Range-Wide Phylogeography and Ecological Niche Modeling Provide Insights into the Evolutionary History of the Mongolian Racerunner (Eremias argus) in Northeast Asia.

The Mongolian racerunner, Eremias argus, is a small lizard endemic to Northeast Asia that can serve as an excellent model for investigating how geography and past climate change have jointly influenced the evolution of biodiversity in this region. To elucidate the processes underlying its diversification and demography, we reconstructed the range-wide phylogeographic pattern and evolutionary trajectory, using phylogenetic, population genetic, landscape genetic, Bayesian phylogeographic reconstruction and ecological niche modeling approaches. Phylogenetic analyses of the mtDNA cyt b gene revealed eight lineages that were unbounded by geographic region. The genetic structure of E. argus was mainly determined by geographic distance. Divergence dating indicated that E. argus and E. brenchleyi diverged during the Mid-Pliocene Warm Period. E. argus was estimated to have coalesced at~0.4351 Ma (Marine Isotope Stage 19). Bayesian phylogeographic diffusion analysis revealed out-of-Inner Mongolia and rapid colonization events from the end of the Last Interglacial to the Last Glacial Maximum, which is consistent with the expanded suitable range of the Last Glacial Maximum. Pre-Last Glacial Maximum growth of population is presented for most lineages of E. argus. The Glacial Maximum contraction model and the previous multiple glacial refugia hypotheses are rejected. This may be due to an increase in the amount of climatically favorable habitats in Northeast Asia. Furthermore, E. argus barbouri most likely represents an invalid taxon. The present study is the first to report a range-wide phylogeography of reptiles over such a large region in Northeast Asia. Our results make a significant contribution towards understanding the biogeography of the entire Northeast Asia.

The complete mitochondrial genome of Rana coreana (Anura: Ranidae).

Rana coreana is a brown frog species native to the Korean Peninsula. We characterized the complete mitochondrial genome of the species. The mitochondrial genome sequence of R. coreana is 22,262 bp and comprises 13 protein-coding genes, two ribosomal RNA (rRNA) genes, 22 transfer RNA (tRNA) genes, and two control regions (CRs). The CR duplication and gene organization were identical to those observed in Rana kunyuensis and Rana amurensis. A total of 13 protein-coding genes were used to examine the phylogenetic relationships between this species and the genus Rana. R. coreana living on the Korean Peninsula, formed a cluster with R. kunyuensis and R. amurensis, with R. coreana showing the closest phylogenetic affinity for R. kunyuensis.

Genetic and morphological variation analyses of Glandirana rugosa with description of a new species (Anura, Ranidae).

Glandirana rugosa is known to include several geographic groups differing in sex chromosomes, and has been proven to be paraphyletic in mitochondrial phylogeny with respect to G. susurra. By analyzing genetic and morphological variation in a large number of individuals of Glandirana, we studied their taxonomic relationships. A mitochondrial DNA phylogeny, with the G. tientaiensis as outgroup, revealed two major lineages containing respectively (1) the East group of G. rugosa, G. susurra, and the Central and Southeast-Kyushu groups of G. rugosa; and (2) G. emeljanovi, and the North and West groups of G. rugosa. In contrast, in a nuclear DNA phylogeny based on SNP data, lineages of (1) G. susurra and East group, and (2) the remaining groups of G. rugosa and G. emeljanovi, were split, indicating a distinct status of the East group among G. rugosa. In adult morphology, there were only minor differences between the East group and the remaining groups of G. rugosa, but in larvae, the East group had significantly more sparse skin glands than the others. The exact type locality of G. rugosa is most probably in western Japan, not including the range of the East group. From these results, we describe the East group as a new species, G. reliquia, distinct from the remaining groups of G. rugosa. The new species with sexually homomorphic chromosomes is thought to represent a basic stock of Japanese Glandirana, which existed far before G. rugosa originated.

Efficient and cost-effective non-invasive population monitoring as a method to assess the genetic diversity of the last remaining population of Amur leopard (Panthera pardus orientalis) in the Russia Far East.

