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.

Combining multiple autosomal introns for studying shallow phylogeny and taxonomy of Laurasiatherian mammals: Application to the tribe Bovini (Cetartiodactyla, Bovidae).

Mitochondrial sequences are widely used for species identification and for studying phylogenetic relationships among closely related species or populations of the same species. However, many studies of mammals have shown that the maternal history of the mitochondrial genome can be discordant with the true evolutionary history of the taxa. In such cases, the analyses of multiple nuclear genes can be more powerful for deciphering interspecific relationships. Here, we designed primers for amplifying 13 new exon-primed intron-crossing (EPIC) autosomal loci for studying shallow phylogeny and taxonomy of Laurasiatherian mammals. Three criteria were used for the selection of the markers: gene orthology, a PCR product length between 600 and 1200 nucleotides, and different chromosomal locations in the bovine genome. Positive PCRs were obtained from different species representing the orders Carnivora, Cetartiodactyla, Chiroptera, Perissodactyla and Pholidota. The newly developed markers were analyzed in a phylogenetic study of the tribe Bovini (the group containing domestic and wild cattle, bison, yak, African buffalo, Asian buffalo, and saola) based on 17 taxa and 18 nuclear genes, representing a total alignment of 13,095 nucleotides. The phylogenetic results were compared to those obtained from analyses of the complete mitochondrial genome and Y chromosomal genes. Our analyses support a basal divergence of the saola (Pseudoryx) and a sister-group relationship between yak and bison. These results contrast with recent molecular studies but are in better agreement with morphology. The comparison of pairwise nucleotide distances shows that our nuDNA dataset provides a good signal for identifying taxonomic levels, such as species, genera, subtribes, tribes and subfamilies, whereas the mtDNA genome fails because of mtDNA introgression and higher levels of homoplasy. Accordingly, we conclude that the genus Bison should be regarded as a synonym of Bos, with the European bison relegated to a subspecies rank within Bos bison. We compared our molecular dating estimates to the fossil record in order to propose a biogeographic scenario for the evolution of Bovini during the Neogene.

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.

Phylogenetic relationships between three Korean pit viper Gloydius (Serpentes: Crotalinae) species using mitochondrial DNA genes.

BACKGROUND: Molecular phylogenetic studies of the Asian pit viper genus Gloydius have been widely published in Asia, but Korea population have not been conducted till date. OBJECTIVE: This study aimed to analyze the phylogenetic relationships of three Gloydius species (G. saxatilis, G. brevicaudus, and G. ussuriensis) from Korea with other Gloydius species, based on Cytochrome b and ND4. METHODS: We compared 160 samples representing the three species with those of 17 reference species and their phylogenetic status and genetic diversity were analyzed with concatenated sequences of two mitochondrial DNA. RESULTS: Korean G. brevicaudus and G. saxatilis showed high haplotype diversity and relatively low and moderate nucleotide diversity, respectively. Although G. ussuriensis showed high genetic diversity, it was low in the Baengnyeong Island population. The phylogenetic tree represented two major lineages. One major lineage comprised G. ussuriensis, G. tsushimaensis, G. blomhoffii, and G. brevicaudus. The Chinese G. ussuriensis belonged to the same clade as the Korean G. ussuriensis and was closely related to the Baengnyeong Island population. Moreover, G. tsushimaensis was closely related to G. ussuriensis from southwestern Korean and Jeju Island populations. The other major lineage comprised the remaining 12 species and G. saxatilis. Korean G. saxatilis was closely related to G. saxatilis, G. shedanoensis, and G. intermedius from China. CONCLUSION: The phylogenetic status of the Korean Gloydius species in comparison with the other Gloydius species was identified. We suggesting the conservation management unit for the Baengnyeong Island population, while the current conservation status of Korean G. saxatilis is suggested to be revised to a higher level.

Whole-genome sequencing revealed different demographic histories among the Korean endemic hill pigeon (Columba rupestris), rock pigeon (Columba livia var. domestica) and oriental turtle dove (Streptopelia orientalis).

