Temporal Changes in Demography and Genetic Diversity of Oriental Storks at the Stage of Long-Term Captive Propagation and Reintroduction Initiation.

Captive propagation and reintroduction are the major steps in the ex-situ conservation of locally extirpated endangered species in a historical region. In a species restoration project conducted in South Korea, we examined temporal changes in demographics and genetic diversity of oriental storks (Ciconia boyciana). Demographic and genetic data from 1996-2018 were analyzed for 80% of all captive and recently reintroduced individuals. Founder establishment and pair formation induced increases in population size and genetic diversity during the early stage of captive propagation. The degree of genetic diversity was found to become saturated and stable with long-term captive propagation. However, this might be a concern for future genetic diversity of both captive and reintroduced populations simultaneously due to the extraction of captive populations at the early stage of reintroduction. Our findings suggest that periodic evaluation of genetic diversity and selection for releasing individuals, using effective genetic markers, would assist in balancing the genetic diversity of the captive and reintroduced oriental storks at the early stage of reintroduction.

Complete mitochondrial genome of two shorebirds (Charadriiformes: Scolopacidae), great knot (Calidris tenuirostris) and bar-tailed godwit (Limosa lapponica).

The mitochondrial genome of Calidris tenuirostris and Limosa lapponica were described using the whole mitochondrial genome obtained from Illumina Next-Generation Sequencing (NGS) technology. Total length of the mitogenome of C. tenuirostris was 16,732bp with slight A+T bias (55.3%). Genome size of L. lapponica was 16,773bp long and A+T biased (56.3%). Both gemones consisting of 2 rRNAs, 13 protein-coding genes, 22 tRNA genes and 1 non-coding regions. This is the first report of complete mitogenomes of these two shorebird species, (C. tenuirostris and of L. lapponica). We observed paraphyletic relationship among the species in the Family Scolopacidae. Also our result showed analogous patterns with the previous studies on the parallel relationships of shorebird species. This study provides basic genetic information for help in understanding phylogenetic relationships . within the Charadriiformes.

A core set of microsatellite loci for yellow-throated marten, Martes flavigula: a case of inferences of family relationships.

BACKGROUND: Microsatellite markers are an ideal molecular marker for population genetic studies such as population structure, pedigree, and kinship. The yellow-throated marten (Martes flavigula) is widely distributed in coniferous and deciduous forests of eastern Asia and plays the role of an indicator and umbrella species in South Korea, given the absence of top predators such as tiger and leopard. OBJECTIVE: The aim of our study was to establish a core set of microsatellite markers that could be used for a population genetics study on M. flavigula. METHODS: We characterized 21 di-motif microsatellites for M. flavigula by Illumina next-generation sequencing. We evaluated them for a population genetics study against five established criteria together with 33 previously developed microsatellites. We calculated relatedness values between individual yellow-throated martens in two groups that were suspected to be siblings using the selected core set of markers to confirm applicability. RESULTS: Twenty-three loci were determined as the core set of microsatellite markers. The probability of identity P(ID) and probability of identity between siblings P(ID)sib of the core set was estimated as 2-15 and 2.2-7, respectively. Relatedness values between individuals in the two groups of M. flavigula revealed that one of the pairs was sisters, confirming that the core set can be applied to kinship studies. CONCLUSION: The developed microsatellite core set in this study is expected to contribute to studies on molecular ecology and population structure of M. flavigula.

Microsatellite DNA analysis reveals lower than expected genetic diversity in the threatened leopard cat (Prionailurus bengalensis) in South Korea.

To optimize conservation efforts, it is necessary to determine the risk of extinction by collecting reliable population information for a given species. We developed eight novel, polymorphic microsatellite markers and used these markers in conjunction with twelve existing markers to measure genetic diversity of South Korean populations of leopard cat (Prionailurus bengalensis), a species for which population size and habitat area data are unknown in the country, to assess its conservation status. The average number of alleles and the observed heterozygosity of the species were 3.8 and 0.41, respectively, and microsatellite diversity was lower than the average genetic diversity of 57 populations of 12 other felid species, and lower than that of other mammal populations occurring in South Korea, including the raccoon dog (Nyctereutes procyonoides), water deer (Hydropotes inermis), and endangered long-tailed goral (Naemorhedus caudatus). Furthermore, analysis of genetic structure in the national leopard cat population showed no clear genetic differentiation, suggesting that it is not necessary to divide the South Korean leopard cat population into multiple management units for the purposes of conservation. These results indicate that the genetic diversity of the leopard cat in South Korea is unexpectedly low, and that the risk of local extinction is, as a result, substantial. Thus, it is necessary to begin appropriate conservation efforts at a national level to conserve the leopard cat population in South Korea.