Small populations of the endangered species are more vulnerable to extinction and hence require periodic genetic monitoring to establish and revisit the conservation strategies. The Amur leopard is critically endangered with about 100 individuals in the wild. In this study, we developed a simple and cost-effective noninvasive genetic monitoring protocol for Amur leopards. Also, we investigated the impact of fecal sample's age, storage, and collection season on microsatellite genotyping success and data quality. We identified 89 leopard scats out of the 342 fecal samples collected from Land of the Leopard between 2014-2019. Microsatellite genotyping using 12 markers optimized in 3 multiplex PCR reactions reveals presence of at least 24 leopard individuals (18 males and 6 females). There was a significant difference in the success rate of genotyping depending on the time from feces deposition to collection (p = 0.014, Fisher's exact test), with better genotyping success for samples having <2 weeks of environmental exposure. Amur leopard genetic diversity was found low (Ho- 0.33, HE- 0.35, and NA- 2.57) with no visible population substructure and recent bottleneck signature. Although a historical bottleneck footprint was observed. Mitochondrial DNA diversity was also found low with two haplotypes differing by a point mutation reported in 1,769 bp of investigated sequence covering parts of cytochrome b gene (846 bp), NADH-5 gene (611 bp) and control region (312 bp). We recommend periodic genetic monitoring of wild Amur leopards following the proposed methodology to achieve cost effectiveness and efficiency.

The complete mitochondrial genome of the Amur soft-shelled turtle (Pelodiscus maackii Brandt, 1858), from South Korea.

In this study, we use a specimen from wild-caught individual to determine the complete mitochondrial genome of the Amur soft-shelled turtle (Pelodiscus maackii). The complete mitogenome of P. maackii has 16,258 bp in length and consists of 13 protein-coding genes (PCGs), 22 tRNAs, two rRNAs, and one control region. The arrangement of genes of P. maackii is identical with previously reported mitogenomes in the family Trionychoidea. According to our result, the ML tree for the phylogenetic reconstruction revealed that the individuals used in present study is closely related with the previously reported sequences of P. sinensis (AY962573 and MG431983) in p-distance 0.7% and 2.5%.

Driven to the edge: Species distribution modeling of a Clawed Salamander (Hynobiidae: Onychodactylus koreanus) predicts range shifts and drastic decrease of suitable habitats in response to climate change.

Climate change is one of the major threats to global amphibian diversity, and consequently, the species distribution is expected to shift considerably in the future. Therefore, predicting such shifts is important to guide conservation and management plans. Here, we used eight independent environmental variables and four representative concentration pathways (RCPs) to model the current and future habitat suitability of the Korean clawed salamander (Onychodactylus koreanus) and then defined the dispersal limits of the species using cost distance analysis. The current habitat suitability model generated using the maximum entropy algorithm was highly consistent with the known distribution of the species and had good predictive performance. Projections onto years 2050 and 2070 predicted a drastic decrease of habitat suitability across all RCPs, with up to 90.1% decrease of suitable area and 98.0% decrease of optimal area predicted from binary presence grids. The models also predicted a northeastward shift of habitat suitability toward high-elevation areas and a persistence of suitability along the central ridge of the Baekdudaegan Range. This area is likely to become a climatic refugium for the species in the future, and it should be considered as an area of conservation priority. Therefore, we urge further ecological studies and population monitoring to be conducted across the range of O. koreanus. The vulnerability to rapid climate change is also shared by other congeneric species, and assessing the impacts of climate change on these other species is needed to better conserve this unique lineage of salamanders.

Whole genome survey of big cats (Genus: Panthera) identifies novel microsatellites of utility in conservation genetic study.

