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The endangered crocodile newt, Echinotriton andersoni, is a relatively large species of the family Salamandridae and is distributed on six islands in the central part of the Ryukyu Archipelago, Japan. Because of an originally small distribution range and recent habitat loss, this species has been steadily declining in number. To elucidate fine-scale population structure, which is essential for effective conservation management, we analyzed genetic diversity and gene flow based on nine microsatellite loci. Our results identified three different island groups (Amamioshima, Tokunoshima, and Okinawajima) and multiple genetic assemblages within the Amami and Okinawa island groups. The gross genetic variation within each island was positively correlated with island size. Population structure followed a latitudinal cline and isolation by distance, even among geographically isolated islands. In northern Okinawajima, relatively complex genetic structure was observed. This unexpected population structure seems to reflect historical migration and distribution expansion through the formation of land bridges and shifted coastlines in the Pleistocene. We also found that small islands showed little genetic variation (Ukeshima, Sesokojima, and Tokashikijima). In particular, our findings revealed that the Tokashikijima population is at greater risk for extinction than the other populations because it has the smallest effective population size.

The Anderson's crocodile newt, Echinotriton andersoni, is considered a relic and endangered species distributed in the Central Ryukyus. To elucidate phylogenetic relationships and detailed genetic structures among populations, we analyzed variation in the mitochondrial cytochrome b gene. Results strongly support a primary dichotomy between populations from the Amami and Okinawa Island Groups with substantial genetic divergence, favoring a primary divergence between the two island groups. Within the latter, populations from the southern part of Okinawajima Island are shown to be more closely related to those from Tokashikijima Island than to those from the northern and central parts of Okinawajima. The prominent genetic divergence between the two island groups of the Central Ryukyus seems to have initiated in the Miocene, i.e., prior to formation of the strait that has consistently separated these island groups since the Pleistocene. The ancestor of the southern Okinawajima-Tokashikijima is estimated to have migrated from the northern and central parts of Okinawajima into southern Okinawajima at the Pleistocene, and dispersed into Tokashikijima subsequently.

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.

Crocodile newts, which constitute the genera Echinotriton and Tylototriton, are known as living fossils, and these genera comprise many endangered species. To id entify 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 ende mic to the Ryukyu Islands, we performed molecular dating analysis using whole and partial mt genomic data. The estimated di vergence 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 pa rtial mt sequence data also suggests the existence of at least two more undescribed species in the genus Ty lototriton. We also found unusual repeat s equences 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. v errucosus mtDNA. Similar repeat sequences were found in t wo other Tylototriton species. The Ty lototriton taxa with the repeats become a monophyletic group, indicating a single origin of the repeat sequences. The intraa nd inter-specific comparisons of the repeat sequences suggest the occurrences of homologous recombination-based concerted evolution among the repeat sequences.

We observed male courtship behavior of Tylototriton (Echinotriton) andersoni in the laboratory. In some males, the cloaca swelled and became wet with mucous secretions from December to May. A male in this condition crept around a female, sniffing around her body and drawing a thread of mucus from his cloaca, so that the female was surrounded by spiderweb-like string of mucous attached to the substrate. The male then deposited a spermatophore by rubbing his cloaca against the substratum while swaying his body to and fro. The male behaved similarly towards females from different localities, though we could not observe any female reaction to the male. Under a phase-contrast microscope, the gross morphology of sperms from a male from Amamioshima Island was similar to that reported for males from Okinawajima.