Phylogeography of the Asian lesser white-toothed shrew, Crocidura shantungensis, in East Asia: role of the Korean Peninsula as refugium for small mammals.

Many peninsulas in the temperate zone played an important role as refugia of various flora and fauna, and the southern Korean Peninsula also served as a refugium for many small mammals in East Asia during the Pleistocene. The Asian lesser white-toothed shrew, Crocidura shantungensis, is a widely distributed species in East Asia, and is an appropriate model organism for exploring the role of the Korean Peninsula as a refugium of small mammals. Here, we investigated phylogenetic relationships and genetic diversity based on the entire sequence of the mitochondrial cytochrome b gene (1140 bp). A Bayesian tree for 98 haplotypes detected in 228 C. shantungensis specimens from East Asia revealed the presence of three major groups with at least 5 subgroups. Most haplotypes were distributed according to their geographic proximity. Pairwise FST's and analysis of molecular variance (AMOVA) revealed a high degree of genetic differentiation and variance among regions as well as among populations within region, implying little gene flow among local populations. Genetic evidence from South Korean islands, Jeju-do Island of South Korea, and Taiwan leads us to reject the hypothesis of recent population expansion. We observed unique island-type genetic characteristics consistent with geographic isolation and resultant genetic drift. Phylogeographic inference, together with estimates of genetic differentiation and diversity, suggest that the southern most part the Korean Peninsula, including offshore islands, played an important role as a refugium for C. shantungensis during the Pleistocene. However, the presence of several refugia on the mainland of northeast Asia is also proposed.

Complete mitochondrial genome of the Northern Long-eared Owl (Asio otus Linnaeus, 1758) determined using next-generation sequencing.

In this study, the mitogenome of Asio otus, the Northern Long-eared Owl, was analysed using Illumina next-generation sequencing. The mitogenome was found to be a circular molecule, 17,735 bp long with a slight AT bias (53.0%). The gene arrangement pattern was the same as that of a typical vertebrate, containing 37 genes (13 protein-coding genes (PCGs), 22 transfer RNA genes, two ribosomal RNA genes, and a non-coding control region). In the putative control region (1984 bp), there were two types of tandem repeats at the end of the region. A phylogenetic tree was constructed using the 13 PCG sequences discovered in this study and those of that have been previously published of other Strigidae species and revealed a close relationship between A. otus and A. flammeus. The newly generated mitogenome from this study enriches the genomic resources available for future evolutionary studies and promotes conservation genetics of this species.

Complete mitochondrial genome and phylogenetic analysis of Callorhinus ursinus: an endangered species from South Korea.

We present the complete mitochondrial genome and a phylogenetic analysis of Callorhinus ursinus, the northern fur seal, determined using Illumina next-generation sequencing (NGS) technology. The total length of the mitogenome was 17,154 bp, which consisted of 13 protein-coding genes, two ribosomal RNA genes, 22 tRNA genes, and one control region. The base composition of the entire mitogenome was 33.5% (A), 26.3% (C), 13.9% (G), and 26.3% (T) with an A + T bias of 59.8%. The control region contained two types of tandem repeats. A neighbour-joining (NJ) tree was constructed and comprised two clades with C. ursinus forming a monophyletic group. Data produced in this study will aid exploration of the genetic diversity of endangered C. ursinus and contribute to molecular identification of this species.

The mitochondrial genome of Milvus migrans (Aves, Accipitriformes, Accipitridae), an endangered species from South Korea.