Big cats (Genus: Panthera) are among the most threatened mammal groups of the world, owing to hunting, habitat loss, and illegal transnational trade. Conservation genetic studies and effective curbs on poaching are important for the conservation of these charismatic apex predators. A limited number of microsatellite markers exists for Panthera species and researchers often cross-amplify domestic cat microsatellites to study these species. We conducted data mining of seven Panthera genome sequences to discover microsatellites for conservation genetic studies of four threatened big cat species. A total of 32 polymorphic microsatellite loci were identified in silico and tested with 152 big cats, and were found polymorphic in most of the tested species. We propose a set of 12 novel microsatellite markers for use in conservation genetics and wildlife forensic investigations of big cat species. Cumulatively, these markers have a high discriminatory power of one in a million for unrelated individuals and one in a thousand for siblings. Similar PCR conditions of these markers increase the prospects of achieving efficient multiplex PCR assays. This study is a pioneering attempt to synthesise genome wide microsatellite markers for big cats.

The Asian plethodontid salamander preserves historical genetic imprints of recent northern expansion.

The Korean Peninsula, located at the southern tip of Northeast Asia, has never been covered by ice sheets and was a temperate refugium during the Pleistocene. Karsenia koreana, the sole Asian plethodontid salamander species, occurs only on the southern half of the Korean Peninsula and is thought to have found various climatic refugia. Despite its phylogenetic and biogeographic importance, no population-level genetic analysis has been performed on this species. Here we study the population genetic structure of K. koreana using mitochondrial and microsatellite loci to understand the recent historical dispersion process that shaped its current distribution. Overall, the genetic distance between populations correlated well with the spatial distance, and the genetic structure among populations showed signs of a unilateral northward expansion from a southernmost refugium population. Given the distinct genetic structure formed among the populations, the level of historical gene flow among populations appears to have been very low. As the estimated effective population size of K. koreana was also small, these results suggest that the small, restricted populations of K. koreana are extremely vulnerable to environmental changes that may require high levels of genetic diversity to cope with. Thus, special management strategies are needed to preserve these remnant populations.

The complete mitochondrial genome information of Rana uenoi (Amphibia, Anura, Ranidae) and the phylogenetic implication.

We determined the complete mitochondrial genome of Rana uenoi (Anura: Ranidae) for the first time. The whole sequences were 17,370 bp and included 13 protein-coding genes, 2 ribosomal RNA genes, and 22 transfer RNA genes. The gene arrangement was completely identical to those observed from other Ranidae species. We used 11 protein-coding genes to examine the phylogenetic placement of this species in the genus Rana. Rana dybowskii was the closest sister species to R. uenoi. The clade of R. uenoi and R. dybowskii formed a cluster with Rana huarensis, which had a sister relationship with the group of Rana amurensis, Rana coreana, and Rana kunyuensis.

Disentangling the Impacts of Speciation, Sympatry and the Island Effect on the Morphology of Seven Hynobius sp. Salamanders.

Closely related individuals from different areas can see their morphologies change based on differences between clades, but also ecological variables such as the island effect or sympatry. This is the case of salamanders, which have adapted to a broad range of ecological niches, ranging from underground dwellers in xeric landscape to tropical arboreal habitats. On the Korean Peninsula, salamanders from the Hynobius clade are widespread on the mainland and islands, with several partially sympatric clades and candidate species. Currently, seven lineages have been identified based on mtDNA, four of them matching named species and three others for which the species status remains untested. While the morphology of Korean Hynobius is known to be variable between genetically segregated clades, we hypothesise that (1) the candidate species are morphologically different, and that (2) the island effect and (3) the sympatric status have significant impacts on the morphology of individuals within the genus. Here we measured 329 Hynobius salamanders from all seven clades, in areas of sympatry and allopatry, and on islands and on the mainland (Graphical Abstract A). We determined that the island effect had a significant impact on the morphology of the genus, with mainland individuals generally displaying a broader range of morphology than islandic individuals (Graphical Abstract B). We also determined that sympatry had an impact on morphology, with the sizes of individuals from clades in sympatric areas diverging from each other (Graphical Abstract C). Finally, we demonstrated that all seven clades have significantly different morphologies, and we described the three candidate species that had already been isolated based on mtDNA and microsatellite data: Hynobius notialis sp. nov., Hynobius geojeensis sp. nov. and Hynobius perplicatus sp. nov. We conclude that looking at morphology alone would be misleading about the true diversity of Hynobius species, and species in general, because of the island and patry effects.

