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Japanese giant salamander (Andrias japonicus) is one of the largest amphibian species in the world and an iconic species in Japan. However, as its distribution has recently declined across the country, rapid and extensive monitoring of the distribution is urgently needed for its efficient conservation. Here, we used environmental DNA (eDNA) analysis to assess the Japanese giant salamander's distribution in western Japan and, for that purpose, we collected 410 water samples from 12 rivers. We then developed a new eDNA assay for multi-copy nuclear DNA (nuDNA) of the giant salamander and compared the eDNA detectability of the nuDNA marker with that of a previous mitochondrial DNA (mtDNA) marker. Throughout the survey, we detected target eDNA from 162 water samples using either of the markers, which generally corresponded to the known natural distribution of the species. Additionally, the use of the nuDNA marker allowed for higher detection rate of target eDNA than the mtDNA marker. Moreover, the detection rate of target eDNA decreased substantially in water samples with higher conductivity and also partly in those with higher pH, suggesting their negative impacts on the salamander's ecology. Our results demonstrated that eDNA analysis with multi-copy nuDNA marker is highly useful for efficient and sensitive surveillance of Japanese giant salamander's distribution. Our study provided the methodology for efficiently monitoring the Japanese giant salamander's distribution via eDNA analysis and facilitating conservation activities for them. [ABSTRACT FROM AUTHOR]

The giant water bug Kirkaldyia deyrolli is one of the most well‐known endangered aquatic insects in Japan. The population of this species in Kyushu, one of the four major islands of the Japanese Archipelago, is extremely limited. In the present study, we identified a new population of K. deyrolli and undertook population genetic structure analyses based on genome‐wide nuclear DNA single nucleotide polymorphisms (SNPs). We collected samples using a nonlethal sampling method and obtained 59,279 SNPs. The results of our study confirm that sufficient SNP data for population genetic structure analysis can be obtained through nonlethal sampling. We determined that the newly identified population is genetically differentiated from a previously identified population on Kyushu. Therefore, this population is considered highly important for the conservation of K. deyrolli in Kyushu. [ABSTRACT FROM AUTHOR]

Vicia sepium (bush vetch) is a perennial legume widely distributed throughout the Eurasian continent. However, its distribution in Japan is limited to Mt. Ibuki and small parts of central and southern Hokkaido. Therefore, each Japanese V. sepium lineage has been considered to have been introduced separately from Europe. Here, we examined whether the species was introduced or not on the basis of cpDNA sequences and genome-wide SNPs from Japanese and overseas samples. Both the cpDNA haplotype network and the nuclear DNA phylogenetic tree showed that Japanese V. sepium is monophyletic. Furthermore, although the nuclear DNA phylogenetic tree also showed that each lineage is clearly monophyletic, genetic admixture of the genetic cluster dominated in the Hokkaido lineage was also detected in the Mt. Ibuki lineage. Population divergence analysis showed that the two lineages diverged during the last glacial period. The Mt. Ibuki lineage showed a sudden population decline 300–400 years ago, indicating that some anthropogenic activity might be involved, while the Hokkaido lineage showed a gradual population decline from 5000 years ago. Consequently, these two lineages show low current genetic diversity compared with overseas lineages. These results show that the Japanese V. sepium is not introduced but is native. [ABSTRACT FROM AUTHOR]

Alisma L. is a genus of aquatic and wetland plants belonging to family Alismataceae. At present, it is thought to contain ten species. Variation in ploidy level is known in the genus, with diploids, tetraploids and hexaploids recorded. Previous molecular phylogenetic studies of Alisma have generated a robust backbone that reveals important aspects of the evolutionary history of this cosmopolitan genus, yet questions remain unresolved about the formation of the polyploid taxa and the taxonomy of one particularly challenging, widely distributed species complex. Here we directly sequenced, or cloned and sequenced, nuclear DNA (nrITS and phyA) and chloroplast DNA (matK, ndhF, psbA-trnH and rbcL) of multiple samples of six putative species and two varieties, and conducted molecular phylogenetic analyses. Alisma canaliculatum and its two varieties known in East Asia and A. rariflorum endemic to Japan possess closely related but heterogeneous genomes, strongly indicating that the two species were generated from two diploid progenitors, and are possibly siblings of one another. This evolutionary event may have occurred in Japan. Alisma canaliculatum var. canaliculatum is segregated into two types, each of which are geographically slightly differentiated in Japan. We reconstructed a single phylogeny based on the multi-locus data using Homologizer and then applied species delimitation analysis (STACEY). This allowed us to discern A. orientale as apparently endemic to the Southeast Asian Massif and distinct from the widespread A. plantago-aquatica. The former species was most likely formed through parapatric speciation at the southern edge of the distribution of the latter. [ABSTRACT FROM AUTHOR]

Wetland biodiversity is currently declining on a global scale. Wetland biodiversity understanding is critical for determining the wetlands' conservation value. In this study, Macroplea Samouelle, 1819 (Coleoptera: Chrysomelidae) was discovered in Aomori Prefecture, Honshu Island, Japan. Only two Macroplea species have been recorded in Japan, M. japana (Jacoby, 1885) and M. mutica (Fabricius, 1792). Macroplea japana had been unrecorded for 60 years before being rediscovered in Honshu Island in 2022, and a single adult M. mutica female was discovered in Hokkaido Prefecture in 2003. The discovered individuals were concluded to be M. mutica based on morphological and molecular analyses. Although morphological differences were observed with the Eurasian M. mutica individuals, the male genitalia was nearly identical to M. mutica. For the molecular phylogenetic analysis based on COI and 28S sequences, Macroplea individuals in Japan were clustered with M. mutica on the Eurasian Continent. This is the first record of this species on Honshu Island (and the second in Japan), as well as the first record of adult males. This species would require conservation policies and additional distributional surveys. [ABSTRACT FROM AUTHOR]

Elucidating the mechanism of formation of biota on islands will contribute to our understanding of the evolutionary mechanisms that generate biodiversity. In this study, we focus on the evolutionary history of Ephemera orientalis , which is distributed in East Asia. Phylogenetic analyses were performed using the mitochondrial DNA 16S rRNA, cytochrome oxidase subunit I and the nuclear DNA histone H3 regions. We found that these mayflies exhibit genetic differentiation between the Japanese and Korean populations. The Tsushima population, which is located between the Japanese Islands and the Korean Peninsula, was positioned genetically as a continental clade. The populations of the Fuji-Goko Lakes and Lake Ashino-ko in Central Japan were genetically differentiated from the other populations. The genetic structures over a wide area of the Japanese Islands were confirmed and a loose genetic regionality was observed, which was thought to result from secondary contact of the north-eastern and south-western lineages after their long separation by a geohistorical division event. We have gathered important knowledge regarding the evolutionary history of organisms on islands and investigated the colonization and isolation of biological populations within a species with high dispersal ability. [ABSTRACT FROM AUTHOR]

SUMMARY: Green algal blooms by Cladophora species are primarily reported in freshwater and coastal regions and cause severe ecological problems. A taxonomic report of the geographic distribution of Cladophora blooms is essential to explore the cause and impact of macroalgal blooms and for eco‐physiological studies. The identification of blooming species is necessary for monitoring and controlling algal blooms. Genetic information of DNA sequences is useful for identifying species in the genus Cladophora. In the coastal area of Sangrok, Korea, a large‐scale Cladophora bloom was reported for the first time in September 2015. In the present study, we identified the taxonomic entity of Cladophora oligocladoidea (Ulvophyceae, Cladophorales) in Korea. We report for the first time a green algal bloom by this species globally. This is the second report about genetic diversity of C. oligocladoidea since the species was established in Japan. Four ribotypes in the internal transcribed spacer (ITS) region of the nuclear ribosomal DNA represent the genetic diversity of Korean C. oligocladoidea. The results from this taxonomic report and our analysis of the green algal bloom of Korean C. oligocladoidea can provide valuable data to understand the geographic distribution and the genetic diversity of the species. [ABSTRACT FROM AUTHOR]

