Phylogeography of the endangered tideland snail Batillaria zonalis in the Japanese and Ryukyu Islands

The phylogeography of the endangered tideland snail Batillaria zonalis in the Japanese and Ryukyu Islands was analyzed on the basis of nucleotide sequences of a mitochondrial gene for cytochrome oxidase c subunit I (COI). Extremely low genetic diversity was found in populations at both the northern and southern boundaries of the geographic distribution of this species in Japan, i.e., Sendai Bay and Iriomotejima Island, respectively, which might be attributed to the population bottleneck due to historical environmental variations and/or the recent foundation of populations in the marginal part of the inhabitable range. Most populations contained unique rare haplotypes, and significant genetic differentiation on the whole was shown, while no clear geographic genetic structure was detected between the Japanese and Ryukyu Islands or over the distribution area of B. zonalis in Japan, with the exception of significant genetic divergences in Ago Bay in the central part of Honshu and the southern part of Okinawajima Island.     

First record of Leptospira borgpetersenii isolation in the Amami Islands, Japan

In 2003, a Leptospira survey was performed on Yoroshima Island of the Amami Islands located in the southwestern part of Japan. Seven Leptospira strains were isolated from the field rat Rattus rattus, which were identified as L. borgpetersenii by flaB sequencing, 16S rDNA sequencing and gyrB sequencing, and serovar Javanica was determined by a microscopic agglutination test. NotI-long restriction fragment analysis indicated that these isolates were genetically indistinguishable from an isolate from the Okinawa Islands. The present results suggest that L. borgpetersenii is migrating into the Amami Islands in Japan.     

Alien parasitic infections in the endangered Ryukyu long-furred rat (Diplothrix legata) on Amami-Oshima Island,Japan

There is limited knowledge about parasites of the endangered Ryukyu long-furred rat, Diplothrix legata (Murinae, Rodentia) endemic to Okinawa, Tokunoshima, and Amami-Oshima Islands in Japan. In the present study, postmortem histopathological examination of an individual found on Amami-Oshima Island revealed a mixed helminth infection of Calodium hepaticum, Hydatigera taeniaeformis, and Angiostrongylus cantonensis. These helminths are considered non-native to Amami-Oshima Island and are maintained by invasive mammals, such as non-native rats and outdoor cats. This observation presents a new host record for C. hepaticum and H. taeniaeformis and the first record of A. cantonensis in Ryukyu long-furred rat on Amami-Oshima Island.     

Genetic variations and phylogeography of the swallowtail butterfly Papilio machaon on the Japanese Islands

The swallowtail butterfly Papilio machaon Linnaeus, 1758 is widely distributed in the Holarctic region, including all of the main islands of Japan, as well as Sakhalin, and on other smaller islands south to Yakushima Island. The Japanese population is situated at the margin of the Eurasian distribution range of this species. It is morphologically different from other populations and has been classified as the subspecies hippocrates C. & R. Felder, 1864. The population of the Japanese Islands is considered to be genetically distinct from the continental populations in relation to the geographical history of the Japanese Islands. Therefore, we examined a part of the ND5 gene sequence of the mitochondrial DNA for P. machaon individuals of various localities in Japan and some nearby countries, and found 68 haplotypes in 400 individuals from the Japanese Islands and Sakhalin. A DNA polymorphism analysis revealed that the genetic structure of the Hokkaido population was significantly different from that of the southern populations on the main Japanese islands. These results imply that P. machaon expanded its range from the Amur region of Russia southward through Sakhalin to the Japanese Islands, and that the Tsugaru Strait between Hokkaido and Honshu may have subsequently limited their gene flow as a geographical barrier.     