BACKGROUND: The family Columbidae is known as the pigeon family and contains approximately 351 species and 50 genera. Compared to the wealth of biological and genomic information on these Columba livia var. domesteca, information on Columba rupestris and Streptopelia orientalis has been rather limited. The C. rupestris population size is decreasing in Korea. OBJECTIVES: Whole-genome sequencing and identification of population characterization of each species based genome variation on 9 Korean pigeon and dove samples, namely, six hill pigeon (C. rupestris), one rock pigeon (C. livia var. domestica) and two oriental turtle dove (S. orientalis) samples. RESULTS: The whole genome of 9 genotypes were sequenced and mapped to the C. livia reference genome. Sequence alignment showed over 96% identity in C. rupestris and 94% identity in S. orientalis to the reference genome (GenBank assembly accession: GCA_001887795.1). Sequence variations, including single nucleotide polymorphisms (SNPs), insertions and deletions (InDels), and structural variations, revealed that intergenus (Columba vs. Streptopelia) variations were approximately four times higher than intragenus variations (C. livia vs. C. rupestris). Of the two Columba species, C. livia var. domestica is closer to S. orientalis than C. rupestris. Pairwise sequentially Markovian coalescent (PSMC) demographic history analysis revealed that the three species underwent a common population bottleneck between 105 and 120 Kya; since then, the effective population sizes of the rock pigeon and oriental turtle dove have increased. CONCLUSION: The effective population size of the hill pigeon, an Endangered Species of Grade II in Korea, has increased slowly from the second severe bottleneck that occurred approximately 0.5-1.4 × 104 years ago. Our results showed no relationship between copy number variation in the Norrie disease protein (NDP) regulatory regions and plumage color patterns. We report the first comparative analysis of three pigeon genomes.

Development of InDel markers for interspecific hybridization between hill pigeons and feral pigeons based on whole-genome re-sequencing.

Interspecific hybridization occurs among birds, and closely related sister taxa tend to hybridize at a high rate. Genomic hybridization markers are useful for understanding the patterns and processes of hybridization and for conserving endangered species in captivity and the wild. In this study, we developed genomic hybridization markers for the F1 progeny of the sister taxa feral pigeons (Columba livia var. domestica) and endangered hill pigeons (Columba rupestris) (family Columbidae). Using whole-genome re-sequencing data, we performed genome-wide analysis for insertion/deletion (InDel) polymorphisms and validated using primers. We conducted polymerase chain reaction (PCR) and agarose gel electrophoresis to identify species-specific InDels. We produced eight F1 hybrids of hill and feral pigeons, and their samples were tested by re-performing analyses and sequencing using 11 species-specific InDel polymorphisms. Eight InDel markers simultaneously amplified two DNA fragments from all F1 hybrids, and there was no abnormality in the sequencing results. The application of genomic tools to detect hybrids can play a crucial role in the assessment of hybridization frequency in the wild. Moreover, systematic captive propagation efforts with hybrids can help control the population decline of hill pigeons.

Complete mitochondrial genome of Gloydius saxatilis (Viperidae: Crotalinae) from Korea.

In this study, we sequenced the complete mitochondrial genome of Gloydius saxatilis using Illumina next-generation sequencing. The total length of the mitogenome was 17,223 bp, and contained 13 protein-coding genes (PCGs), two ribosomal RNAs (rRNAs), 22 transfer RNAs (tRNAs), two non-coding control regions (CRs), and the origin of light (OL)-strand replication. The genome structure and order of the genes were similar to other Crotalinae species. Phylogenetic analysis based on the 13 concatenated PCGs indicated that G. saxatilis closely related to G. intermedius and, G. shedaoensis.

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.

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.

Microsatellite loci analysis for the genetic variability and the parentage test of five dog breeds in South Korea.

