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.

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.

On the phylogeny of Mustelidae subfamilies: analysis of seventeen nuclear non-coding loci and mitochondrial complete genomes.

BACKGROUND: Mustelidae, as the largest and most-diverse family of order Carnivora, comprises eight subfamilies. Phylogenetic relationships among these Mustelidae subfamilies remain argumentative subjects in recent years. One of the main reasons is that the mustelids represent a typical example of rapid evolutionary radiation and recent speciation event. Prior investigation has been concentrated on the application of different mitochondrial (mt) sequence and nuclear protein-coding data, herein we employ 17 nuclear non-coding loci (>15 kb), in conjunction with mt complete genome data (>16 kb), to clarify these enigmatic problems. RESULTS: The combined nuclear intron and mt genome analyses both robustly support that Taxidiinae diverged first, followed by Melinae. Lutrinae and Mustelinae are grouped together in all analyses with strong supports. The position of Helictidinae, however, is enigmatic because the mt genome analysis places it to the clade uniting Lutrinae and Mustelinae, whereas the nuclear intron analysis favors a novel view supporting a closer relationship of Helictidinae to Martinae. This finding emphasizes a need to add more data and include more taxa to resolve this problem. In addition, the molecular dating provides insights into the time scale of the origin and diversification of the Mustelidae subfamilies. Finally, the phylogenetic performances and limits of nuclear introns and mt genes are discussed in the context of Mustelidae phylogeny. CONCLUSION: Our study not only brings new perspectives on the previously obscured phylogenetic relationships among Mustelidae subfamilies, but also provides another example demonstrating the effectiveness of nuclear non-coding loci for reconstructing evolutionary histories in a group that has undergone rapid bursts of speciation.

Characterization of TRPC2, an essential genetic component of VNS chemoreception, provides insights into the evolution of pheromonal olfaction in secondary-adapted marine mammals.

Pheromones are chemical cues released and sensed by individuals of the same species, which are of major importance in regulating reproductive and social behaviors of mammals. Generally, they are detected by the vomeronasal system (VNS). Here, we first investigated and compared an essential genetic component of vomeronasal chemoreception, that is, TRPC2 gene, of four marine mammals varying the degree of aquatic specialization and related terrestrial species in order to provide insights into the evolution of pheromonal olfaction in the mammalian transition from land to water. Our results based on sequence characterizations and evolutionary analyses, for the first time, show the evidence for the ancestral impairment of vomeronasal pheromone signal transduction pathway in fully aquatic cetaceans, supporting a reduced or absent dependence on olfaction as a result of the complete adaptation to the marine habitat, whereas the amphibious California sea lion was found to have a putatively functional TRPC2 gene, which is still under strong selective pressures, reflecting the reliance of terrestrial environment on chemical recognition among the semiadapted marine mammals. Interestingly, our study found that, unlike that of the California sea lion, TRPC2 genes of the harbor seal and the river otter, both of which are also semiaquatic, are pseudogenes. Our data suggest that other unknown selective pressures or sensory modalities might have promoted the independent absence of a functional VNS in these two species. In this respect, the evolution of pheromonal olfaction in marine mammals appears to be more complex and confusing than has been previously thought. Our study makes a useful contribution to the current understanding of the evolution of pheromone perception of mammals in response to selective pressures from an aquatic environment.

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.

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.

New insights into the evolution of intronic sequences of the beta-fibrinogen gene and their application in reconstructing mustelid phylogeny.

Mustelidae is the largest and most diverse family in the order Carnivora. The phylogenetic relationships among the subfamilies have especially long been a focus of study. Herein we are among the first to employ two new introns (4 and 7) of the nuclear beta-fibrinogen gene to clarify these enigmatic problems. In addition, two previously available nuclear (IRBP exon 1 and TTR intron 1) and one mt (ND2) data sets were also combined and analyzed simultaneously with the newly obtained sequence data in this study. Detailed characterizations of the two intronic regions not only reveal the remarkable occurrences of short interspersed element (SINE) insertion events, providing a new example supporting the attractive hypothesis that attrition of an earlier retroposition may offer a proper environment for successive retropositions by forming a "dimer-like" structure, but also demonstrate their utility in the resolution of mustelid phylogeny. All of our analyses confirm the assemblage of Mustelinae, Lutrinae, and Melinae with confidence; moreover, two clades within Mustelinae were clearly recognized, i.e., genera Mustela and Martes. Notably, genus Martes of Mustelinae was found to branch off first, followed by Melinae and then a clade containing Lutrinae and genus Mustela of Mustelinae, indicating paraphyly of Mustelinae. In addition, Mephitinae diverges before the other mustelids and the monophyletic Procyonidae in all cases, supporting its elevation to a separate family. Additional independent genetic markers are still in need to resolve the trichotomy among Mephitinae and the other two carnivoran clades, Ailuridae and Procyonidae/non-mephitine Mustelidae.

Isolation of a recent Korean epizootic strain of Newcastle disease virus from Eurasian Scops Owls affected with severe diarrhea.

Velogenic Newcastle disease virus (NDV) was recovered from two dead Eurasian Scops Owls (Otus scops) from a wildlife rescue center in Korea during 2005. Phylogenetic analysis based on the sequence of the partial fusion (F) protein revealed that the isolates had the highest level of homology to recent Korean NDV strains from poultry.

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.

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.

A molecular genetic approach for species identification of mammals and sex determination of birds in a forensic case of poaching from South Korea.

DNA-based analysis was performed using partial mitochondrial cytochrome b genes of five mammalian specimens and Chromo-Helicase-DNA-binding (CHD) genes of five pheasants to determine whether specimens were from illegally hunted animals. Mammalian specimens were identified as being those of horse, roe deer, and cow through gene amplification using cytb981f and cytb981r primer set and sequencing. CHD genes were revealed to be those of three male and two female pheasants through polymerase chain reaction amplification. Because hunting of roe deers and female pheasants is prohibited in Korea, these results provided forensic evidences of illegal wild animal hunting.

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.

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.

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.

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.