Morphometric analyses of non-invasive fecal samples of the Korean long-tailed goral (Naemorhedus caudatus) for species and age identification

The Korean long-tailed goral (Naemorhedus caudatus) is at risk of population decline due to habitat loss and fragmentation. Therefore, it is essential to ascertain its presence and/or the identity of individuals of the goral using non-invasive fecal samples for its conservation and management. In this study, we examined the morphology of fecal samples to provide the baseline data that can be used to distinguish species and age of goral individuals. We detected a significant difference in the length-to-width ratios of feces among the five ungulate species found in Korea. Also, we detected a significant difference in the length-to-width ratios of feces of gorals depending on the age groups. To assess the accuracy of species and age identification based on the fecal morphology, we conducted a series of blind comparison between the mean length-to-width ratios of the fecal pellets and the reference mean ratio values of the fecal pellets. Using 20 fecal pellets, our results showed 73%–86% probability of correct identification of the three species (gorals, goats, and roe deer), and 83%–90% probability of correct identification of >5 year-old goral individuals. The use of fecal morphometric analyses will be useful for the studies of Korean ungulate species, particularly the endangered gorals.     

Genetic diversity and genetic structure of the endangered Manchurian trout, <Emphasis Type="Italic">Brachymystax lenok tsinlingensis</Emphasis>, at its southern range margin: conservation implications for future restoration

The Manchurian trout, Brachymystax lenok tsinlingensis (family: Salmonidae), is a cold freshwater fish endemic to Northeast Asia. South Korean populations of this species, which comprise its southern range limit, have recently decreased markedly in size and are now becoming critically endangered. We assessed the current population status of this species in South Korea by estimating the levels of genetic diversity and genetic structure of five natural and four restored populations using mitochondrial DNA (mtDNA) control region sequences and eight nuclear microsatellite loci. Levels of within-population genetic diversity were low, suggesting that past effective population sizes (N _e) have been small. Each population had one or a maximum of two mtDNA haplotypes. Microsatellite allelic richness (AR) was significantly higher for natural populations (mean AR?=?3.51; 95% confidence interval, 3.00–4.03) than for restored populations (mean AR?=?2.61; 2.38–2.98). South Korean populations were significantly genetically isolated from one another, with private mtDNA haplotypes and microsatellite alleles, suggesting that limited gene flow has been occurring among populations. A mtDNA phylogeny revealed that South Korean lineages were more closely related to those of China than to those of North Korea and Russia. Overall, we suggest that future restoration efforts aimed at South Korean populations should consider the genetic characteristics reported here, which should help to fulfil effective conservation strategies for this highly cherished species. Our results will inform other conservation efforts, including assisted migration of freshwater fish populations at the equatorial end of the geographical range limit of the species.     

Microsatellite DNA analysis reveals lower than expected genetic diversity in the threatened leopard cat (<Emphasis Type="Italic">Prionailurus bengalensis</Emphasis>) 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.     

Considering threats to population viability of the endangered Korean long-tailed goral (Naemorhedus caudatus) using VORTEX

Population viability analysis (PVA) has frequently been used in conservation biology to predict extinction rates for threatened or endangered species. In this study, we used VORTEX to model Korean long-tailed goral (Naemorhedus caudatus) using previously collected ecological data. We focused on modelling population extinction, mean population size and heterozygosity. The minimum viable population size was found to be at least 50 gorals for 100 years, regardless of carrying capacity. However, populations with fewer than 50 gorals could not remain successful in the model. Inbreeding depression, catastrophes and supplementation also affected patterns of population extinction, mean population size and heterozygosity. Supplementation with new individuals had the strongest effect on extinction, mean population size and heterozygosity, followed by initial population size, inbreeding, catastrophes and carrying capacity. These results suggest that a supplementation by extra goral individuals from goral proliferation facilities would be the most helpful means for the restoration programme. More Korean goral-specific information regarding demographic and habitat parameters is needed for further PVA of the species.     

Isolation and characterization of 15 microsatellite loci in the Korean goral (Nemorhaedus caudatus)

Microsatellites have been applied in a variety of fields, including conservation genetics. Species‐specific microsatellites are considered as more powerful genetic markers to generate an accurate genetic composition of a species itself. Accordingly, we characterized 15 polymorphic microsatellite loci from Korean goral, Nemorhaedus caudatus, one of the most endangered species in South Korea. The new markers should benefit future studies of the endangered species of other Asian gorals and their relatives for the study of genetic diversity and potential conservation management.     