Allozyme diversity ofPittosporum (Pittosporaceae) on the Bonin (Ogasawara) Islands

The genusPittosporum includes about 160 species. Four species ofPittosporum occur in the Bonin Islands, and all of these are endemic to the islands. Electrophoretic studies of the four endemic species,P. tobira, from the Japanese mainland, andP. lutchuense var.denudatum from the Ryukyu Islands, were used to determine the origin and speciation pattern of the endemic species. 259 individuals were sampled from ten populations. Twenty loci in nine enzyme systems were resolved and used to calculate the gene frequencies for each population. A low genetic diversity was observed in three of the Bonin Island species, as is reported for other oceanic island plants. The exception,P. boninense, has the largest population size and widest distribution. A dendrogram generated by the UPGMA method shows two clusters. One consists of only the Bonin endemics, suggesting a monophyletic origin for these species.     

Distribution and molecular characterization of distinct Asian populations of Bemisia tabaci (Hemiptera: Aleyrodidae) in Japan

Bemisia tabaci (Gennadius) is considered to be the most economically important pest insect worldwide. The invasive variant, the Q biotype of B. tabaci was first identified in 2004, and has caused significant crop yield losses in Japan. The distribution and molecular characterization of the different biotypes of B. tabaci in Japan have been little investigated. In this study, B. tabaci populations were sampled from the Japanese Archipelago, the Amami Archipelago and the Ryukyu Islands between 2004 and 2008, and the nucleotide sequences of their mitochondrial cytochrome oxidase I genes were determined. Bayesian phylogenetic relationship analysis provided the first molecular evidence that the indigenous Japanese populations could be separated into four distinct genetic groups. One major native population from the Japanese Archipelago, given the genetic group name Lonicera japonica, was separated into an independent group, distinct from the other genetic groups. The second major population, the Nauru biotype in the Asia II genetic group, was identified in the Amami Archipelago and the Ryukyu Islands. Two distinct minor genetic groups, the Asia I and the China, were also identified. One invasive B‐related population belonging to the Mediterranean/Asia Minor/Africa genetic group has been identified in Honshu. All lineages generated by the phylogenetic analyses were supported by high posterior probabilities. These distinct indigenous B. tabaci populations developed in Japan under geographical and/or biological isolation, prior to recent invasions of the B and Q biotypes.     

Mitochondrial DNA divergence in yoshinobori gobies (Rhinogobius species complex) between the Bonin Islands and the Japan–Ryukyu Archipelago

To investigate the genetic differentiation between the Bonin (Ogasawara) Islands' freshwater goby Rhinogobius sp. Bonin Island (BI) form (Ogasawara-yoshinobori) and the Japan–Ryukyu Archipelago relatives, the mitochondrial DNA (mtDNA) phylogeny of Japanese Rhinogobius species was inferred from partial nucleotide sequences of the mitochondrial NADH dehydrogenase 5 subunit (ND5) gene (945 bp). The resultant tree showed that the Bonin Islands group separated first from the other Japanese lineage, and a test calculation indicated the divergence date to be approximately 3 million years BP. Although it is necessary to use a more reliable estimate to confirm the divergence date, Rhinogobius sp. BI has retained its mtDNA lineage in the islands for millions of years.     

Fine-Scale Genetic Structure and Demographic History in the Miyako Islands of the Ryukyu Archipelago

The Ryukyu Archipelago is located in the southwest of the Japanese islands and is composed of dozens of islands, grouped into the Miyako Islands, Yaeyama Islands, and Okinawa Islands. Based on the results of principal component analysis on genome-wide single-nucleotide polymorphisms, genetic differentiation was observed among the island groups of the Ryukyu Archipelago. However, a detailed population structure analysis of the Ryukyu Archipelago has not yet been completed. We obtained genomic DNA samples from 1,240 individuals living in the Miyako Islands, and we genotyped 665,326 single-nucleotide polymorphisms to infer population history within the Miyako Islands, including Miyakojima, Irabu, and Ikema islands. The haplotype-based analysis showed that populations in the Miyako Islands were divided into three subpopulations located on Miyakojima northeast, Miyakojima southwest, and Irabu/Ikema. The results of haplotype sharing and the D statistics analyses showed that the Irabu/Ikema subpopulation received gene flows different from those of the Miyakojima subpopulations, which may be related with the historically attested immigration during the Gusuku period (900 − 500 BP). A coalescent-based demographic inference suggests that the Irabu/Ikema population firstly split away from the ancestral Ryukyu population about 41 generations ago, followed by a split of the Miyako southwest population from the ancestral Ryukyu population (about 16 generations ago), and the differentiation of the ancestral Ryukyu population into two populations (Miyako northeast and Okinawajima populations) about seven generations ago. Such genetic information is useful for explaining the population history of modern Miyako people and must be taken into account when performing disease association studies.     