Phylogeographic study of the Bufo gargarizans species complex, with emphasis on Northeast Asia.

We conduct a phylogeographic and population genetic study of the Asiatic toad (Bufo gargarizans) to understand its evolutionary history, and the influence of geology and climate. A total of 292 individuals from 94 locations were genotyped for two mitochondrial loci (cytb, ND2) and five nuclear introns (Sox9-2, Rho-3, CCNB2-3, UCH-2, and DBI-2), and we performed a suite of phylogenetic, population genetic, and divergence dating analyses. The phylogenetic trees constructed using mitochondrial loci inferred B. gargarizans being divided into two major groups: China mainland and Northeast Asia (Northeast China, Russia, and Korean Peninsula). As with previous studies of this species, we recover population genetic structure not tied to geographic region. Additionally, we discover a new genetic clade restricted to Northeast Asia that points towards the Korean Peninsula being a glacial refugium during the Pleistocene. The weak phylogeographic pattern of B. gargarizans is likely the result of multiple biological, anthropogenic, and historical factors - robust dispersal abilities as a consequence of physiological adaptations, human translocation, geologic activity, and glacial cycles of the Pleistocene. We highlight the complex geologic and climatic history of Northeast Asia and encourage further research to understand its impact on the biodiversity in the region.

Asia-wide phylogeography of wild boar (Sus scrofa) based on mitochondrial DNA and Y-chromosome: Revising the migration routes of wild boar in Asia.

Genetics of pigs has been well studied in Europe and Asia, but most of previous studies of molecular phylogeny of Sus scrofa have been based on sequences of both wild and domestic forms. In this study we analysed genetic traits of Sus scrofa from 13 regions in Asia (including previously undisclosed Eastern Caucasus and Trans-Baikal regions) using purely wild boar samples. Mitochondrial control region and Y-chromosome genes (AMELY & USP9Y) were employed to resolve phylogeographic relationships. We discussed spatio-temporal dynamics of wild boar distribution and compared molecular data to morphological and cytogenetic data on wild boar variability and taxonomy. A total of 51 haplotypes were detected in mtDNA control region and five haplotypes were found in combined sequences of Y-chromosome genes. The phylogeography of Asia-wide wild boars supported a hypothesis of migration from South-East Asia to South Asia, followed by migration to East and West Asia. We present a hypothesis about independent dispersal of wild boars into West Asia from South and North-East Asia. Mitochondrial DNA phylogeny generally fits the morphologically based intraspecies taxonomy. Distribution of chromosomal variants of wild boar presently does not show clear correlation with mtDNA clades.

Phylogenetic relationships between different raccoon dog (Nyctereutes procyonoides) populations based on four nuclear and Y genes.

BACKGROUND: The raccoon dog (Nyctereutes procyonoides), endemic to East Asia, is classified as six subspecies according to their geographical distribution including a population introduced to Europe. Studies on phylogenetic relationship or population genetics in both native and introduced areas have been carried out recently. Lately, opinions that Japanese raccoon dogs should be classified as a different species were asserted based on several studies using karyotypes, morphometric characters, mtDNA, and microsatellites analysis. However, no data pertaining to the nuclear DNA (nDNA) or Y chromosome are available. OBJECTIVE: To estimate the relationship among the species using different genes is necessary in understanding of the history of this species. METHOD: Therefore, we investigated nDNA and Y chromosomes in our study to define relationships: (1) between continental raccoon dog populations, (2) between original and introduced groups, and (3) between continental and Japanese groups. RESULTS: The analysis of four nuclear (CHRNA1, VTN, TRSP, WT1) and ZFY genes indicated that there had been no genetic differentiation among the continental populations. However, significant differences were observed between continental and Japanese raccoon dogs in VTN and ZFY genes implying genetic differentiation has been going between them. CONCLUSION: To better understand the phylogenetic relationship among raccoon dog populations, further study will be necessary.