Molecular phylogenetic status of the Korean goral and Japanese serow based on partial sequences of the mitochondrial cytochrome b gene

To investigate the molecular phylogenetic status of the Korean goral, Nemorhaedus caudatus raddeanus, and Japanese serow, Capricornis crispus, we determined partial sequences of the mitochondrial cytochrome b gene of twelve Korean gorals and sixteen Japanese serows, and compared them with those of the major lineages of Rupicaprini species including two other Nemorhaedus species and two other Capricornis species. The Korean gorals examined possessed two haplotypes with only one nucleotide difference between them, while the Japanese serows showed slightly higher sequence diversity with five haplotypes. Genetic distances and molecular phylogenetic trees indicated that there is considerable genetic divergence between the Korean goral and N. caudatus (the Chinese goral) [Groves and Shields (1996)], but virtually none between Korean and Russian gorals. The Korean and Russian gorals may therefore be distinct from the Chinese goral. The data highlight the importance of conservation of the goral populations of these regions, and the need to reconsider the taxonomic status of Korean and Russian gorals. Our study also clearly demonstrated sufficient genetic distance between serows and gorals to justify their assignment to separate genera. Of the three species of Capricornis, the Formosan serow, C. swinhoei is more closely related to C. sumatraensis than to the Japanese serow, suggesting that the Formosan serow is a distinct species. Preliminary data on intraspecific genetic variation in the Japanese serow are also presented.     

Genetic characteristics of Y-chromosome short tandem repeat haplotypes from cigarette butt samples presumed to be smoked by North Korean men

Korea has been divided into South Korea and North Korea for over 70 years. DNA profiles of the North Korean population have never been reported in the Y-chromosome STR Haplotype Reference Database (YHRD; https://yhrd.org). To investigate genetic features of Y-chromosome STR haplotypes of the North Korean population for the first time. Genomic DNA was isolated from 838 cigarette butts assumed to have been smoked by North Korean men and amplified with PowerPlex Y23 (PPY23) kit. Statistical parameters were calculated using Nei’s formula and analysis of molecular variance (AMOVA). Multidimensional scaling (MDS) plot was constructed by the AMOVA tool and neighbor-joining (NJ) tree was constructed by MEGA 6.06. A total of 121 haplotypes were analyzed for PPY23 loci from a sample population. Haplotype diversity and discrimination capacity were 0.9992 and 0.9837, respectively. Genetic diversities ranged from 0.2981 to 0.9716. For the 16 Y-filer loci and eight minimal loci, respectively 90.9 and 82.6% of the matched haplotypes were estimated to belong to haplogroup O, representing the Southeast and East Asian type. The MDS plot and NJ tree indicated that the samples are most closely related to South Korean. In addition, p-value in the pairwise comparison to the South Korean was slightly above statistical significance (p?=?0.0534). The Y-STR haplotypes of the samples were unique and highly genetically polymorphic. Despite the separation between North and South Korea for 70 years, they can still be considered a single genetic population, based on Y-STR haplotypes.     

Cross-species amplification of Bovidae microsatellites and low diversity of the endangered Korean goral

The Korean goral (Nemorhaedus caudatus) is an endangered species of wild goat. The conservation and management of this species could benefit from a better understanding of its genetic diversity and structure. Cross-species amplification of 34 Bovidae microsatellite loci was tested on a panel of 6 Korean gorals and 10 domestic goats. After polymerase chain reaction (PCR) optimization, 29 (85.3%) microsatellite loci amplified successfully for the Korean gorals and 27 (79.4%) for the domestic goats. Of the amplified products, 16 (55.2%) were polymorphic in the Korean goral and 22 (81.5%) in domestic goats. Nei's unbiased mean heterozygosity and mean allele number per locus were, respectively, 0.356 and 2.6 in the Korean goral and 0.636 and 4.8 in domestic goats. Low genetic diversity in the Korean gorals observed in this preliminary microsatellite survey suggests an urgent need for further detailed study of genetic diversity in Korean goral populations and a population management strategy based on these studies. Current results of cross-species amplification of domestic Bovidae microsatellites could be employed for the necessary population genetic studies on the Korean goral and other endangered Caprinae species.     