The genus Aquarius, of the subfamily Gerrinae, was formerly divided into two species and one subspecies in Japan (A. paludum paludum, A. paludum amamiensis and A. elongatus, respectively). Aquarius haliplous Yasunaga et al. 2018 has recently been described as a new species, which occurs in brackish waters of Nagasaki Prefecture and is found sympatrically with the widely distributed species, A. p. paludum. However, they are considered to be speciated, with no cross‐breeding. In this study, molecular phylogenetic and morphological analyses were undertaken using the mitochondrial DNA COI (529 bp) and nuclear DNA histone H3 (314 bp) gene regions to investigate the genetic and morphological differentiation in the genus Aquarius, including A. haliplous, throughout Japan (including the islands). The results show that, unlike A. p. paludum, A. p. amamiensis and A. haliplous, A. elongatus displays substantial genetic differentiation. Considering that, although morphological differences exist in the antennae of A. p. paludum and A. p. amamiensis, A. haliplous has no clear genetic or morphological difference from A. p. paludum, and hence cannot be classified as a distinct species. Therefore, we propose that A. haliplous be synonymized with A. paludum. [ABSTRACT FROM AUTHOR]

Understandings of fish spawning are important for the effective riverine ecosystem and fisheries resources management. Application of environmental DNA (eDNA) analysis would allow fish spawning evaluation at a high temporal and spatial resolution due to its less-labor intensive property, compared to the conventional collection methods. However, the relative performance of the two eDNA-based methods used to evaluate fish spawning, pre and post comparisons of eDNA concentrations and nuclear DNA to mitochondrial DNA ratio, has not been compared in the field. We evaluated whether spawning events of Ayu Plecoglossus altivelis could be identified from day (pre-spawning) to night (post-spawning) changes in eDNA concentrations using nuclear DNA (RAG1, nuDNA) and nuDNA/mitochondrial DNA ratio in comparison to the conventional mitochondrial DNA (cyt b, mtDNA). In addition, the relative sensitivity of nuDNA concentrations in spawning evaluation was compared with mtDNA concentrations. Water samples were collected biweekly in the lower Takatsu River, western Japan, between early October and early December 2020, covering the spawning season of the fish species. Generalized linear mixed-effects models (GLMM) showed that Ayu spawning could be detected by nuDNA concentrations likewise by mtDNA concentrations, while nuDNA/mtDNA ratio could not detect spawning earlier in the spawning season. GLS models showed that the day-to-night increase in the eDNA concentrations was more profound for nuDNA than for mtDNA during the main spawning period of the fish. Our findings insist that the application of nuDNA concentrations better serves as a tool for evaluating Ayu spawning intensity in eDNA analysis. [ABSTRACT FROM AUTHOR]

The evolutionary histories of ornamental plants have been receiving only limited attention. We examined the origin and divergence processes of an East Asian endemic ornamental plant, Conandron ramondioides. C. ramondioides is an understory herb occurring in primary forests, which has been grouped into two varieties. We reconstructed the evolutionary and population demography history of C. ramondioides to infer its divergence process. Nuclear and chloroplast DNA sequences were obtained from 21 Conandron populations on both sides of the East China Sea (ECS) to explore its genetic diversity, structure, and population differentiation. Interestingly, the reconstructed phylogeny indicated that the populations should be classified into three clades corresponding to geographical regions: the Japan (Honshu+Shikoku) clade, the Taiwan–Iriomote clade, and the Southeast China clade. Lineage divergence between the Japan clade and the Taiwan–Iriomote and Southeast China clades occured 1.14 MYA (95% HPD: 0.82–3.86), followed by divergence between the Taiwan–Iriomote and Southeast China clades approximately 0.75 MYA (95% HPD: 0.45–1.3). Furthermore, corolla traits (floral lobe length to tube length ratios) correlated with geographical distributions. Moreover, restricted gene flow was detected among clades. Lastly, the lack of potential dispersal routes across an exposed ECS seafloor during the last glacial maximum suggests that migration among the Conandron clades was unlikely. In summary, the extant Conandron exhibits a disjunct distribution pattern as a result of vicariance rather than long-distance dispersal. We propose that allopatric divergence has occurred in C. ramondioides since the Pleistocene. Our findings highlight the critical influence of species' biological characteristics on shaping lineage diversification of East Asian relic herb species during climate oscillations since the Quaternary. [ABSTRACT FROM AUTHOR]

Anthropogenic activities, such as the movement of plants through greening, can result in genetic disturbance that can interfere with local adaptation in wild populations. Although research is underway to prevent genetic disturbance associated with greening, genetic disturbance of intraspecific polyploidy, which is estimated to be present in 24% of vascular plants, has not been well studied. Liriope muscari is a polyploid complex with known diploid (2n = 36), tetraploid (2n = 72), and hexaploid (2n = 108) forms. The plants of this species tolerate dry and hot conditions and are therefore frequently used for greening and gardening. However, the distribution of this polyploid in Japan, its genetic structure, and genetic disturbance are not known. In this study, we investigated the polyploidy distribution and genetic structure in naturally distributed L. muscari in Japan using chloroplast DNA (cpDNA) haplotypes and nuclear DNA (nDNA). Commercially produced individuals were also studied and compared with natural populations to assess any genetic disturbance of the ploidy complex in this species. Chromosome counts, cpDNA, and nDNA results showed three genetically and cytologically distinct groups in Japan: first, a tetraploid group in mainland Japan; second, a hexaploid group in the Ryukyu Islands; and third, a diploid and tetraploid group in the Ryukyu Islands. Significant isolation by distance was also detected within the three groups (p = 0.001). Genetic disturbance due to greening and gardening should be avoided among the three groups. Genetic disturbance can be reduced by using individuals derived from natural populations that are close to the sites used for greening and gardening. For commercially produced individuals, genetic disturbance is unlikely in the Kanto region, an area of high usage, while genetic disturbance is thought possible in the Ryukyu Islands. [ABSTRACT FROM AUTHOR]

Kickxellomycotina encompasses two fungal groups: a saprobic group in excrement and soil and an arthropod gut-inhabiting group. The evolutionary transition between these two lifestyles is unclear due to the lack of knowledge on intermediate forms and lifestyles. Here, we describe a new species, Unguispora rhaphidophoridarum, that was isolated from the excrement of cave crickets (Rhaphidophoridae) in Japan. This species has a novel lifestyle that is intermediate between the saprobic and gut-inhabiting groups. The new genus Unguispora is a member of the Kickxellales and characterized by the sterile appendages born on the sporocladium and by the claw-like ornamentation of the sporangiole. Phylogenetic analysis based on 18S and 28S nuclear ribosomal DNA showed that this fungus is distinct from all known kickxellalean genera and is sister to Linderina. The sporangiospore of the new species germinated only in anaerobiosis and grew in a yeast-like form. The yeast-like cells, defined as "secondary spores," germinated into hyphae in aerobiosis. In the alimentary tract of cave crickets, the sporangiola are attached to the proventriculus (foregut) by the claw-like ornamentation and multiplicate in the same yeast-like form as under culture. We introduce a new term, "amphibious fungi," to describe fungi that have two life stages, one outside and the other inside the host gut, like U. rhaphidophoridarum. The discovery of an amphibious fungus in Kickxellales, which was formerly considered to be only saprobic, suggests that Kickxellomycotina has evolved in association with the animal gut. [ABSTRACT FROM AUTHOR]

The diversification of phenotypes and species has been a major concern in evolutionary biology. Both resource competition and predator–prey interactions have been suggested as mechanisms that drive phenotypic divergence and speciation, but the latter has attracted much less attention than the former. The Karaftohelix land snails in Northeast Asia provide an excellent model to investigate the effects of competition and predation on the divergence process. This is because (1) all species—especially Karaftohelix editha and Karaftohelix gainesi in northern Japan—are closely related and sometimes indistinguishable by genital morphologies and neutral nuclear and mitochondrial DNA markers, despite the distinct phenotypes among species; (2) two different anti‐predatory strategies (i.e., passive and active defenses) are observed for each species in this group; and (3) multiple species have generally overlapping distributions and were often observed at exactly the same time and place. In this study, we conducted quantitative quadrat surveys and carbon/nitrogen stable isotope analyses to compare the microhabitat and trophic niches among Karaftohelix land snails and estimated the cause of adaptive radiation in this taxon. All the results indicated that the differences in microhabitats and trophic niches among Karaftohelix land snails—especially between the most closely related sister species, K. editha and K. gainesi in northern Japan—were too small to promote the adaptive radiation in this group. Therefore, it is difficult to explain the extreme divergence of phenotypes and species among Karaftohelix land snails in Northeast Asia based on environmental adaptation through resource competition and/or reproductive interference. [ABSTRACT FROM AUTHOR]