The complete mitochondrial genome sequence of black kite, Milvus migrans, one of the most common diurnal raptor, was characterized using next generation sequencing. The whole genome size was 18,016 bp and consisted of 13 protein-coding genes, 22 tRNAs, 2 rRNAs, a putative control region (CR), and a second control region (pseudo-CR). A frameshift mutation was found in the ND3 gene. Phylogenetic analysis illustrated monophyly of the subfamily Melieraxinae with high statistical support. The genetic resource obtained here will help to resolve taxonomic issues related to subspecies of M. migrans and will act as a starting point for conservation genetics.

Phylogenetic structure and ancestry of Korean clawed salamander, Onychodactylus koreanus (Caudata: Hynobiidae).

Onychodactylus koreanus, a hynobiid salamander species endemic to the Korean Peninsula, can be regarded as a strict ecological specialist, probably vulnerable to anthropogenic environmental modifications and climate change. We used mitochondrial cytochrome b gene to analyze the genetic diversity and phylogenetic structure of O. koreanus from 19 populations collected in an attempt to cover its major distribution within South Korea. A total of 76 haplotypes of O. koreanus obtained in our analyses could be subdivided into three phylogenetic clades, KR, NE and SE. Clade KR haplotypes occur in most of the regions throughout the Korean Peninsula with four distinct subclades (KR I-IV). Clade NE and SE haplotypes were only observed in two populations YY and YS, respectively. Haplotype sharing was scarce even among populations in geographical proximity, and most of the populations were represented by a single clade or subclade, indicating the low level of gene flow among populations. O. koreanus likely originated from the historical southward dispersal of its ancestral lineages following divergence from Chinese O. zhaoermii that was recovered as the sister of O. koreanus in our phylogenetic analysis. Our results have critical implications for the taxonomic status of O. koreanus and its long-term management plan.

The mitochondrial genome of the long-billed plover, Charadrius placidus (Charadriiformes: Charadriidae).

The present study provides the first full mitochondrial genome sequence of a long-billed plover from South Korea. This mitogenome is 16,895 bp in length and consists of 13 protein-coding genes, 2 ribosomal-RNA genes, 22 transfer-RNA genes, and a non-coding control region. Overall, base composition was: A, 31.4%; C, 31.1%; G, 13.8%; and T, 23.8%. A frameshift mutation in the ND3 gene was identified and a tandem repeat (AACA) was observed in the D-loop region. The phylogenetic analysis based on concatenated coding genes indicated Charadriidae's monophyly. These results contribute to further understanding of long-billed plover phylogenetic relationships and species identification.

Complete mitochondrial genome of Haematopus ostralegus (Charadriiformes: Haematopodidae).

The Eurasian oystercatcher (Haematopus ostralegus), Near Threatened in the IUCN red list, was designated classified endangered species II by the Ministry of Environment of Korea and a Natural Monument (No. 326) by the Cultural Heritage Administration of Korea. In this study, the complete mitochondrial genome (16,798 bp) of H. ostralegus was determined for the first time, including 13 protein-coding genes, 2 ribosomal RNA genes, 22 transfer RNA genes, and 1 non-coding region. The overall base composition was A (31.4%), C (31.3%), G (13.8%), and T (23.5%), so the percentage of A and T (54.9%) was slightly higher than that of G and C. A phylogenetic analysis using concatenated mitogenomes revealed that the family Haematopodidae has a closer relationship with the family Recurvirostridae and H. ostralegus and H. ater clustered together. The results are expected to provide useful resources for species identification and further phylogenetic studies of genus Haematopus.

Sequencing and analysis of the complete mitochondrial genome of Hyla suweonensis (Anura: Hylidae).

The present study reports the complete mitochondrial genome of the Suwon tree frog Hyla suweonensis from South Korea. This endangered species is endemic to Korea. The circular mitogenome of H. suweonensis includes 16,895 bp length and contains 13 protein-coding genes, 2 ribosomal RNA genes, 22 transfer RNA genes, and a non-coding region, which is the typical gene arrangement found in the available Hylinae mitogenomes. Phylogenetic analysis of six Hyla spp. mitochondrial genomes revealed that H. suweonensis is closer to H. tsinlingensis.

Mitochondrial genome analysis of the spoon-billed sandpiper (Eurynorhynchus pygmeus).