Genetic diversity and population demography of narrow‐ridged finless porpoises from South Korea on the basis of mitochondrial DNA variation: Implications for its conservation in East Asia

The narrow‐ridged finless porpoise, Neophocaena asiaeorientalis, is a small cetacean inhabiting the coastal waters of Taiwan, East China, Korea, and Japan, and is threatened by anthropogenic pressures. We analyzed the mitochondrial control region of N. asiaeorientalis from Korean waters to better understand the genetic diversity, population structure, and demographic history through comparison with previously reported sequences from Japan and China. The network and phylogenetic analyses illustrated that few common haplotypes were centrally located, and that most of the unique haplotypes were on the edges of the branches. Moreover, the estimates of fixation indices and gene flow revealed significant genetic differentiation among the regions in East Asia. The mismatch distribution suggested a population expansion in the late Pleistocene. We propose that the current genetic variation in N. asiaeorientalis in East Asia was shaped by historical demographic expansion during the late Pleistocene. The N. asiaeorientalis of Korean waters has the highest diversity observed with the sampling available at this time. However, the abundance of N. asiaeorientalis in Korean waters has rapidly declined and the species needs protection from bycatch.     

Upwelling and eddies affect connectivity among local populations of the goldeye rockfish,Sebastes thompsoni (Pisces,Scorpaenoidei)

The goldeye rockfish, Sebastes thompsoni, commercial rockfish catch in the Northwest Pacific Ocean, may influence its population structure. To clarify the population genetic structure of Korean S. thompsoni and its degree of hybridization with the most close species, Sebastes joyneri, we analyzed a mitochondrial (mt) DNA control region and eleven polymorphic microsatellite (ms) loci. S. joyneri individuals were clearly distinguished from S. thompsoni by the mtDNA control region and ms loci results, with single interspecific hybridization between two species suggesting no impact on genetic structure of S. thompsoni. Analysis of mtDNA revealed no population structure within S. thompsoni, suggesting the survival of a single population in southern refugia during the glacial period. The ms loci results, in contrast, showed two genetically distinct clusters within S. thompsoni: One was predominant throughout Korean coasts (from the Yellow Sea, via the Korea Strait to the East Sea); the other was predominant at Dokdo Island in the East Sea; and both occurred in similar ratios at Wangdolcho Reef in the East Sea. A possible factor that restricts gene flow between Korean coastal and offshore populations in the East Sea may be related to the complex oceanic current patterns such as eddies and upwelling, which represent impermeable barriers to population connectivity for this species. Our findings highlight that these two populations might be representative of two separate stock within Korean waters and maintain their geographically related genetic structure.     

Genetic divergence between the South Korean and Mongolian populations of the dung beetle,Gymnopleurus mopsus (Coleoptera: Scarabaeidae) based on mitochondrial cytochrome c oxidase subunit I (COI) gene sequences

The locally extinct dung beetle, Gymnopleurus mopsus Pallas, 1781 (Coleoptera: Scarabaeidae), has not been found in South Korea since the 1970s. This research was conducted to understand the genetic divergence between the South Korean and Mongolian populations of G. mopsus as a part of its reintroduction program in South Korea. The genetic distance and diversity were determined using the mitochondrial cytochrome c oxidase subunit I (COI) gene sequence (658 bp ) corresponding to the DNA barcode region. The mitochondrial COI gene sequences of 64 individuals of G. mopsus collected in South Korea (7 individuals) and Mongolia (57 individuals) showed a relatively high level of genetic diversity (nucleotide diversity, 0.0078 ± 0.0007; Haplotype diversity, 0.965 ± 0.017). The genetic distances between the South Korean and Mongolian populations lay within the intraspecific level and the phylogenetic reconstruction using the neighbor‐joining (NJ) method showed that all individuals belonged to a single clade. This result indicates that the current Mongolian population of G. mopsus is a good candidate source population to restore the locally extinct population of the species in South Korea.     

Genetic diversity of the dung beetle,Copris tripartitus (Coleoptera: Scarabaeidae), that is endangered in Korea

The dung beetle, Copris tripartitus (Coleoptera: Scarabaeidae), is an endangered insect in Korea. In order to establish a conservation strategy, a preliminary investigation on the genetic diversity of Korean populations was performed using mitochondrial COI (658 bp), CytB (433 bp), and nuclear ITS2 (411 nucleotide positions). Sequencing of 69 individuals collected from five localities showed substantially higher variability (5.02% for COI, 4.62% for CytB, and 8.03% for ITS2). The resulting networks for mitochondrial DNA (mtDNA) haplotypes exhibited two star‐like phylogenies, which might indicate that Korean populations have recently expended from two small populations. The ITS2 network, which was presented in the form of a star‐like phylogeny, confirmed that a recent population expansion occurred. Considering the high genetic diversity and gene flow in C. tripartitus populations, one issue regarding conservation seems to be the recovery of previous habitats.     