Large-scale farming of Undaria pinnatifida is conducted in northern China. Conspicuous natural populations of this alga are distributed on rocky shores in this region. However, the genetic relationship between U. pinnatifida from China and native populations in other countries remains largely uncertain. We obtained sequences for the mitochondrial cox3 and tatC–tLeu regions and the internal transcribed spacer one of nuclear ribosomal DNA from representative natural and farmed populations of U. pinnatifida in China. We analyzed genetic diversity, and evaluated the genetic relationship between Chinese populations and Japanese and Korean populations. The mitochondrial and nuclear DNA sequences revealed high genetic diversity in most Chinese populations. Unique mitochondrial haplotypes were detected in the Gouqi Island population consistent with historical records of a native population on the island. Phylogenetic analyses derived from the mitochondrial DNA sequences revealed that the Chinese samples were classifiable as the Continental and Northern Japan types. All natural populations from rocky reefs in northern China were grouped with the Continental type and all farmed populations with the Northern Japan type. Mitochondrial and nuclear DNA sequences revealed significant genetic differentiation between the farmed populations and adjacent natural populations from rocky reefs, in agreement with previous results obtained using microsatellites. [ABSTRACT FROM AUTHOR]

Aim: Although the evolution of island endemic plants has long been investigated, the majority of such studies have focused on species with remarkable levels of morphological variation and on islands substantially far from the mainland. Endemic plants on nearshore oceanic islands have received less attention. We investigate the population genetic structure and dynamics in plants endemic to nearshore and recently formed oceanic islands and examined the possibility of multiple colonizations onto the islands. Location: Japanese mainland Honshu and the adjacent Izu Islands. Taxon Solidago virgaurea (Asteraceae). Methods: Sixteen and nine populations of S. virgaurea complex were sampled from the mainland and islands respectively; phylogeographical and population genetics analyses, including Bayesian Phylogeographic and Ecological Clustering (BPEC) analysis and Discrete Phylogeographic Approach (DPA) to trace the history of colonization events onto the islands, were performed using plastid DNA and nuclear microsatellite DNA variations. Results: Phylogenetically close plastid DNA haplotypes were shared between the mainland and islands, although the populations of S. virgaurea from different islands tended to exhibit phylogenetically distinct haplotypes. Admixture analyses based on nuclear DNA variations revealed distinct genetic structures between the mainland and island populations. Gene flow among islands is restricted but may partially offset genetic drift on each island. Main conclusions The genetic structure observed in this study may not have originated from a single dispersal event and successive expansion but rather from at least three colonization events and subsequent gene flow among island populations. Based on the nuclear DNA variations, the Izu Island populations of S. virgaurea are genetically distinct from the mainland ones. Repeated colonization events may have provided sufficient genetic diversity, which would generally be susceptible to founder effects and exert a driving force for evolutionary adaptation, to these oceanic island populations. [ABSTRACT FROM AUTHOR]

Species of Broussonetia have been essential in the development of papermaking technology. In Japan and Korea, a hybrid between B. monoica and B. papyrifera (= B. × kazinoki) known as kōzo and daknamu is still the major source of raw materials for making traditional paper washi and hanji, respectively. Despite their cultural and practical significance, however, the origin and taxonomy of kōzo and daknamu remain controversial. Additionally, the long-held generic concept of Broussonetia s.l., which included Sect. Allaeanthus and Sect. Broussonetia, was challenged as phylogenetic analyses showed Malaisia is sister to the latter section. To re-examine the taxonomic proposition that recognizes Allaeanthus, Broussonetia, and Malaisia (i.e., Broussonetia alliance), plastome and nuclear ribosomal DNA (nrDNA) sequences of six species of the alliance were assembled. Characterized by the canonical quadripartite structure, genome alignments and contents of the six plastomes (160,121–162,594 bp) are highly conserved, except for the pseudogenization and/or loss of the rpl22 gene. Relationships of the Broussonetia alliance are identical between plastome and nrDNA trees, supporting the maintenance of Malaisia and the resurrection of Allaeanthus. The phylogenomic relationships also indicate that the monoecy in B. monoica is a derived state, possibly resulting from hybridization between the dioecious B. kaempferi (♀) and B. papyrifera (♂). Based on the hypervariable ndhF-rpl32 intergenic spacer selected by sliding window analysis, phylogeographic analysis indicates that B. monoica is the sole maternal parent of B. × kazinoki and that daknamu carries multiple haplotypes, while only one haplotype was detected in kōzo. Because hybridizations between B. monoica and B. papyrifera are unidirectional and have occurred rarely in nature, our data suggest that daknamu might have originated via deliberate hybrid breeding selected for making hanji in Korea. On the contrary, kōzo appears to have a single origin and the possibility of a Korean origin cannot be ruled out. [ABSTRACT FROM AUTHOR]

Background: Fairy shrimps belong to order Anostraca, class Branchiopoda, subphylum Crustacea, and phylum Arthropoda. Three fairy shrimp species (Eubranchipus uchidai, E. asanumai, and E. hatanakai) that inhabit snowmelt pools are currently known in Japan. Whole mitochondrial genomes are useful genetic information for conducting phylogenetic analyses. Mitochondrial genome sequences for Branchiopoda members are gradually being collated. Results: Six whole mitochondrial genomes from the three Eubranchipus species are presented here. Eubranchipus species share the anostracan pattern of gene arrangement in their mitochondrial genomes. The mitochondrial genomes of the Eubranchipus species have a higher GC content than those of other anostracans. Accelerated substitution rates in the lineage of Eubranchipus species were observed. Conclusion: This study is the first to obtain whole mitochondrial genomes for Far Eastern Eubranchipus species. We show that the nucleotide sequences of cytochrome oxidase subunit I and the 16S ribosomal RNA of E. asanumai presented in a previous study were nuclear mitochondrial DNA segments. Higher GC contents and accelerated substitution rates are specific characteristics of the mitochondrial genomes of Far Eastern Eubranchipus. The results will be useful for further investigations of the evolution of Anostraca as well as Branchiopoda. [ABSTRACT FROM AUTHOR]

We developed eight novel microsatellite markers for the salamander Hynobius tsuensis, which is endemic to Tsushima Island, Japan. In H. tsuensis, the number of alleles, observed heterozygosity, and expected heterozygosity in each locus ranged from 1 to 10, 0.00 to 0.76, and 0.00 to 0.76, respectively. In cross-species amplification, five and four of the eight loci were successfully amplified for all individuals of Hynobius sp. from Tsushima and H. nebulosus, respectively. Three species of Hynobius were clearly split from each other in the STRUCTURE analysis, indicating genetic differentiation in nuclear DNA among three species. This is the first report on genetic variation between two close relatives, Hynobius sp. from Tsushima and H. nebulosus based on nuclear loci. These novel markers will be useful for elucidating intraspecific variations and inferring conservation units within H. tsuensis, and studying intra- and inter-specific variations in other Hynobius. [ABSTRACT FROM AUTHOR]