To investigate the population structure of five dog breeds in South Korea and to validate polymorphic microsatellite markers for the parentage test, microsatellite loci analyses were conducted for two Korean native dog breeds, Poongsan and Jindo, and three imported dog breeds, German Shepherd, Beagle and Greyhound. Overall genetic diversity was high across all dog breeds (expected heterozygosity range: 0.71 to 0.85), although breeds differed in deviations from Hardy-Weinberg equilibrium (HWE). Significant reduction of heterozygosity in the Poongsan and Greyhound breeds was caused by non-random mating and population substructure within these breeds (the Wahlund effects). The close relationship and high degree of genetic diversity for two Korean native dog breeds were substantial. The mean polymorphism information content value was highest in Jindos (0.82) and Poongsans (0.81), followed by Beagles (0.74), Greyhounds (0.72), and German Shepherds (0.66). Accumulated exclusion power values, as an indication of marker validity for parentage tests, were varied but very high across breeds, 0.9999 for Jindos, Poongsans, and Beagles, 0.9997 for Greyhounds, and 0.9995 for German Shepherds. Taken together, the microsatellite loci investigated in this study can serve as suitable markers for the parentage test and as individual identification to establish a reliable pedigree verification system of dog breeds in South Korea. This study also stresses that the population subdivision within breeds can become an important cause of deviation from HWE in dog breeds.

The complete mitochondrial genome information of Phoxinus phoxinus (Cypriniformes: Cyprinidae) on the Korean Peninsula and the phylogenetic implication.

Phoxinus phoxinus is a small Leuciscinae species predominantly found in cool and well-oxygenated streams throughout a wide area encompassing Europe, Siberia and East Asia. It is believed that the populations in Korea hold important clues to how the species has been distributed south along the Eurasian continent to the Korean Peninsula. We characterized the complete mitochondrial genomes of two individual fin-clip samples collected from the two Korean river systems. The whole sequences were 17,665 and 18,220 bp, respectively, and included 13 protein-coding genes, 2 ribosomal RNA genes and 22 transfer RNA genes. The genome size difference was due to the considerably different sizes of the control region. The overall genome structures were identical to those observed in other Leuciscinae species.

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.

Species and sex identification of the Korean goral (Nemorhaedus caudatus) by molecular analysis of non-invasive samples.

Korean long-tailed goral (Nemorhaedus caudatus) is one of the most endangered species in South Korea. However, detailed species distribution and sex ratio data on the elusive goral are still lacking due to difficulty of identification of the species and sex in the field. The primary aim of this study was to develop an economical PCR-RFLP method to identify species using invasive or non-invasive samples from five Korean ungulates: goral (N. caudatus), roe deer (Capreolus pygargus), feral goat (Capra hircus), water deer (Hydropotes inermis) and musk deer (Moschus moschiferus). The secondary aim was to find more efficient molecular sexing techniques that may be applied to invasive or non-invasive samples of ungulate species. We successfully utilized PCR-RFLP of partial mitochondrial cytochrome b gene (376 bp) for species identification, and sex-specific amplification of ZFX/Y and AMELX/Y genes for sexing. Three species (goral, goat and water deer) showed distinctive band patterns by using three restriction enzymes (XbaI, StuI or SspI). Three different sexing primer sets (LGL331/335 for ZFX/Y gene; SE47/48 or SE47/53 for AMELX/Y gene) produced sex-specific band patterns in goral, goat and roe deer. Our results suggest that the molecular analyses of non-invasive samples might provide us with potential tools for the further genetic and ecological study of Korean goral and related species.

Complete mitochondrial genome sequence of Macromia daimoji Okumura, 1949 (Odonata: Macromiidae).

The dragonfly Macromia daimoji Okumura, 1949 (Odonata: Macromiidae) has been listed as an Endangered insect in South Korea. We sequenced the complete 15,198 bp mitochondrial genome (mitogenome) of this organism, which is the first mitogenome sequence reported from the family Macromiidae. The genome includes a typical set of genes [13 protein-coding genes (PCGs), 2 rRNA genes, and 22 tRNA genes) and one non-coding region with an arrangement identical to that observed in most insect genomes. Phylogenetic analyses using concatenated sequences of the 13 PCGs and 2 rRNA genes using the Bayesian inference (BI) method placed Macromiidae, represented by M. daimoji, as a sister group to Libellulidae with the highest nodal support [Bayesian posterior probabilities (BPP) = 1]. Unlike conventional phylogenetic analysis, the suborders Anisozygoptera and Zygoptera formed a strong sister group (BPP =1), justifying the use of different molecular markers for phylogenetic analysis.

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.

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.