Genetic variation and Wolbachia infection status of the lycaenid butterfly Zizina emelina (Lepidoptera,Lycaenidae) from Korean populations

The lycaenid butterfly Zizina emelina emelina (de l’Orza) (previously Zizina otis emelina; Lepidoptera, Lycaenidae) has populations distributed across Japan and Korea. Zizina emelina is listed on Japan’s Red List as an endangered species, owing to the loss of its principal plant food and habitat. Researchers previously investigated the genetic diversity and structure in Japanese populations by using mitochondrial and nuclear DNA analyses alongside their Wolbachia infection status to consider the conservation strategy. In this study, we investigated the genetic variation and Wolbachia infection status of Korean populations and compared our findings with these traits in the Japanese populations. Four novel mitochondrial haplotypes were found in the Korean populations, whereas nuclear DNA analysis revealed one new haplotype and another that matched the one reported previously in Japan. The two known strains of Wolbachia in the Japanese populations were also found in the Korean ones. Mitochondrial DNA analysis demonstrated that no gene flow had occurred between the Japanese and Korean populations. The fact that they share the same nuclear haplotype and Wolbachia strains, however, indicates a low level of population differentiation between the two.     

Gelechiid fauna of Baengnyeongdo,Daecheongdo, and Yeonpyeongdo in the West Sea near North Korea,with description of two new species (Lepidoptera,Gelechioidea)

In the faunistic survey on three islands (Baengnyeongdo, Decheongdo, and Yeonpyeongdo) in the northern West Sea of the Korean Peninsula, 37 species of Gelechiidae (Gelechioidea) are recognized. Of them, Anarsia asymmetrodes Park, sp. nov. and Aristotelia nesiotatos Park, sp. nov. are described as new to science and Stenolechia kodamai Okada is reported for the first time in Korea.     

Genetic variation of <Emphasis Type="Italic">Picea jezoensis</Emphasis> populations in South Korea revealed by chloroplast,mitochondrial and nuclear DNA markers

Genetic variation associated with Picea jezoensis populations of South Korea was investigated using chloroplast (cp), mitochondrial (mt) and nuclear DNA markers. In South Korea, P. jezoensis is distributed across a very restricted area, being found on the summits of three mountains: Mts. Jiri, Dokyu and Gyebang. Examination of five region restriction enzyme combinations for mtDNA and four for cpDNA revealed haplotypes endemic to South Korea. The Gyebang population, the most northerly and most isolated, was genetically distinct from the other populations. Nuclear microsatellite markers indicated, overall, a low level of genetic diversity (H _e = 0.406) in South Korea; this could be attributed to genetic drift and/or founder effects associated with historical events. The Wilcoxon sign-rank test did not indicate a recent bottleneck in any of the populations irrespective of the model considered (infinite allele model, two-phased model of mutation, and stepwise mutation model). Microsatellite markers also demonstrated that the Gyebang population was distinct from the others. The results of this study could be used as the basis for conservation guidelines for the management of this species in South Korea.     

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.     

Mitochondrial DNA Distinction of Northeastern China Roe Deer, Siberian Roe Deer, and European Roe Deer, to Clarify the Taxonomic Status of Northeastern China Roe Deer

Partial sequences of the mitochondrial control region of northeastern China roe deer were analyzed to determine the degree of genetic diversity. Fourteen haplotypes were observed. The haplotype diversity was high (h = 0.872), nucleotide diversity was medium (p _i = 0.0108), and the average Tamura–Nei nucleotide distance among them was 1.9%, indicating that genetic diversity of roe deer from northeastern China was relatively high and that the effective population size was large historically. To clarify the northeastern China roe deer's taxonomic status, these 14 haplotypes were compared with 31 haplotypes published in Genbank from Europe, Siberia, and Korea. The average genetic distance between haplogroups of northeastern China and European roe deer (5.8%) was more than twice that between northeastern China and Siberian roe deer (2.7%), indicating sufficient variation to consider roe deer of northeastern China and Siberia as a single species (Capreolus pygargus), distinct from European roe deer (Capreolus capreolus). This is the first presentation of mtDNA data for roe deer in northeastern China, which will be helpful in investigations of genetic diversity and clarifications of the taxonomic status of roe deer in the whole of China.     