To clarify phylogenetic relationships among Japanese Dioscorea taxa and to re-examine taxonomy based on these relationships, we conducted molecular phylogenetic analyses of four plastid regions [ trnK (including matK), rbcL , atpB and trnL-F ] and two nuclear regions (PHYC and 18S rDNA) for all Dioscorea taxa from Japan and selected taxa from adjacent regions. Although species are often sympatric and variation in leaf shape is relatively wide within species and not clearly demarcated between species, our results suggest that Japanese Dioscorea spp. do not hybridize. Our results also resolve several taxonomic issues surrounding Japanese species. We confirm that D. japonica , D. polystachya , D. pseudojaponica and D. tabatae , which are morphologically similar, are distinct species. Further, plants from the Daito Islands identified as D. luzonensis , which have never been observed to flower to our knowledge, are attributable to D. pseudojaponica. Dioscorea zentaroana , often treated as a synonym of D. asclepiadea , was found to be distinct from the latter. Dioscorea izuensis should be treated as D. collettii var. izuensis. In conclusion, 16 species and three varieties of native Dioscorea should be recognized in Japan. [ABSTRACT FROM AUTHOR]

Phylogeography infers the demographic history of various species by resolving genetic relationships among populations across a geographic range. Comparison of phylogeographic patterns of multiple species with similar geographic distributions, i.e., comparative phylogeography, allows us to assess the roles of regional historical events and species-specific ecological factors in shaping intraspecific genetic diversity, providing model systems for studying the processes and genetic basis of adaptive evolution and speciation. In addition, these patterns can also be compared among species from different regions to infer the impact of global-scale historical and present events, such as Pleistocene glacial cycles, on current biodiversity. In this review, I summarize and integrate recent findings of phylogeographic studies for two geographic lineages, Pacific Ocean (PO) and Sea of Japan (SJ) lineages, of five coastal gobies distributed around the Japanese Archipelago. Using mitochondrial and nuclear DNA markers, these studies revealed genetic divergences of the PO and SJ lineages in the coastal gobies. However, the two types of markers showed discordant patterns for genetic divergences between the two lineages (mitonuclear discordance), suggesting different coalescent processes between these genetic markers. Ecological features that depend on shallow coastal resources may contribute to restricted gene flow and be responsible for the existence of the two lineages that are predominantly found in the coastal gobies. All coastal gobies shared sharp contrasts in genetic diversity between the two lineages, with the SJ lineages rapidly expanding during Pleistocene interglacial periods. Such rapid population expansions can provide alternative ways to obtain recent calibration points to estimate divergence times of the two lineages. A comparison of the geographic distributions between the two lineages indicated that the centers of their secondary contact zones vary, which may appear to represent the continuous transition process of the two lineage distributions. Compared with results of phylogeographic studies from other oceans, multiple divergence times, sharply contrasting demographic histories, and two independent secondary contact zones are unique characteristics of the divergence between the PO and SJ lineages. These characteristics will allow us to test the continuous processes of genomic divergence during geographical isolation with environmental fluctuations and the consequences of hybridization of divergent lineages in independent secondary contact zones with different environmental settings. [ABSTRACT FROM AUTHOR]

In this study, we describe the ferns Diplazium ×kanayamaense hyb. nov. and D. ×tsukushiense hyb. nov. and further compare them to parental species D. chinense, D. deciduum and D. fauriei in terms of morphological characteristics, plastids and nuclear DNA markers. These new hybrids have been determined to be endemic to western Japan. The International Union for Conservation of Nature and Natural Resources status was evaluated for D. × kanayamaense as endangered (EN) and D. ×tsukushiense as critically endangered (CR). [ABSTRACT FROM AUTHOR]

Within their natural distribution ranges, plant species exhibit a genetic structure that has been created by global climate change and natural selection over long periods. This genetic structure needs to be conserved for sustainable use of genetic resources. To conserve local forests with different genetic structures, genetic guidelines for seed and seedling transfer in individual species are necessary. Genetic guidelines have been published for 43 Japanese tree species using population genetic data; however, for practical use, more detailed genetic borders between important genetic lineages should be clarified to inform seed collection and planting. Thus, we investigated in detail the genetic borders between two important Japanese oak species, Quercus serrata and Quercus crispula, in the Chubu region of Japan using chloroplast and nuclear DNA markers, and we discuss the factors that influenced border creation using the results of species distribution modeling (SDM). The chloroplast DNA (cpDNA) haplotype was clearly different within the Chubu region of Japan but the difference in nuclear DNA between northern and southern haplotype populations was very small, both in Q. serrata and Q. crispula. The results of SDM showed that during the last glacial maximum (LGM) Q. serrata was distributed mostly along the coastline but Q. crispula was distributed not only along the coast but also in mountainous areas further inland. The cpDNA genetic borders of these two oak species are complex and seem to have been influenced by topography and their distribution during the LGM. We propose and discuss genetic guidelines for these two oak species based on the results of this study. [ABSTRACT FROM AUTHOR]

PREMISE: Orchids depend primarily on mycorrhizal fungi to obtain nutrients throughout their life cycle. Epiphytic orchids account for 69% of orchid diversity. The unstable availability of water and nutrients in their arboreal habitats often results in severe water and nutrient stresses. Consequently, mycorrhizal associations may be important for the survival of epiphytic orchids, but our understanding thereof remains limited. Here, we investigated the mycorrhizal community in a single epiphytic orchid species, using more samples than in any previous study. METHODS: We assessed the mycorrhizal communities of Thrixspermum japonicum, one of the most common epiphytic orchids in the temperate region of Japan. In total, 144 individuals were collected from 28 host tree species at 20 sites across 1300 km. The mycorrhizal fungi were identified based on nuclear ribosomal DNA internal transcribed spacer sequences and assigned operational taxonomic units (OTUs) based on 97% sequence similarity. RESULTS: We obtained 24 OTUs; 9 belonged to the Ceratobasidiaceae and 15 to the Tulasnellaceae. These OTUs are widely distributed throughout the phylogenetic trees of the two fungal families. However, a single Ceratobasidiaceae OTU accounted for 49.7% of all fungal sequences and was predominant in samples from 15 host tree species and 12 sites. CONCLUSIONS: Our results imply that despite having a broad range of mycorrhizal partners, T. japonicum was predominantly associated with a single fungal taxon at most of the sites among the host‐tree species investigated. These findings contribute to elucidating mycorrhizal symbiosis in epiphytic habitats. [ABSTRACT FROM AUTHOR]

I describe Deparia ×nanakuraensis hyb. nov. and discuss differences in morphological characteristics between parental species D. pterorachis and D. viridifrons with chromosome counting, plastid, and nuclear DNA markers. The new hybrid is endemic to the eastern and northern parts of Japan. Based on the criteria of the International Union for Conservation of Nature and Natural Resources, this new species is here considered Data Deficient. The ploidy level is diploid sterile. [ABSTRACT FROM AUTHOR]

An endangered tetraploid spined loach species, Cobitis takenoi (Cypriniformes: Cobitidae; hereafter called Tango loach) is known to inhabit only a single river in Kyoto Prefecture, Japan. Since Tango loach was discovered recently, in 2010, and only described in 2016, its morphology, ecology, and genetics are not well studied. Another tetraploid spined loach species Cobitis sp. BIWAE type A (hereafter, called Ohshima loach) inhabits the same river. The two loaches are reported as morphologically distinguishable from each other. Although the habitats of the two species in the river are segregated (Ohshima loach and Tango loach inhabit the upper and lower reaches, respectively), they overlap to a small degree in the boundary area. Recently, some individuals with morphological characteristics that are intermediate between the two species were found in the overlap zone. It was suspected that hybrids between the two species were produced since breeding seasons of the two species overlapped. To investigate whether the two species produce hybrids, we performed mitochondrial and nuclear DNA analyses on the unidentifiable individuals. Eight individuals unidentifiable to the species level collected in the river between 2017 and 2018 were examined and compared with the Tango and Ohshima loach species. Using mitochondrial DNA (mtDNA) cytochrome b analysis, we found that six individuals had mtDNA types identical to Tango loach and two individuals had mtDNA types identical to Ohshima loach. Furthermore, sequencing analysis of nuclear recombination activating gene 1 (RAG‐1) revealed that each species had species‐specific alleles. The phylogenetic analysis indicated that alleles in Tango loach were divided into two clusters and those from Ohshima loach formed a single cluster. There were no discrepancies in the combination between mtDNA and nuclear DNA species types within each specimen. DNA fingerprinting analysis (AFLP) showed that the species‐unidentifiable individuals exhibited distinctly segregated genetic groups corresponding with Tango and Ohshima loaches. In summary, no hybrids were detected from among any unidentifiable individual examined in this study. New conventional genetic method for discriminating the two sympatric loach species developed here can be effective tool for the conservation of the Tango loach since there was no strict diagnostic morphological character between them. [ABSTRACT FROM AUTHOR]