Yellow sea mediated segregation between North East Asian Dryophytes species.

While comparatively few amphibian species have been described on the North East Asian mainland in the last decades, several species have been the subject of taxonomical debates in relation to the Yellow sea. Here, we sampled Dryophytes sp. treefrogs from the Republic of Korea, the Democratic People's Republic of Korea and the People's Republic of China to clarify the status of this clade around the Yellow sea and determine the impact of sea level change on treefrogs' phylogenetic relationships. Based on genetics, call properties, adult morphology, tadpole morphology and niche modelling, we determined the segregated status species of D. suweonensis and D. immaculatus. We then proceeded to describe a new treefrog species, D. flaviventris sp. nov., from the central lowlands of the Republic of Korea. The new species is geographically segregated from D. suweonensis by the Chilgap mountain range and known to occur only in the area of Buyeo, Nonsan and Iksan in the Republic of Korea. While the Yellow sea is the principal element to the current isolation of the three clades, the paleorivers of the Yellow sea basin are likely to have been the major factor for the divergences within this clade. We recommend conducting rapid conservation assessments as these species are present on very narrow and declining ranges.

Phylogenetic study of extirpated Korean leopard using mitochondrial DNA from an old skin specimen in South Korea.

The leopard, Panthera pardus, is a threatened species in its range throughout the world. Although, historically, the Korean Peninsula had a high population density of leopards, they were extirpated from South Korea by 1970, leaving almost no genetic specimens. Traditionally, Korean leopards are classified as Panthera pardus orientalis; however, their classification is based only on locality and morphology. Therefore, there is a need for genetic studies to identify the phylogenetic status of Korean leopards at the subspecies level. Presently, no extant wild specimen is available from South Korea; therefore, we extracted genetic material from the old skin of a leopard captured in Jirisan, South Korea in the 1930s and conducted the first phylogenetic study of the South Korean leopard. A total of 726 bp of mitochondrial DNA, including segments of the NADH5 and control region, were amplified by PCR. A phylogenetic analysis of the fragment, along with sequences of nine leopard subspecies from GenBank revealed that the extinct South Korean leopard belonged to the Asian leopard group and in the same clade as the Amur leopard (Panthera pardus orientalis). Thus, the leopard that inhabited South Korea in the past was of the same subspecies as the Amur leopard population currently inhabiting the transboundary region of Russia, China, and North Korea. These results emphasize the importance of conserving the endangered wild Amur leopard population (estimated to be about 60-80 individuals) in Russia and China, for future restoration of leopards in the Korean Peninsula.

Genetic diversity and inferred dispersal history of the Schlegel's Japanese Gecko (Gekko japonicus) in Northeast Asia based on population genetic analyses and paleo-species distribution modelling.

To understand the genetic diversity and dispersal history of Schlegel's Japanese gecko (Gekko japonicus), we performed genetic analyses and paleo-species distributional modelling. For the genetic analysis, we analyzed mitochondrial DNA (mtDNA) (cytochrome b [Cytb] and NADH dehydrogenase 2 [ND2]) and seven microsatellite loci of 353 individuals from 11 populations (2 east coast China, 4 west and central coast Japan and 5 Korea). For the paleo-species distribution modelling, we used 432 occurrence data points (125 China, 291 Japan and 16 Korea) over the Pleistocene and Holocene. China is inferred to be the source population, which had higher genetic diversity (mtDNA) and more private alleles (mtDNA) compared to Japanese and Korean populations. Differences between the three counties were very small in the mtDNA haplotype network despite some genetic structure among the three countries. Microsatellite analysis inferred that genetic exchange has actively occurred among the Chinese, Japanese and Korean populations. Suitable habitats in Japan should have been plentiful by the mid-Holocene, but have only recently become available in Korea. These results suggest that dispersal of G. japonicus occurred after the Holocene warming from the east coast of China to the west and central coasts of Japan and Korea, and gene flow is actively occurring among the three countries.