The complete mitogenome sequence of Spoon-billed sandpiper (Eurynorhynchus pygmeus) was reported in this study. The mitogenome was a circular molecule (16,709 bp) with a typical vertebrate mitogenome arrangement, which consisted of 13 protein-coding genes, two ribosomal RNA genes, 22 transfer RNA genes, and one non-coding region (D-loop). The overall base composition was 31.3% A, 29.9% C, 13.8% G, and 25.0% T. The length of D-loop is 1155 bp in length, containing tandem repeats. Phylogenetic analyses based on the concatenated protein-coding genes indicated that the South Korean and Chinese E. pygmeus formed a group that was most closely related to Arenaria interpres.

Sequencing and analyzing complete mitochondrial genome of Anser cygnoides (Anserini: Anserinae).

The complete mitogenome sequence of Anser cygnoides was determined and compared with those previously released. The whole mitogenome was 16,740 bp, with 13 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes, and 1 putative non-coding region (1178 bp). The overall nucleotide composition was 30.2%, 32.0%, 15.1%, and 22.7% for A, C, G, and T, respectively. Phylogenetic analyses strongly supported two clades in Anserini and demonstrated that A. cygnoides is not monophylogenetic. This division of A. cygnoides indicates that more research is necessary to clarify phylogenetic relationships for A. cygnoides and determine how they relate to morphological traits.

Complete mitochondrial genome of Ciconia nigra (Ciconiiformes: Ciconiidae).

The complete mitogenome of the Black Stork Ciconia nigra, obtained using a PCR-based method, is reported. It is 17,905 bp, slightly A + T biased (30.8% A, 31.5% C, 14.1% G, and 23.6% T), and comprises 13 protein-coding genes, 2 ribosomal RNA genes, 22 transfer RNA genes, and 1 putative non-coding gene. The two kinds of tandem repeat units found in D-loop (2316 bp) generated a length polymorphism between this and the previously reported D-Loop of C. nigra from China (2206 bp). A frameshift mutation was observed in ND3. The phylogenetic analysis clustered C. nigra with other Ciconia species.

Complete mitochondrial genome of Siberian musk deer Moschus moschiferus (Artiodactyla: Moschidae) and phylogenetic relationship with other moschus species.

The Siberian musk deer, Moschus moschiferus, is an Endangered species in South Korea due to its decreasing population size caused by illegal hunting and habitat destruction. In this study, the complete mitochondrial genome of M. moschiferus was determined using next-generation sequencing. Total length of its mitogenome is 16,356 bp in length, encoding 13 protein-coding genes, 22 transfer RNA genes, two ribosomal RNA genes, and one control region. Its AT contents are 62.4%, which are higher than its GC contents (37.7%) (A, 34.1%; C, 24.9%; G, 12.8%; and T, 28.3%). Phylogenetic relationship of genus Moschus showed topologies similar to those reported in previous studies. Sequence comparison between two M. moschiferus from South Korea indicated high sequence variations with 122 nucleotide differences. These results provide useful information necessary for further phylogenetic studies of Moschus species.

Complete mitochondrial genome sequence of Emberiza sulphurata (Emberizidae: Emberiza).

The Japanese yellow bunting (Emberiza sulphurata) is considered to be an endangered species. Here, the complete mitochondrial genome of E. sulphurata (16,797 bp in length) was determined. The genome consists of 37 genes (13 protein-coding genes, two ribosomal RNA genes, and 22 transfer RNA genes) and one control region (D-loop), and so is highly similar in architecture to the typical vertebrate mtDNA genome. The base composition of the mtDNA was A (29.9%), C (32.6%), G (14.5%), and T (23.0%), so the percentage of A and T (52.9%) was slightly higher than that of G and C. All the genes in E. sulphurata were encoded on the H-strand, except for the genes for the ND6 subunit and eight tRNAs, which were encoded on the L-stand. Phylogenetic analysis using Emberizidae mitogenomes revealed that E. sulphurata was grouped into the family Emberizidae and that E. spodocephala is the most closely related species.

Genetic diversity and population structure of the long-tailed goral, Naemorhedus caudatus, in South Korea.