Wolbachia, cytoplasmically inherited endosymbionts of arthropods, are known to hijack their host reproduction in various ways to increase their own vertical transmission. This may lead to the selective sweep of associated mitochondria, which can have a large impact on the evolution of mitochondrial lineages. In Japan, two different Wolbacahia strains (wCI and wFem) are found in two sister species of pierid butterflies, Eurema mandarina and Eurema hecabe. In both species, females infected with wCI (C females) produce offspring with a nearly 1:1 sex ratio, while females infected with both wCI and wFem (CF females) produce all‐female offspring. Previous studies have suggested the historical occurrence of hybrid introgression in C individuals between the two species. Furthermore, hybrid introgression in CF individuals is suggested by the distinct mitochondrial lineages between C females and CF females of E. mandarina. In this study, we performed phylogenetic analyses based on nuclear DNA and mitochondrial DNA markers of E. hecabe with previously published data on E. mandarina. We found that the nuclear DNA of this species significantly diverged from that of E. mandarina. By contrast, mitochondrial DNA haplotypes comprised two clades, mostly reflecting Wolbachia infection status rather than the individual species. Collectively, our results support the previously suggested occurrence of two independent historical events wherein the cytoplasms of CF females and C females moved between E. hecabe and E. mandarina through hybrid introgression. [ABSTRACT FROM AUTHOR]

Rhodeus ocellatus kurumeus is a small cyprinid fish endemic to the Japanese archipelago. This fish frequently hybridizes with an exotic subspecies Rhodeus ocellatus ocellatus, and non-introgressed populations of R. o. kurumeus have greatly decreased. Previous studies based on mitochondrial and nuclear DNA identified introgressed populations and inferred phylogenetic relationships, but these approaches may lead to underestimates of introgression or ambiguous results owing to the small number of genetic markers used. The present study applied double digest restriction site-associated DNA (ddRAD) sequencing to assignment tests and phylogenetic inferences. Data matrices can vary in size and content depending on the strategies used to process ddRAD sequences; therefore, we prepared 25 data matrices with various processing strategies. The assignment tests based on the 25 data matrices resulted in similar assignment patterns and provided evidence for introgression in some populations that had been identified as non-introgressed populations in previous studies. The maximum-likelihood (ML) phylogenetic trees differed in robustness and topology among the 25 matrices; interestingly, ML trees with low statistical support reflected geographical distributions of fish better than ML trees with high statistical support. These results indicate that ddRAD sequencing can detect introgression with greater sensitivity than conventional DNA markers, that ddRAD sequencing is useful for phylogenetic inference among closely related populations within a subspecies, and that statistically robust ML trees do not necessarily reflect the true phylogeny. Application of ddRAD data to conservation genetics and evolutionary history of this subspecies are also discussed. [ABSTRACT FROM AUTHOR]

Multiclavula petricola is described as a new species based on morphological characters, its unique habitat on rocks, and the result of molecular phylogenetic analyses of the sequence of internal transcribed spacer of the nuclear ribosomal DNA. The fruiting bodies of M. petricola were dispersed on wet rock surface with numerous lichenized globules. The globular thallus was organized by several green algal photobiont cells with pyrenoids surrounded by mycobiont hyphae. The phylogenetic analyses indicated that its affiliation was in Multiclavula. The basidiospores of M. petricola germinated on water agar, and the brief descriptions of the colony morphology are also provided. • Multiclavula petricola , a new clavarioid fungus, is described from Japan. • This species is distinguishable by the substrate and morpho-anatomical features. • The lichenized thallus of this species has a globular structure. • The photobiont of this species is a green alga containing a pyrenoid. [ABSTRACT FROM AUTHOR]

Isaria macroscyticola is a hypocrealean pathogen of Cydnidae first described by Dr. Yosio Kobayasi in 1941. Since then, records of this species have been limited, and therefore no cultures have been established and its phylogenetic position is unknown. Recently, synnemata on Macroscytus japonensis were collected from Kanagawa Prefecture, Japan. These were identified as I. macroscyticola based on the morphologies of the synnemata, conidiophores, and conidia. Since cultures established by conidial isolations exhibited pale purplish colonies and Acremonium -like synanamorph, this species was suggested to belong to Purpureocillium (Ophiocordycipitaceae) rather than Isaria (Cordycipitaceae). The sequence similarities of the internal transcribed spacer region and 28S of nuclear ribosomal DNA and the elongation factor 1-alpha gene sequences of I. macroscyticola and P. lilacinum were 99–100%. Furthermore, I. macroscyticola was phylogenetically nested within the P. lilacinum clade. Accordingly, we conclude that I. macroscyticola is a synnematous form and synonym of P. lilacinum. • Isaria macroscyticola , a pathogen of Cydnidae, was rediscovered in Japan. • The species is characterized by ochraceous synnemata covered with purplish conidia. • The isolate formed pale-purplish colonies and Acremonium -like synanamorph. • I. macroscyticola was phylogenetically nested in Purpureocillium lilacinum. • I. macroscyticola is considered to be a synnematous form of P. lilacinum. [ABSTRACT FROM AUTHOR]

The anti-equatorial labrid Pseudojuloides elongatus has a wide but disjunct distribution across the Western Pacific and Eastern Indian Oceans, with populations occurring in Western Australia, southern Japan, and the southwest Pacific Ocean. Principal component analysis of morphological characters and coalescent-based species-tree estimates of mitochondrial and nuclear DNA markers suggest that these populations are under incipient stages of divergence. The three allopatric populations differ strongly in coloration patterns of both sexes, particularly in terminal males, suggestive of reproductive isolation. We redescribe Pseudojuloides elongatus on the basis of nine paratypes and two additional specimens from eastern Australia and Norfolk Island, and describe two new species, Pseudojuloides crux, new species, from Western Australia, and P. paradiseus, new species, from southern Japan. The complex is distinguished from other members of the genus in sharing the following combination of characters: body elongate; dorsal-fin rays IX,12; pectoral-fin rays 12; no median predorsal scales; and usually 27 lateral-line scales. We briefly comment on anti-equatorial biogeographical patterns and Pseudojuloides argyreogaster from the Western Indian Ocean. [ABSTRACT FROM AUTHOR]

The evolutionary history and diversity of unionoid mussels in East Asia need to be clarified and would shed light on the formation process of the unique fauna of Japan. Unionoid mussels (Mollusca: Bivalvia) are unique models for understanding the process by which organisms have diversified before and after the formation of the Japanese archipelago. Unionoid mussels have poor dispersal ability, so it is thought that they would have been strongly influenced by the archipelago's formation. Therefore, the speciation and diversification processes of mussels before and after the archipelago's formation were investigated by analyzing the nuclear and mitochondrial DNA of a wide range of species, particularly those inhabiting East Asia. The evolutionary history and divergence time of these mussels were examined. Unionoid mussels were found to have higher endemicity than other freshwater organisms. Although most of the endemic unionoid mussels of Japan are likely to have diverged before the formation of the Japanese archipelago, some other Japanese unionoid mussel species, including species endemic to Lake Biwa, an ancient lake in Japan, potentially diverged after the Japanese archipelago began to separate from the continent. This suggest that adaptation to the unique habitat of the ancient lake has caused diversification in the mussels endemic to it. [ABSTRACT FROM AUTHOR]