Genetic differentiation within and among island populations of the endangered plant Aster miyagii (Asteraceae), an endemic to the Ryukyu Islands

Genetic diversity was examined at 17 putative allozyme loci in 18 populations of the insular endemic plant Aster miyagii (Asteraceae). This species is geographically restricted to only three islands of the Ryukyu Islands and is on the federal list of threatened plants. Genetic differentiation within an island is small, suggesting that gene flow among populations on the same island is sufficiently large to prevent divergence. By contrast, genetic differentiation among islands is large, especially between Amamioshima Island and the other two islands, suggesting that gene flow between the islands is highly restricted. Two unique alleles are nearly fixed in populations on Amamioshima Island, which is the southernmost island of the three. Comparatively, genetic diversity is the smallest on Amamioshima Island. This genetic paucity on Amamioshima Island is probably a result of a population bottleneck at colonization or the small effective population size on this island. Genetic diversity at the species level of A. miyagii is larger than those of the species with a similar life history and of the congeneric widespread species, suggesting that the species has an old origin as an insular endemic species.     

Historical effects on local variation in walnut-feeding behavior by the Japanese wood mouse, Apodemus speciosus

The Japanese wood mouse Apodemus speciosus eats large, hard-walled walnuts of Juglans ailanthifolia immediately after finding or after hoarding them. However, not all individuals can efficiently eat the nuts. In this study, to examine local variation in the ability to eat walnuts, feeding behavior was compared among nine wood mouse populations, four from mainland Honshu, where the walnut tree is distributed, and five from the Izu Islands (30-100 km south of Honshu), which lack the tree species. To avoid the effects of pre-capture experience with walnuts, mice from areas lacking the walnut trees were used for testing, even in the Honshu sites. Most mice from Honshu were able to eat walnuts after a 14-day training period, whereas most insular mice could not, with the exception of mice on Kouzushima Island. An analysis of the population genetic structures of these mice based on sequences of the mitochondrial control (D-loop) region revealed that the four insular populations are genetically distinct from the mainland populations, whereas the Kouzushima population remains genetically similar to the mainland populations. The relatively recent colonization of Kouzushima may explain why mice from this island were able to feed on walnuts despite the lack of walnut trees on the island. Thus, walnut-feeding ability appears to have some innate basis in the Japanese wood mouse, and this trait would be selected for in a walnut-available environment as it would better enable mice to survive during food shortages.     

Intersectional gene flow between insular endemics of Ilex (Aquifoliaceae) on the Bonin Islands and the Ryukyu Islands

Hybridization and introgression play important roles in plant evolution, and their occurrence on the oceanic islands provides good examples of plant speciation and diversification. Restriction fragment length polymorphisms (RFLPs) and trnL (UAA) 3'exon-trnF (GAA) intergenic spacer (IGS) sequences of chloroplast DNA (cpDNA), and the sequences of internal transcribed spacer (ITS) of nuclear ribosomal DNA were examined to investigate the occurrence of gene transfer in Ilex species on the Bonin Islands and the Ryukyu Islands in Japan. A gene phylogeny for the plastid genome is in agreement with the morphologically based taxonomy, whereas the nuclear genome phylogeny clusters putatively unrelated endemics both on the Bonin and the Ryukyu Islands. Intersectional hybridization and nuclear gene flow were independently observed in insular endemics of Ilex on both sets of islands without evidence of plastid introgression. Gene flow observed in these island systems can be explained by ecological features of insular endemics, i.e., limits of distribution range or sympatric distribution in a small land area.     

