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.

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.

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 tRNAThr 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 intraand inter-specific comparisons of the repeat sequences suggest the occurrences of homologous recombination-based concerted evolution among the repeat sequences.

No abstract

Simple SummaryWe naturally bred the endangered Anderson’s crocodile newt (Echinotriton andersoni) and tested a laboratory farming technique using near-biotopic breeding cages with several male and female pairs collected from Okinawa, Amami, and Tokunoshima Islands. This is the first published report of successfully propagating an endangered species by using breeding cages in a laboratory setting for captive breeding. Our findings on the natural breeding and raising of larvae and adults are useful in breeding this endangered species, and can be applied to the preservation of other similarly wild and endangered species.AbstractAnderson’s crocodile newt (Echinotriton andersoni) is distributed in the Central Ryukyu Islands of southern Japan, but environmental degradation and illegal collection over the last several decades have devastated the local populations. It has therefore been listed as a class B1 endangered species in the IUCN Red List, indicating that it is at high risk of extinction in the wild. The species is also protected by law in both Okinawa and Kagoshima prefectures. An artificial insemination technique using hormonal injections could not be applied to the breeding of this species in the laboratory. In this study we naturally bred the species, and tested a laboratory farming technique using several male and female E. andersoni pairs collected from Okinawa, Amami, and Tokunoshima Islands and subsequently maintained in near-biotopic breeding cages. Among 378 eggs derived from 17 females, 319 (84.4%) became normal tailbud embryos, 274 (72.5%) hatched normally, 213 (56.3%) metamorphosed normally, and 141 (37.3%) became normal two-month-old newts; in addition, 77 one- to three-year-old Tokunoshima newts and 32 Amami larvae are currently still growing normally. Over the last five breeding seasons, eggs were laid in-cage on slopes near the waterfront. Larvae were raised in nets maintained in a temperature-controlled water bath at 20 °C and fed live Tubifex. Metamorphosed newts were transferred to plastic containers containing wet sponges kept in a temperature-controlled incubator at 22.5 °C and fed a cricket diet to promote healthy growth. This is the first published report of successfully propagating an endangered species by using breeding cages in a laboratory setting for captive breeding. Our findings on the natural breeding and raising of larvae and adults are useful in breeding this endangered species and can be applied to the preservation of other similarly wild and endangered species such as E. chinhaiensis.