Pandanus boninensis, endemic to the Ogasawara Islands, Japan, is distributed on both the older Bonin and younger Volcano Islands. In this study, we conducted population genetic analyses of P. boninensis on these islands to examine the population diversity and structure across old and young islands, to assess potential differences in population demography with island age, and to collect any evidence of migration between old and young islands. We found that the genetic diversity of expressed sequence tag (EST)–based microsatellite (SSR) markers, the nucleotide diversity of nuclear DNA sequences, and the haplotype diversity of chloroplast DNA on young islands were lower than those on old islands. Clustering analyses of EST‐SSR indicated that populations on old islands were strongly diverged from those on young islands. Approximate Bayesian computation analysis of EST‐SSR suggested that population expansion occurred on old islands while population reduction occurred on young islands. We also found evidence of migration among old islands (mostly from south to north), while it appears that there have been very few migration events between old and young islands. These differences could be due to the fact that young islands tend to be geographically isolated and support smaller populations that began a shorter time ago from limited founders. The P. boninensis populations on the Volcano Islands are interesting from an evolutionary perspective as they constitute a classic example of the early stages of progressive colonization on oceanic islands with small effective population sizes and low genetic diversity. [ABSTRACT FROM AUTHOR]

Plagiothecium longisetum was described by Lindberg in 1872, based on Maximowicz materials from Japan. In the 1970s, this species was synonymized with P. nemorale. However, a polyphasic approach applied to the investigation of the P. nemorale sensu lato showed a clear separation between the specimens of former P. longisetum and the type of P. nemorale. Morphological features and molecular analyses provide evidence that those two groups are distinct, as well as allowed to describe the new species. The results are strongly supported by the statistical analyses of morphometric features and phylogenetic analyses based on concatenated nuclear and chloroplast DNA markers. The maximum likelihood (ML) and Bayesian inference (BI) analyses of ITS, rps4 and rpl16 regions place both species outside the P. nemorale group. The distinctions between individual species, reflected by the morphological features—easy to observe—and the molecular data, provide a scientific foundation for the resurrection of P. longisetum Lindb. and establishment of a new species–P. angusticellum sp. nov. [ABSTRACT FROM AUTHOR]

Ascomata of a Metarhizium species were collected from Tochigi Prefecture, Japan. The ascomata were similar to M. kalasinense in its host preference for elaterids, olive-green clavate stromata, and obliquely immersed perithecia, but the asci and ascospores were significantly shorter in length than those of the latter species. Furthermore, phylogenetic analyses based on the internal transcribed spacer region of nuclear ribosomal DNA and the elongation factor 1-alpha gene showed that the Japanese species was phylogenetically distinctive in the M. anisopliae lineage, including M. kalasinense. Accordingly, we describe a new species, M. brachyspermum. Isolates of this species produced olive-green conidial masses on the surface of the colony. Also, hyphal anastomoses between two metulae were often observed on conidiophores. • Metarhizium brachyspermum , a new pathogen of elaterid, is described from Japan. • This species is distinguishable from similar species by the size of ascospores. • Isolates of this species produced olive-green conidial masses. • Metarhizium brachyspermum belongs to the M. anisopliae lineage. [ABSTRACT FROM AUTHOR]

Several individuals of an unknown taxon of Goodyera were discovered on Kozu Island, Japan. Detailed morphological investigation suggests that this taxon is considered as a natural hybrid between G. maximowicziana and G. velutina based on morphological characteristics, such as length of peduncle. Since the natural hybrid between G. maximowicziana and G. velutina was recently reported in Jeju Island, South Korea and described as G. xmaximo-velutina, here we recorded the first occurrence of G. xmaximo-velutina from Japan. Investigation on chromosome number among G. maximowicziana, G. velutina, and G. xmaximo-velutina provides the further support of hybrid status in G. xmaximo-velutina (Orchidaceae) from Kozu Island, Japan. We also discussed the utility of the internal transcribed spacer (ITS) regions of the nuclear ribosomal DNA for detecting the hybrid between G. maximowicziana and G. velutina. [ABSTRACT FROM AUTHOR]

To determine the origin and genetic consequences of anagenesis in Rubus takesimensis on Ulleung Island, Korea, we compared the genetic diversity and population structure of R. takesimensis with those of its continental progenitor R. crataegifolius. We broadly sampled a total of 315 accessions in 35 populations and sequenced five noncoding regions of chloroplast DNA. Rubus takesimensis emerged as nonmonophyletic and several geographically diverse continental populations were likely responsible for the origin of R. takesimensis; the majority of R. takesimensis accessions were sisters to the clade containing accessions of R. crataegifolius, primarily from the Korean peninsula, while rare accessions from three populations shared common ancestors with the ones from the southern part of the Korean peninsula, Jeju Island, and Japan. A few accessions from the Chusan population originated independently from the Korean peninsula. Of 129 haplotypes, 81 and 48 were found exclusively in R. crataegifolius and R. takesimensis, respectively. We found unusually high genetic diversity in two regions on Ulleung Island and no geographic population structure. For R. crataegifolius, two major haplotype groups were found; one for the northern mainland Korean peninsula, and the other for the southern Korean peninsula and the Japanese archipelago. Compared with populations of R. crataegifolius sampled from Japan, much higher haplotype diversity was found in populations from the Korean peninsula. The patterns of genetic consequences in R. takesimensis need to be verified for other endemic species based on chloroplast DNA and independent nuclear markers to synthesize emerging patterns of anagenetic speciation on Ulleung Island. [ABSTRACT FROM AUTHOR]

The DNA-based identification of aquatic insects, which show high species diversity, is becoming increasingly popular because these insects are excellent indicators of the condition of the aquatic environment. Spiny crawler mayflies (Ephemerellidae) are aquatic insects known for their altitudinal stratified distribution patterns and niche partitioning at the microhabitat scale. For these reasons, they are a good bioindicator of the condition of an aquatic ecosystem. Here, we report the results of a study in which we obtained DNA barcoding data on various genotypes of spiny crawler mayfly species collected in various areas. In addition, we discuss the importance of accumulating genetic data collected from a wide area. We conducted molecular analyses of Drunella mayflies based on the mitochondrial DNA COI region and the nuclear DNA H3 region. A total of 101 specimens of 8 spiny crawler mayfly species were collected between 2013 and 2016 from 75 locations of the East Asian region, including the Japanese Archipelago, the Korean Peninsula, and the Russian Far East. We found a total of 86 haplotypes and 57 genotypes of East Asian Drunella mayflies and reinterpreted two cryptic Drunella lineages (Drunella basalis clades I and II and Drunella ishiyamana clades I and II). [ABSTRACT FROM AUTHOR]

• No significant difference in nuclear DNA between Lepus mandshuricus and L. coreanus. • Recurrent mitochondrial introgression from L. timidus to the L. mandshuricus complex. • Long independent history of L. brachyurus in the Japanese Archipelago. • Reticulate evolution of continental hares, but no involvement of insular hares. Hares of the genus Lepus are distributed worldwide, and introgressive hybridization is thought to be pervasive among species, leading to reticulate evolution and taxonomic confusion. Here, we performed phylogeographic analyses of the following species of hare across East Asia: L. timidus , L. mandshuricus , L. coreanus , and L. brachyurus collected from far-eastern Russia, South Korea, and Japan. Nucleotide sequences of one mitochondrial DNA and eight nuclear gene loci were examined, adding sequences of hares in China from databases. All nuclear DNA analyses supported the clear separation of three phylogroups: L. timidus , L. brachyurus , and the L. mandshuricus complex containing L. coreanus. On the other hand, massive mitochondrial introgression from two L. timidus lineages to the L. mandshuricus complex was suggested in continental East Asia. The northern population of the L. mandshuricus complex was mainly associated with introgression from the continental lineage of L. timidus , possibly since the last glacial period, whereas the southern population of the L. mandshuricus complex experienced introgression from another L. timidus lineage related to the Hokkaido population, possibly before the last glacial period. In contrast to continental hares, no evidence of introgression was found in L. brachyurus in the Japanese Archipelago, which showed the oldest divergence amongst East Asian hare lineages. Our findings suggest that glacial–interglacial climate changes in the circum-Japan Sea region promoted distribution shifts and introgressive hybridization among continental hare species, while the geographic structure of the region contributed to long-term isolation of hares on the islands, preventing inter-species gene flow. [ABSTRACT FROM AUTHOR]