Molecular phylogeny of Japanese Leporidae,the Amami rabbit Pentalagus furnessi,the Japanese hare Lepus brachyurus,and the mountain hare Lepus timidus,inferred from mitochondrial DNA sequences

We determined mitochondrial 12S ribosomal RNA (rRNA) and cytochrome b (cyt b) gene sequences in three leporid species of Japan, the Amami rabbit Pentalagus furnessi from the Ryukyu Islands, the Japanese hare Lepus brachyurus from Honshu, and a Japanese form of the mountain hare Lepus timidus ainu from Hokkaido. We compared the sequences with those of other taxa of leporids available in databases. Phylogenetic trees of the 12S rRNA gene sequences indicated that the lineage of P. furnessi diversified during the generic radiation of the leporids at an ancient time, which was estimated to have been the middle Miocene. Cyt-b gene trees revealed that the lineage of L. brachyurus branched off at an early stage in the speciation of Lepus, probably at the beginning of the Pliocene. The cyt b sequences of L. t. ainu were somewhat distinct from those of continental conspecific populations; this lineage divergence is likely to have occurred during the middle or late Pleistocene. The results show that the three regions of the Japanese archipelago, Ryukyu, Honshu-Shikoku-Kyushu, and Hokkaido, now preserve their own leporid taxa, each with a different extent of genetic endemicity. It is possible that the zoogeographic traits of the Japanese leporids are a consequence of the evolutionary dynamics of leporids in East Asia, in that the radiation centers of leporids are likely to have shifted from tropical, through temperate, to arctic zones.     

Vicariant versus dispersal processes in the settlement of Pseudamnicola (Caenogastropoda,Hydrobiidae) in the Mediterranean Balearic Islands

The genus Pseudamnicola comprises a group of tiny springsnails inhabiting several continental and insular regions of the Mediterranean basin. Given the limited dispersal capabilities of these animals, it is difficult to explain the wide distribution range of the genus and, more specifically, its presence in isolated habitats, such as on islands. Thus, to investigate the process(es) that may explain these distribution patterns, we morphologically re‐described and genetically analysed the six Pseudamnicola (Pseudamnicola) species occurring in the Iberian Peninsula and the nearby Balearic Islands. Genetic relationships were explored by sequencing two mitochondrial (cytochrome c oxidase subunit I and 16S rRNA) and one nuclear (28S rRNA) gene in 19 populations. Our morphological study confirmed the presence of previously described species, whereas our phylogenetic results revealed three lineages within the subgenus: one clade grouping the species from Minorca Island with an Iberian Peninsula species, a second clade grouping the three species from Majorca Island, and a third clade that consists of a single species, which occurs in both the Iberian Peninsula and Ibiza Island. Calculated speciation times show that the cladogenetic events involving the insular species seem to have occurred after the current conformation of the Balearic Islands (c. 20 Mya). Therefore, the speciation process may have been related to subsequent transmarine colonizations, probably during the Messinian Salinity Crisis, and the Pleistocene glaciations when landmass corridors connected the islands with the continent. © 2014 The Linnean Society of London     

Population structure of an endangered frog (<Emphasis Type="Italic">Babina subaspera</Emphasis>) endemic to the Amami Islands: possible impacts of invasive predators on gene flow

