Development of 12 Microsatellite Markers in Dorcus titanus castanicolor (Motschulsky, 1861) (Lucanidae, Coleoptera) from Korea Using Next-Generation Sequencing.

In the present study, we used next-generation sequencing to develop 12 novel microsatellite markers for genetic structural analysis of Dorcus titanus castanicolor (Lucanidae; Coleoptera), a popular pet insect in China, Korea, and Japan. We identified 52,357 microsatellite loci in 339,287,381 bp of genomic sequence and selected 19 of the loci based on their PCR amplification efficiency and polymorphism. The 19 selected markers were then tested for the presence of null alleles and linkage disequilibrium. We did not detect any evidence of null alleles; however, four pairs of loci (DT03 and DT11, DT05 and DT26, DT08 and DT26, DT26 and DT35) exhibited linkage disequilibrium. Thus, we assessed the genetic diversity of a D. titanus castanicolor population from the Daejeon region of Korea (n = 22) using 13 markers. Among them, one marker (DT17) deviated from Hardy-Weinberg equilibrium. Therefore, 12 markers may be useful for further analyzing the genetic diversity of D. titanus castanicolor.

DNA Barcoding of the Korean Lymantria Hübner, 1819 (Lepidoptera: Erebidae: Lymantriinae) for Quarantine Inspection.

DNA barcoding and morphological analyses of Korean Lymantria (Erebidae, Lepidoptera) were conducted for quarantine inspection. In DNA barcoding, Lymantria dispar identified through quarantine inspection was distinguished as three species, L. dispar asiatica, L. albescens, and L. xylina. Lymantria monacha, which is known as a single species in Korea, is revealed as containing three species, L. monacha, L. minomonis, and L. sugii. At the subspecies level, L. dispar dispar formed a single cluster, whereas L. d. asiatica and L. d. japonica formed a cluster containing both subspecies. In morphological re-examination on DNA barcoding results, L. dispar was distinguished from L. albescens by wing pattern, and from L. xylina by papillae anale. L. monacha and the related species were hard to be distinct from each other by using wing pattern, but it was easily distinct through comparison of genitalia. Therefore, DNA barcoding led to accurate identification in species level, but in subspecies level, only a taxon showing geographically far distance was discriminated from the others. These results may provide a taxonomic outline of the Korean Lymantria fauna and may be used as an identification reference for Lymantria species during quarantine inspection.

Development of Seven Microsatellite Markers Using Next Generation Sequencing for the Conservation on the Korean Population of Dorcus hopei (E. Saunders, 1854) (Coleoptera, Lucanidae).

We developed microsatellite markers for genetic structural analyses of Dorcus hopei, a stag beetle species, using next generation sequencing and polymerase chain reaction (PCR)-based genotyping for regional populations. A total of 407,070,351 base pairs of genomic DNA containing >4000 microsatellite loci except AT repeats were sequenced. From 76 loci selected for primer design, 27 were polymorphic. Of these 27 markers, 10 were tested on three regional populations: two Chinese (Shichuan and Guangxi) and one Korean (Wanju). Three markers were excluded due to inconsistent amplification, genotyping errors, and Hardy-Weinberg equilibrium (HWE). By multi-locus genotyping, the allele number, observed heterozygosity and polymorphism information content of seven microsatellite loci were ranged 2-10, 0.1333-1.0000, and 0.1228-0.8509, respectively. In an analysis on the genetic differentiation among regional populations including one Japanese population and one cross-breeding population, the individual colored bar-plots showed that both Chinese populations were closer to each other than to the Far East Asian populations. In Far East Asian populations, Wanju and Nirasaki populations could not be distinguished from each other because the frequency of genetic contents was very similar in some individuals of two populations. Moreover, the cross-breeding population contained all patterns of genetic contents shown in Chinese, Korean, and Japanese populations, compared with the genetic content frequency of each regional population. As a result, we examined whether the cross-breeding population might be a hybrid population, and might contain a possibility of interbreeding with Chinese populations in parental generations. Therefore, these markers will be useful for analyses of genetic diversity in populations, genetic relationships between regional populations, genetic structure analyses, and origin tests.