A recent phylogenetic study based only on chloroplast DNA (cpDNA) variation revealed that populations of an Isodon species are frequently embedded paraphyletically among other Isodon species. This phylogenetic discrepancy between species taxonomy and molecular phylogeny was considered to have resulted from chloroplast DNA captures and/or incomplete lineage sorting. To elucidate which of these factors was mainly responsible for the observed phylogenetic pattern, we performed phylogenetic analyses of multiple populations of Isodon species in Japan using cpDNA variation, three single‐copy nuclear genes, and double‐digest restriction‐site‐associated DNA sequencing (ddRAD‐seq). Although a species often shared chlorotypes with other species, our phylogenetical analyses based on variation in the three single‐copy nuclear genes and the ddRAD‐seq data showed that most populations belonging to the same species were monophyletic at the species level, suggesting that chloroplast capture may have frequently occurred between Isodon species. Some populations of an intraspecific taxon were embedded paraphyletically within the species, regardless of the large amount of phylogenetic information in nuclear DNA; this incongruity may have resulted from incomplete lineage sorting. [ABSTRACT FROM AUTHOR]

The genetic diversity and structure of Pulsatilla cernua, a continental‐grassland relict, were investigated using variations in chloroplast DNA (cpDNA) and microsatellites of nuclear DNA. In the analyses of three cpDNA regions, 17 haplotypes were found in 24 populations of P. cernua from Japan, Korea, and Russia. Although the route and time of migration between the continent of Asia and Japan could not be well resolved, the cpDNA haplotype network suggests the existence of several ancient lineages in Japan and a recent secondary migration from Japan to the continent. Microsatellite analyses did not indicate genetic structure among the Japanese populations, indicating the existence of gene flow across the distribution area until recently. These results indicate that the present fragmentation of P. cernua in Japan may reflect a rapid, recent reduction from a previously large, continuous distribution. [ABSTRACT FROM AUTHOR]

Aim: Vicariance events have been proposed as a major source of lineage divergence on continental islands, whereas dispersal events followed by isolation have been proposed as the major cause on oceanic islands. However, organisms on continental islands may include taxa with characteristics similar to those on oceanic islands. Lineage divergence unassociated with the geological events that separated islands may also have occurred. This study addresses these possibilities through morphological and molecular phylogeographic analyses of land snails (Bradybaena) on continental islands. Location: Samples were mainly collected on the Ryukyu and Izu Islands in Japan. Methods: Molecular phylogenetic analyses using mtDNA (16S rRNA+COI) and nDNA (18S rRNA+ITS1+5.8S rRNA+ITS2+28S rRNA) were conducted for 225 individuals comprising B. circulus, B. phaeogramma and four of its subspecies endemic to the Ryukyu Islands. Shell and genital morphologies were also examined using qualitative and statistical methods. Results: The Bradybaena populations examined were phylogenetically classified into six major clades whose divergence occurred during either Miocene‐Pliocene or Pliocene‐Pleistocene. Although phylogeographic patterns estimated on the basis of the 16S rRNA gene support the vicariance hypothesis for the origin of the major clades of Bradybaena, the phylogeographic patterns estimated by COI showed that these major clades resulted from diversification before the archipelagos separated from the continent. Both of these phylogeographic patterns suggest that genetic divergence occurred by dispersal from the southern to northern islands after divergence of major clades. The inferred phylogenies document parallel morphological evolution, yielding phylogenetic, morphological and taxonomic incongruences that created mosaic geographical patterns in the distribution of nominal taxa. Main conclusions: Lineage diversification in endemic species on continental islands can occur before archipelago separation or subsequently via dispersal, as is seen in species on oceanic islands. Mechanisms other than vicariance should also be considered when estimating processes that created geographical patterns of genetic variations on continental islands. [ABSTRACT FROM AUTHOR]

Vitex rotundifolia is an important coastal and medicinal plant, and is recorded in the List of the Important Wild Plants for Conservation in China and Japan. However, an effective conservation strategy is lacking. In the present study, the genetic diversity and population structure were analyzed using phylogeographical methods based on the trnH-psbA and trnG-trnS intergenic spacers of the chloroplast DNA (cpDNA) sequences from 157 individuals from 25 sampling sites for V. rotundifolia and V. trifolia plus the internal transcribed spacer (ITS) of the nuclear ribosomal DNA (nrDNA) sequences of 177 individuals from 27 sampling sites. The results showed that V. rotundifolia and V. trifolia had eight cpDNA and two nrDNA haplotypes, respectively, and the V. rotundifolia has a low level of genetic diversity (haplotype diversity hd,cp D 0.360, hd,nr D 0.440), a more pronounced genetic differentiation among populations (population differentiation at the species level (GST)D0.201, population differentiation at the allele level (NST) D 0.462), and an insignificantly different phylogeographical structure (NST > GST, P >0:05). In addition, haplotype network analyses indicated that V. rotundifolia and V. trifolia have distinct haplotypes. Divergence dating based on BEAST software analyses showed that most cpDNA clades diverged in the late Pleistocene era. Demographic analysis indicated that V. rotundifolia underwent a rapid demographic expansion. Some scientific strategies are suggested for resource conservation of V. rotundifolia based on its genetic diversity and population structure. [ABSTRACT FROM AUTHOR]

[Display omitted] • Pheretimoid megascolecids are the major component of earthworm fauna in Japan. • Their phylogenetic relationships were studied using mitogenome sequence data. • Multiple Japan-endemic groups originated from Taiwan Island and continental China. • Distribution ranges within the Japanese Archipelago differed among the endemic groups. • The endemic groups may have diversified during the Miocene or later. Megascolecid earthworms of the pheretimoid group are dominant detritivores of soil ecosystems in the Japanese Archipelago and East Asia. However, their diversity and phylogenetic relationships are poorly understood. We assembled whole mitogenome sequences for 197 megascolecid earthworms collected throughout Japan to study the phylogenetic relationships, phylogeography, divergence times, and diversification of important morphological characteristics among pheretimoid earthworms. Using 197 mitogenome sequences and 24 published mitogenome sequences from the East Asian mainland (221 sequences in total), we constructed a maximum likelihood tree and found that the pheretimoid earthworms currently assigned to Amynthas , Metaphire , Duplodicodrilus , and Manus are involved in the most senior genus Amynthas ; thus, Amynthas can be treated as the sole genus encompassing all of the above genera. Within the Amynthas group, we identified three major lineages that led to four groups of endemic species in Japan. These lineages originated from different lineages on the East Asian mainland and Taiwan Island, indicating multiple colonization events from the East Asian mainland by different ancestral lineages, possibly after the Miocene. We also assembled nuclear ribosomal DNA sequences encompassing the 18S to 28S rRNA genes. The nuclear gene tree showed major groups consistent with the mitogenome tree except for different (and not well-resolved) relationships among major clades. Our molecular data covered 115–158 native and 7 non-native Amynthas group species in Japan in terms of DNA-based species delimitation. Our findings provide a basis for understanding the evolutionary relationships among diversified megascolecid earthworms in the Amynthas group in Japan and adjacent regions. [ABSTRACT FROM AUTHOR]