The otton frog (Babina subaspera) is an endangered species endemic to the Amami Islands, Japan. High predation pressure from an introduced carnivore, the mongoose, has caused declines in the frog populations and created a large habitat gap around an urban area. To promote effective conservation, we investigated the genetic status of the species and examined the effect of the habitat gap on gene flow among populations. Using five polymorphic microsatellite loci and mitochondrial DNA sequences, we investigated genetic diversity, genetic structure and gene flow in B. subaspera populations on the islands of Amami-Oshima and Kakeroma-jima. The expected heterozygosity (H _E) within each locality was generally high (range: 0.67–0.85), indicating that B. subaspera maintains high genetic diversity. However, genetic differentiation was observed, and the two populations, TAG and KAR, showed little gene flow with other populations. The clustering and F _ST analyses also predicted that these two populations were clearly distinct. According to the mitochondrial DNA analysis, the observed genetic differentiation occurred relatively recently. Possible barriers such as mountain ridges, rivers or roads did not result in genetic separation of the populations. These data support the hypothesis that the habitat gap created by an introduced predator prevented the gene flow among B. subaspera populations. When developing conservation strategies for B. subaspera, focus should be directed to these two isolated populations; careful monitoring of population size and genetic diversity should be conducted along with the mongoose elimination project ensues.     

Phylogeography of Cycas revoluta Thunb. (Cycadaceae) on the Ryukyu Islands: very low genetic diversity and geographical structure

Variations in the chloroplast and mitochondrial DNA of Cycas revoluta Thunb. (Cycadaceae) were examined in 22 populations distributed across the Ryukyu Islands and southern Kyushu. Among the 14,130 bp of sequence examined, only one site mutation and one indel were polymorphic. The identified polymorphisms were located in the spacers between trnS (UGA) and trnfM (CAU) of the chloroplast DNA and between nad1 exon B and exon C of the mitochondrial DNA, respectively. Three haplotypes were identified from the Ryukyu Islands and southern Kyushu. The areas of distribution of the three haplotypes were highly geographically structured. The boundaries of two of the three haplotypes were demarcated by Okinoerabujima Island in the middle Ryukyus. The northern type and southern types lay north and south of the island, respectively. The third haplotype was almost sympatrically distributed with the southern type. The genetic variation within C. revoluta was estimated to be very low (h = 0.641, π = 0.00071) in comparison to its relative in Taiwan, C. taitungensis, which possesses 97 cpDNA haplotypes and 55 mtDNA haplotypes from two relic populations. A reasonable explanation for the low genetic diversity of the cycad on the Ryukyu Islands could be severe bottleneck effects, resulting from the submersion of low islands and the diminished landmass of islands in the interglacial age in the Quaternary period. The geographically restricted nature of the haplotypes could be attributed to vicariance resulting from the land configuration of the Ryukyu Islands, including changes in geography during the interglacial age in the Quaternary.     

Genetic variation of the white-spotted longicorn beetle <Emphasis Type="Italic">Anoplophora</Emphasis> spp. (Coleoptera: Cerambycidae) in Japan detected by mitochondrial DNA sequence

Genetic variation of the white-spotted longicorn beetle Anoplophora spp., distributed on the Japanese mainland and the Ryukyu Islands, was examined using nucleotide sequences of mitochondrial DNA. Two fragments, a 1.2-kb-long fragment containing portions of the cytochrome oxidase subunit I and II genes and a 1.4-kb-long fragment containing portions of 16S and 12S rDNAs, were sequenced. In phylogenetic analyses based on the sequences, 294 individuals collected at 75 localities were divided into two major groups (groups A and B) and then split into seven subgroups (A1–A4 and B1–B3). In group A, the closely related subgroups A1, A2, and A3 were mainly distributed on the Japanese mainland and were roughly separated among geographic areas, although the range of A3 spread significantly from Kyushu to the Central Ryukyu Islands. The sequence of A4, detected from one individual collected in eastern Honshu, was almost the same as that reported for A. chinensis (Forster) on the Chinese continent. In group B, subgroups B1 and B2 were restricted to the Central and Southern Ryukyu Islands, respectively, while B3 was distributed widely in both regions. Based on the results, we discuss the geographic origin of the haplotypes and movement of the insects between geographic areas.     