The long-tailed goral, Naemorhedus caudatus, is an internationally endangered species. This species is distributed throughout Northeastern Asia including Northeastern China, the Russian Far East and the Korean peninsula. The population size of long-tailed gorals is currently decreasing in South Korea, and thus effective conservation of the animal is urgently needed. Although the evolution and phylogeny of this animal have been studied, population genetic studies are needed to design effective conservation and management strategies. To evaluate the present status of genetic diversity and genetic structure of long-tailed gorals in South Korea, we investigated genetic variability among 68 goral individuals from different regions, including 11 captive zoo animals, at 12 microsatellite loci. The level of genetic diversity was moderate in wild goral populations, but lower in the captive group. The goral population from the lower northeast region of South Korea was distinct from the upper northeast population, probably due to the natural climatic and geographic conditions. The genetic characteristics of the captive group were more similar to those of the upper northeast population than the lower northeast, confirming that the zoo animals originated in the Seorak Mountain range. Direct translocations between the upper and lower northeast populations are not currently recommended considering the natural population structure and the moderate levels of genetic diversity in the two populations. This study highlights the importance of genetic information in designing effective conservation strategies and translocations of endangered animals, including the Korean goral.

Mitochondrial DNA data unveil highly divergent populations within the genus Hynobius (Caudata: Hynobiidae) in South Korea.

Korean salamanders of the genus Hynobius are currently classified into 3 species, H. leechii, H. quelpaertensis, and H. yangi. To investigate the phylogenetic relationship of these species, we analyzed the partial sequence of mitochondrial cytochrome b gene (907 bp) of 197 specimens from 43 regions in South Korea. Of these specimens, 93 were additionally examined with 12S rRNA (799 bp). Based on the partial sequence of the mitochondrial cytochrome b gene and 12S rRNA, 89 and 36 haplotypes were defined, respectively, consisting of six subclades (H. leechii, H. quelpaertensis, H. yangi, HC1, HC2, and HC3). Among these subclades, the three subclades (HC1, HC2, and HC3) were clearly separated from the 3 previously reported species in the genus Hynobius. Pairwise sequence divergence between the six subclades ranged from 6.3 to 11.2% in cytochrome b gene and 2.0 to 4.3% in 12S rRNA. These results indicate there may be more divergent populations than the three currently described. Moreover, the estimation of divergence time revealed that the Hynobius species in South Korea diverged during the Miocene epoch, approximately 9 - 5 MYA. In addition, we confirmed the distribution of the three known species (H. leechii, H. quelpaertensis, and H. yangi) and determined the distributions of new, distinct groups (or subclades; HC1, HC1, and HC3). To more accurately establish the taxonomic status and population structure, further genetic, morphological, and ecological studies will be needed.

Mitochondrial cytochrome b sequence variations and population structure of Siberian chipmunk (Tamias sibiricus) in Northeastern Asia and population substructure in South Korea.

Twenty-five chipmunk species occur in the world, of which only the Siberian chipmunk, Tamias sibiricus, inhabits Asia. To investigate mitochondrial cytochrome b sequence variations and population structure of the Siberian chipmunk in northeastern Asia, we examined mitochondrial cytochrome b sequences (1140 bp) from 3 countries. Analyses of 41 individuals from South Korea and 33 individuals from Russia and northeast China resulted in 37 haplotypes and 27 haplotypes, respectively. There were no shared haplotypes between South Korea and Russia--northeast China. Phylogenetic trees and network analysis showed 2 major maternal lineages for haplotypes, referred to as the S and R lineages. Haplotype grouping in each cluster was nearly coincident with its geographic affinity. In particular, 3 distinct groups were found that mostly clustered in the northern, central and southern parts of South Korea. Nucleotide diversity of the S lineage was twice that of lineage R. The divergence between S and R lineages was estimated to be 2.98-0.98 Myr. During the ice age, there may have been at least 2 refuges in South Korea and Russia--northeast China. The sequence variation between the S and R lineages was 11.3% (K2P), which is indicative of specific recognition in rodents. These results suggest that T. sibiricus from South Korea could be considered a separate species. However, additional information, such as details of distribution, nuclear genes data or morphology, is required to strengthen this hypothesis.