[Display omitted] • There are inconsistencies in phylogenies based on nuclear and mitochondrial DNA. • Two well-supported genome DNA-based clades show the presence of cryptic species. • Molecular clock shows Beringiana and Sinanodonta diverged >35 million years ago. • Ball-like shell morphology repeatedly appeared in the two genera in Lake Biwa. • The mtDNA-based classification of unionid mussels requires careful consideration. Accurate species identification is of primary importance in ecology and evolutionary biology. For a long time, the unionid mussels Beringiana and Sinanodonta have puzzled researchers trying to unravel their diversity because of their poorly discernible morphologies. A recent study conducted species delineation of unionid mussels based on mitochondrial DNA variation, opening up a new avenue to grasp species diversity of the mussels. However, mtDNA-based classification may not align with species boundaries because mtDNA is prone to introgression and incomplete lineage sorting that cause discordance between species affiliation and gene phylogeny. In this study, we evaluated the validity of the mtDNA-based classification of unionid mussels Beringiana and Sinanodonta in Japan using mitochondrial sequence data, double digest restriction site-associated DNA library (ddRAD) sequencing, and morphological data. We found significant inconsistencies in the mitochondrial and nuclear DNA phylogenies, casting doubt on the reliability of the mtDNA-based classification in this group. In addition, nuclear DNA phylogeny revealed that there are at least two unionid lineages hidden in the mtDNA phylogeny. Although molecular dating technique indicates that Beringiana and Sinanodonta diverged >35 million years ago, their shell morphologies are often indistinguishable. Specifically, morphological analyses exhibited the parallel appearance of nearly identical ball-like shell forms in the two genera in Lake Biwa, which further complicates species identification and the morphological evolution of unionid mussels. Our study adds to a growing body of literature that accurate species identification of unionid mussels is difficult when using morphological characters alone. Although mtDNA-based classification is a simple and convenient way to classify unionid mussels, considerable caution is warranted for its application in ecological and evolutionary studies. [ABSTRACT FROM AUTHOR]

The genus Rhabdias Stiles & Hassall, 1905 comprises lung parasites of amphibians and reptiles worldwide. In Japan, 9 species have been recorded, including Rhabdias incerta Wilkie, 1930 which has been reported only in Bufo species. In this study, to assess the diversity of R. incerta , we performed molecular analyses of Rhabdias species sampled from three species/subspecies of Japanese toads namely Bufo japonicus , B. japonicus formosus , and B. torrenticola , collected in various regions of Honshu, Shikoku, and Kyushu, Japan. DNA sequence divergence was compared using mtDNA (COI) and nuclear DNA (28S) to identify possible cryptic species. Morphological analysis was performed through light microscopy and scanning electron microscopy (SEM). The results revealed that Bufo spp. serve as hosts for at least three Rhabdias species. Morphologically, most samples were identified as R. incerta but with a longer body and larger buccal cavity than originally described. Rhabdias incerta appears to be specific to the genus Bufo and is further subdivided into two or three phylogroups based on subspecies divisions and biogeography of their host. Some Rhabdias specimens collected in this study resemble R. tokyoensis Wilkie, 1930, parasitic in hosts from the order Caudata, which suggests host switching. Both molecular and morphological analyses suggested the presence of undescribed and cryptic Rhabdias species within toads collected in Japan. This study was the first to molecularly characterize Rhabdias species in Japan, including novel sequences of R. incerta and two undescribed species. [Display omitted] • Three species of Rhabdias were identified from 3 species/subspecies of Bufo in Japan. • Rhabdias incerta is specific to Bufo and divided depending on its host. • There are possibly 2 crpytic species of Rhabdias within the Japanese toads. • First to generate DNA sequences from R. incerta and R. tokyoensis. [ABSTRACT FROM AUTHOR]

Little is known about the spatiotemporal dynamics of gray wolves in the Pleistocene across low-latitude regions of Eurasia. In Japan, a small-bodied endemic subspecies of Japanese wolves existed and went extinct in the early 1900s. The fossil record indicates that a giant wolf, which reached 70 cm in body height, inhabited Japan during the Pleistocene, but its evolutionary relationship, if any, with the Japanese wolf remains uncertain. Here, to reveal the genetic origin of the Japanese wolf, we analyzed ancient DNA from remains (recovered in Japan) of one Pleistocene wolf that lived 35,000 years ago and one Holocene wolf from 5,000 years ago. The analysis of the mitochondrial DNA revealed that the Pleistocene wolf was not part of the Japanese wolf clade but rather an earlier-diverging lineage. The analysis of the nuclear DNA of the Holocene Japanese wolf revealed that it was an admixture of the Japanese Pleistocene wolf and continental wolf lineages. These findings suggest that the Japanese wolf originated via waves of colonization of multiple Pleistocene wolf populations at 57–35 and 37–14 ka, respectively, followed by interpopulation hybridization. [Display omitted] • Ancient DNA of 35,000- and 5,000-year-old wolf fossils found in Japan are analyzed • The Pleistocene giant wolf was one of the early-diverging Pleistocene wolf lineages • An admixed genome of the Japanese wolf suggests its unique origin via hybridization • Repeated wolf expansions caused a complex colonization history in Japanese islands Segawa et al. report ancient DNA from fossils of a giant Pleistocene wolf that lived 35,000 years ago and a Japanese wolf (Canis lupus hodophilax) that lived 5,000 years ago. This small-bodied extinct subspecies was an admixture of the Pleistocene wolf in Japan and continental wolf lineages. Repeated migration of ancient wolves resulted in a complex colonization history. [ABSTRACT FROM AUTHOR]

The genetic constitution and phylogenetic relationships among the proposed species and subspecies of the crucian carp complex in Japan ( Carassius cuvieri, C. auratus subspp. 1 and 2, C. a. grandoculis, C. a. buergeri, and C. a. langsdorfii) were investigated based on analyses of the partial nucleotide sequences of the mitochondrial DNA control region and amplified fragment length polymorphisms of nuclear DNA. Our results demonstrate that C. cuvieri and C. auratus are different entities. However, although several distinct lineages were observed for C. auratus, none corresponded to the proposed subspecies. Moreover, the five subspecies of C. auratus were not necessarily separated genetically from each other. Triploid fish, which are currently classified as a single subspecies ( C. a. langsdorfii) but are sometimes treated as an independent species, fell into various clades along with diploid individuals of other subspecies that have the same or similar mitochondrial haplotypes. This suggests that gynogenetic triploid crucian carps distributed throughout the Japanese Archipelago have polyphyletic maternal origins. Our results indicate that Japanese crucian carps are a much more complex assemblage than previously believed. [ABSTRACT FROM AUTHOR]

Betula maximowicziana is an ecologically and economically important tree species in Japan. In order to examine the phylogeographical pattern of the species in detail, maternally inherited chloroplast (cp) DNA variations of 25 natural populations of Betula maximowicziana and a total of 12 populations of three related species were evaluated by PCR-RFLP analysis. Two main haplotypic groups of B. maximowicziana populations (northern and southern) were detected, with the main boundary passing through the Tohoku region in northeastern Japan; in addition there was high genetic differentiation among the 25 populations studied ( GST = 0.950, $$ G_{\text{ST}}^{\prime } = 0. 9 7 7 $$). The phylogeographical pattern exhibited by B. maximowicziana was much more similar to that of alpine plants than to that of beech and oak. Comparison of the patterns of genetic structure obtained from the cpDNA with previously and newly acquired data on bi-parentally inherited nuclear DNA indicates that the nuclear genome was transferred via pollen from the northern haplotypic group to the southern group more frequently than it moved in the opposite direction. Although common haplotypes were detected among B. maximowicziana and the two related species examined, these haplotypes were not shared sympatrically, suggesting very rare hybridization among the species currently occurring in their natural populations. [ABSTRACT FROM AUTHOR]

Hybrids between native white-spotted charr Salvelinus leucomaenis and non-native brown trout Salmo trutta were identified in streams of Hokkaido, Japan, using both appearance and genetic characters. The DNA analyses indicated that the specimens were hybrids between female S. leucomaenis and male S. trutta. Occurrence of such hybrids implies increased mating opportunities between these species in wild streams. [ABSTRACT FROM AUTHOR]

A scincid lizard of the genus Plestiodon from Kuchinoshima Island in the Tokara Group of the Northern Ryukyus, Japan, has proved to be genetically and morphologically differentiated from any previously recognized species in the genus. We thus describe this island population as a new species, Plestiodon kuchinoshimensis. The new species shows characteristics of the P. latiscutatus species group, but differs from other species of this group by the combination of the following character states: postnasal absent; hatchling with five longitudinal light lines on dorsum; lateral light line on each side passing over ear opening and the sixth to eighth scale rows at midbody; dorsolateral light line beginning from behind supraoculars; patch of enlarged irregular scales on posterior femur absent; scale rows around midbody 27-32; and brownish background on the dorsal surface of the juvenile.