Genetic diversity and phylogeography of Jeju Orthohantavirus (Hantaviridae) in the Republic of Korea.

Orthohantaviruses are negative-sense, single-stranded RNA viruses harbored by rodents, shrews, moles, and bats. Of the shrew-borne orthohantaviruses in the Republic of Korea (ROK), Jeju orthohantavirus (Jeju virus, JJUV) was found on Jeju Island. This small-scale epidemiologic survey investigated the geographic distribution and molecular phylogeny of JJUV in the ROK. In 32 trapping sites, tissues of 84 Crocidura shantungensis were analyzed for JJUV RNA. JJUV RNA was detected in seven (8.3%) shrews captured on the Korean peninsula. The molecular epidemiologic survey demonstrated the prevalence of JJUV by geographic distribution. The RNA loads of JJUV were evaluated in various tissues. Entire coding sequences of tripartite genomes were recovered from two JJUV strains on the mainland. Phylogenetic relationships of the JJUV revealed a distinct geographic lineage of mainland strains from the strains on Jeju Island. This study sheds light on the molecular epidemiology, phylogeographic diversity, and virus-host co-divergence of JJUV, ROK.

Genetic and phylogenetic structure of Hynobius quelpaertensis, an endangered endemic salamander species on the Korean Peninsula.

BACKGROUND: The Korean Peninsula is a small but unique area showing great endemic Hynobius diversity with H. quelpaertensis, H. yangi, H. unisacculus and three species candidates (HC1, HC3 and HC4). H. quelpaertensis is distributed in the southern part and in Jeju Island, while the remaining species have extremely narrow distributions. OBJECTIVES: To examine the genetic structure of H. quelpaertensis and the phylogenetic placement in Hynobius. METHODS: Three mitochondrial and six microsatellite loci were genotyped for 204 Hynobius quelpaertensis, three H. leechii, three H. yangi, three HC1, two H. unisacculus, three HC3, three HC4 and ten Japanses H. lichenatus. RESULTS: A high level of mitochondrial diversity was found in H. quelpaertensis. Our mitochondrial data showed evidence of a historical link between inland and Jeju Island despite the signature of founder effect likely experienced by the early island populations. However, our microsatellite analysis showed the fairly clear signature of isolation history between in- and island populations. Upon phylogenetic analysis, H. quelpaertensis, H. unisacculus and HC1 formed a cluster, whereas H. yangi belonged to a separate cluster. HC3 and HC4 were clustered with either H. quelpaertensis or H. yangi depending on the locus used. CONCLUSION: Our results show at least partially the historical imprints engraved by dispersal of Korean endemic Hynobius during Pleistocene, potentially providing a fundamental basis in determining the conservation units and finding management strategies for these species.

What is the taxonomic status of East Asian otter species based on molecular evidence?: focus on the position of the Japanese otter holotype specimen from museum.

The Japanese otter (Lutra nippon), once inhabited in most islands of Japan, is now considered as an extinct species. Although the Japanese otter is regarded as a distinct species from the Eurasian otter (L. lutra), its phylogeny and taxonomic status are based on limited information on morphological and genetic data, and thus further clarification is required. Here, we assessed the phylogenetic relationship among the genus Lutra and taxonomic status of L. nippon by using the complete sequences of cytochrome b gene of its holotype. The present phylogenic trees supported that the genus Lutra specimens largely formed monophyletic group, with L. sumatrana as a basal to other Lutra species. Within Lutra species, L. nippon was distantly related with L. lutra. The European otter population of L. l. lutra were clustered together with its subspecies, L. l. chinensis rather than the same subspecies, Korean otter population. The discrepancy between the genetic data and traditional taxonomy justifies the necessity of reexamination of the current subspecific classification system of Eurasian otters. Level of genetic divergence between the holotype of L. nippon and L. lutra was two to three-fold lower than those among the other sister species of the Lutrinae. Based on the level of divergence between the L. nippon and L. lutra, and insufficient evidence of morphological difference between them, it is suggested that designation of Japanese otter as a separate species from L. lutra will be reconsidered.