Molecular phylogeny of the genus Pieris (Ericaceae) with special reference to phylogenetic relationships of insular plants on the Ryukyu Islands

Phylogenetic relationships within the genus Pieris (Ericaceae) were investigated based on the rbcL and matK genes along with five spacer sequences of chloroplast DNA to address questions regarding the phylogeography of the genus in association with insular plants on the Ryukyu Islands. The most parsimonious trees indicated that P. floribunda from eastern North America is a sister taxon to the remaining taxa examined, and suggested that the East Asian taxa examined are monophyletic. A morphologically cohesive group, section Pieris, was revealed to be paraphyletic. Within the East Asian clade, insular endemics from the Ryukyu Islands, Taiwan, and mainland Japan formed a sister group to P. formosa from the Himalayas and southern China. Our data suggest that the insular endemics of the Ryukyu Islands and Taiwan arose via allopatric divergence as a result of a paleogeographical land configuration of a landbridge during the early–middle Pleistocene in the Quaternary Period.     

A record of the archerfish (Toxotes jaculator) from Iriomote Island,the Ryukyu Islands

A specimen ofToxotes jaculator (Pallas, 1767) (Pisces: Toxotidae) was collected from the mouth of Urauchi River (24°23′55″N, 123°46′48″E), Iriomote Island, Ryukyu Islands, Japan. This species was recorded for the first time from the Japanese Archipelago. Its occurrence in Japan is the northern-most record of this species.     

Genetic and geographical differentiation of <Emphasis Type="Italic">Pandaka</Emphasis> gobies in Japan

The mitochondrial DNA (mtDNA) phylogeny of Japanese Pandaka species (Perciformes: Gobiidae) was inferred from partial nucleotide sequences of the mitochondrial 12S and 16S rRNA genes (1083bp). The resultant mtDNA tree showed two major clades (clade I and clade II), which were inconsistent with the present taxonomic classification. One of the major clades was further divided into two geographical groups, distributed on the Japanese Major Islands (clade I-A) and from Amami-oshima Island to Iriomote Island (clade I-B). The mtDNA haplotypes in clade II were found only on Iriomote Island. The mtDNA divergences in clade I indicated that the Japanese Major Island (clade I-A) and Ryukyu (clade I-B) groups have been geographically isolated from each other for millions of years, based on the putative molecular divergence rate. The geographical distributions of mtDNA haplotypes in clade I-A and clade I-B also suggested that Pandaka gobies had not dispersed to distant offshore islands, indicating that their geographical differentiation may be closely associated with the geological history of the Japanese and Ryukyu Archipelagos.This revised version was published online in January 2005 with corrections to the repetition of the 1st authors name.     

Phylogeny and phylogeography of the sword-tailed newt,Cynops ensicauda (Amphibia: Caudata), as revealed by nucleotide sequences of mitochondrial DNA

We investigated the phylogenetic relationships and phylogeography among individuals of the endemic newt (Cynops ensicauda) of the Central Ryukyus, Japan based on the mitochondrial cytochrome b gene. The results of phylogenetic analyses showed the presence of remarkable differentiation between assemblages from the Amami and Okinawa Island Groups, supporting the validity of the subspecies C. e. popei described from the latter on morphological grounds. The group of individuals from Okinawa Island Group was further divided into two distinct subgroups unlike the results of previous morphological and allozyme studies. Geographic ranges of these subgroups overlapped in the northern part of Okinawajima Island. The phylogeographic pattern within the Okinawa Island Group suggests an initial division into two geographically isolated population lineages and subsequent secondary sympatry before formation of reproductive isolation. It is also likely that within the Okinawa Island Group emigration occurred from the central and northern parts of Okinawajima Island to its southern part, as well as to several small islets off its western coast. Within the Amami Island Group, recurring restricted gene flow with isolation by distance seems to have occurred around the southwestern region including three islets southwest of Amamioshima Island.