Origin and dispersal of bluegill sunfish, Lepomis macrochirus, in Japan and Korea

The bluegill sunfish, Lepomis macrochirus, is a notorious exotic species in many freshwater ecosystems, currently expanding its distribution worldwide. In 1960, a small group of bluegills captured in the Mississippi River at Guttenberg in Iowa were imported to Japan as a gift from the mayor of Chicago to the Japanese government. The offspring of these fish were released into the wild in Japan and also in Korea. Over 40 years after this first introduction, L. macrochirus now occupies all the freshwater ecosystems of both countries. We compared invading populations of L. macrochirus in Japan and Korea with native populations in the USA, using PCR-RFLP (polymerase chain reaction-restricted fragment length polymorphism) analyses of mitochondrial DNA, to estimate the origin and dispersal of L. macrochirus in Japan and Korea. Five haplotypes of mitochondrial DNA detected in Japanese and Korean populations completely coincided with the haplotypes of the Guttenberg population. Haplotype diversity of invading populations was shown to be highest in populations established in the 1960s, while genetic variability was lower in more recently established populations. Our results suggest that all L. macrochirus in Japan and Korea have originated from the 15 fish first introduced in 1960. Low haplotype diversity in newly established populations is probably due to genetic drift arising from repeated population bottlenecks, while the high similarity of haplotypes among neighbouring populations is considered to reflect the history of transplantation by humans.     

Molecular evidence for the taxonomic identity of Korean Adonis (Ranunculaceae)

 Although Korean Adonis has been traditionally recognized as Adonis amurensis Regel and Radde with various infraspecific taxa described, its taxonomic identity is still in dispute. We investigated the genetic variation in 60 individuals from 12 populations in Korea to elucidate the taxonomic identity of the Korean Adonis complex. Random amplified polymorphic DNA (RAPD) analysis revealed that Korean Adonis comprises three species: A. amurensis, A. pseudoamurensis, and A. multiflora. Adonis amurensis is distributed in northern central inland regions of Korea and A. pseudoamurensis is found in southern parts of the Korean peninsula. Adonis multiflora grows only on Cheju Island, which is the southernmost part of Korea. Phylogenetic analysis of nuclear ribosomal internal transcribed spacer (ITS) sequences partially supported the presence of three Adonis taxa in Korea as detected by RAPD analysis. The Adonis population on Jangbong Island located in the West Sea, which was referred to as A. pseudoamurensis on the basis of morphological examination, was separated from the other populations of A. pseudoamurensis. Otherwise, the molecular evidence is well congruent with the recent morphological study that proposes that Korean Adonis consists of these three species. Received: November 6, 2001 / Accepted: February 26, 2002     

Population Genetic Structure of the Ark Shell <Emphasis Type="Italic">Scapharca broughtonii</Emphasis> Schrenck from Korea,China, and Russia Based on <Emphasis Type="Italic">COI</Emphasis> Gene Sequences

Haplotype distribution, gene flow, and population genetic structure of the ark shell (Scapharca broughtonii) were studied using a partial sequence of a mitochondrial COI gene. The sequence analysis of 100 specimens obtained from a total of seven localities-five in Korea, one in China, and one in Russia- revealed 29 haplotypes, ranging in sequence divergence from 0.1% to 2.1%. Among these, the most frequent haplotype, SB16, was extensively distributed over study areas, especially in all Korean localities. This extensive distribution consequently resulted in the near absence of statistically significant genetic distance. Also, a high rate of gene flow was characteristic among localities in Korea. A test of genetic population structure showed that the ark shell in Korea formed a large genetic group. Moreover, an AMOVA test to determine the allocation of the genetic variance showed that most of the variance was distributed between localities, instead of within localities. However, a significant population differentiation was found between geographic populations [i.e., Jinhae (locality 6) in Korea and Sangdong (locality 5) in China and Vladivostok (locality 7) in Russia] based on geographic distance and population structure. These distinct groups may be associated with geographic characteristics and barriers. The results suggest that most of the ark shell populations in Korea caused considerable distribution to form a genetically homogeneous and intermixing structure, whereas some of the Korean and Chinese and Russian populations had a significantly different genetic structure.