorsal spots and an immaculate ventral surface. Copyright © 2011.


Igawa T.,Hiroshima University | Okuda M.,Hiroshima University | Oumi S.,Section of Agriculture and Forest | Katsuren S.,Okinawa Prefectural Institute of Health and Environment | And 3 more authors.
Conservation Genetics Resources | Year: 2011

Due to an originally small distribution range and over-exploitation of primary forest, Ishikawa's frog (Odorrana ishikawae) has been steadily declining in number. For effective conservation of this species, a greater amount of genetic information of this species is needed. Here, we isolated and characterized 12 microsatellite loci of O. ishikawae using two different methods. Loci were screened from two populations on the Amami (N = 44) and Okinawa (N = 9) Islands. The total number of alleles per locus ranged from 2 to 30, and the observed heterozygosity ranged from 0 to 0.922. Notably, high F ST values for all examined loci were observed between the two populations. Taken together, our findings suggest that these novel loci will be applicable for conservation genetic studies across varying scales. © Springer Science+Business Media B.V. 2010.


Sugawara H.,Hiroshima University | Igawa T.,Hiroshima University | Yokogawa M.,Kyoto University | Okuda M.,Hiroshima University | And 6 more authors.
Conservation Genetics Resources | Year: 2012

Due to an originally small distribution range and recent habitat loss, Anderson's crocodile newt (Echinotriton andersoni) has been steadily declining in number. For effective conservation of this species, a greater amount of genetic information is needed. Here, we isolated ten microsatellite loci of E. andersoni using three different methods, and polymorphism of these 10 loci were evaluated for 27 individuals collected from three islands. The total number of alleles per locus ranged from 3 to 22, and the expected heterozygosity ranged from 0 to 0.876. Taken together, our findings suggest that these novel loci will be applicable for conservation genetic studies across varying scales. © 2012 Springer Science+Business Media B.V.


Kurabayashi A.,Hiroshima University | Yoshikawa N.,Hiroshima University | Sato N.,Hiroshima University | Hayashi Y.,Prefectural University of Hiroshima | And 3 more authors.
Molecular Phylogenetics and Evolution | Year: 2010

We determined the complete nucleotide sequence of the mitochondrial (mt) genome of an endangered Japanese frog, Odorrana ishikawae (family Ranidae). We also sequenced partial mt genomes of three other Odorrana and six ranid species to survey the diversity of genomic organizations and elucidate the phylogenetic problems remaining in this frog family. The O. ishikawae mt genome contained the 37 mt genes and single control region (CR) typically found in vertebrate mtDNAs, but the region of Light-strand replication origin (OL) was triplicated in this species. Four protein-encoding genes (atp6, nd2, nd3, and nd5) were found to have high sequence divergence and to be usable for population genetics studies on this endangered species. Among the surveyed ranids, only two species (Rana and Lithobates) manifested the typical neobatrachian-type mt gene arrangement. In contrast, relatively large gene rearrangements were found in Amolops, Babina, and Staurois species; and translocations of single tRNA genes (trns) were observed in Glandirana and Odorrana species. Though the inter-generic and interspecific relationships of ranid taxa remain to be elucidated based on 12S and 16S rrn sequence data, some of the derived mt gene orders were found to have synapomorphic features useful for solving problematic ranid phylogenies. The tandem duplication and random loss (TDRL) model, the traditional model for mt gene rearrangement, failed to easily explain several of the mt gene rearrangements observed here. Indeed, the recent recombination-based gene rearrangement models seemed to be more suitable for this purpose. The high frequency of gene translocations involving a specific trn block (trnH-trnS1) and several single tRNA genes suggest that there may be a retrotranslocation in ranid mt genomes. © 2010 Elsevier Inc. All rights reserved.


Kakehashi R.,Hiroshima University | Kurabayashi A.,Hiroshima University | Oumi S.,Section of Agriculture and Forest | Katsuren S.,Okinawa Prefectural Institute of Health and Environment | And 3 more authors.
Genes and Genetic Systems | Year: 2013

Genus Babina is a member of Ranidae, a large family of frogs, currently comprising 10 species. Three of them are listed as endangered species. To identify mitochondrial (mt) genes suitable for future population genetic analyses for endangered species, we determined the complete nucleotide sequences of the mt genomes of 3 endangered Japanese Babina frogs, B. holsti, B. okinavana, and B. subaspera and 1 ranid frog Lithobates catesbeianus. The genes of NADH dehydrogenase subunit 5 (nad5) and the control region (CR) were found to have high sequence divergences and to be usable for population genetics studies. At present, no consensus on the phylogenetic position of genus Babina has been reached. To resolve this problem, we performed molecular phylogenetic analyses with the largest dataset used to date (11,345 bp from 2 ribosomal RNA- and 13 proteinencoding genes) in studies dealing with Babina phylogeny. These analyses revealed monophyly of Babina and Odorrana. It is well known that mt gene rearrangements of animals can provide usable phylogenetic information. Thus, we also compared the mt gene arrangements among Babina species and other related genera. Of the surveyed species, only L. catesbeianus manifested typical neobatrachian-type mt gene organization. In the B. okinavana, an additional pseudogene of tRNA-His (trnH) was observed in the CR downstream region. Furthermore, in the B. holsti and B. subaspera, the trnH/nad5 block was translocated from its typical position to the CR downstream region, and the translocated trnH became a pseudogene. The position of the trnH pseudogene is consistent with the translocated trnH position reported in Odorrana. Consequently, the trnH rearrangement seems to be a common ancestry characteristic (synapomorphy) of Babina and Odorrana. Based on the "duplication and deletion" gene rearrangement model, a single genomic duplication event can explain the order of derived mt genes found in Babina and Odorrana.

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