Phylogenetic relationships of Dorcus koreanus Jang and Kawai, 2008 (Coleoptera, Lucanidae): species or subspecies?

Dorcus koreanus Jang and Kawai, 2008 was recently described as a valid species in Haenam, South Korea, based on morphology. However, the taxonomic position and relationships of this new species with the related species Dorcus japonicus and Dorcus carinulatus were not examined in detail. To address this issue, we evaluated the phylogenetic relationships of D. koreanus to its related species based on molecular analyses of mitochondrial 16S rRNA gene sequences. The molecular evidence suggested that D. koreanus and D. carinulatus are more closely related to each other than either is to D. Japonicus. The genetic divergence between D. koreanus and D. carinulatus ranged from 1.2 to 1.6%, whereas that between D. koreanus and D. japonicus ranged from 9.0 to 9.2%. By comparing the range of nucleotide substitutions within Lucanidae, we determined that the sequence distance between D. koreanus and D. carinulatus is smaller than that for required for species-level distinction. Therefore, we reduce D. koreanus to subspecies rank, as Dorcus carinulatus koreanus stat. nov.

DNA barcoding in quarantine inspection: a case study on quarantine insect monitoring for Lepidoptera obtained through quarantine inspection on foreign vessels.

We conducted quarantine insect species diversity monitoring using DNA barcoding with 517 lepidopteran samples that were obtained from quarantine inspections of foreign vessels entering Korea. For species delimitation and species identification of the analyzed samples, we applied a 2% cutoff rule. Consequently, 145 (368 samples) were considered taxonomically identified. Therefore the number of samples that were identified to the species level was relatively low, at approximately 71%. Thirty of 145 species were not known in Korea, three, i.e., Noctua pronuba (Noctuidae), Orthosia hibisci (Noctuidae), and Pieris brassicae (Pieridae), were checked as 'Regulated pests' in Korea.

Dicranomyia crane flies (Diptera: Limoniidae) of Korea.

A total of 38 Korean species of crane flies belonging to the genus Dicranomyia Stephens, 1829 (Diptera: Limoniidae: Limoniinae) are taxonomically revised. No species of Dicranomyia were previously reported from South Korea, and only twelve species were reported from North Korea. New records of Dicranomyia from the Korean Peninsula include: D. (Dicranomyia) depauperata Alexander, 1918, D. (D.) distendens pallida Savchenko, 1983, D. (D.) handlirschi handlirschi Lackschewitz, 1928, D. (D.) hyalinata (Zetterstedt, 1851), D. (D.) kandybinae Savchenko, 1987, D. (D.) longipennis (Schummel, 1829), D. (D.) mesosternatoides Alexander, 1924, D. (D.) modesta (Meigen, 1818), D. (D.) pammelas Alexander, 1925, D. (D.) poli (Alexander, 1941), D. (D.) sera (Walker, 1848), D. (D.) shinanoensis (Alexander, 1933), D. (D.) takeuchii Alexander, 1922, D. (D.) unispinosa Alexander, 1921, D. (Erostrata) globithorax Osten Sacken, 1869, D. (E.) globulithorax Alexander, 1924, D. (E.) yazuensis Kato et al., 2018, D. (Glochina) basifusca Alexander, 1919, D. (Melanolimonia) paramorio platysoma (Alexander, 1933), D. (Numantia) fusca (Meigen, 1804), D. (Sivalimnobia) euphileta (Alexander, 1924). Species D. (D.) byuni Podenas, sp. nov., D. (D.) cornuta Podenas, sp. nov., D. (D.) jirisana Podenas, sp. nov., D. (D.) petrasiuni Podenas, sp. nov., and D. (D.) yankovskyi Podenas, sp. nov. are described. An additional ten new records are reported from North Korea, bringing the total number of Dicranomyia species known from North Korea to 22 species. South Korea has a greater diversity of Dicranomyia with total number of 30 species.        D. (D.) amurensis Alexander, 1925 is synonymised with D. (D.) hyalinata (Zetterstedt, 1851). An illustrated key with redescriptions and photographs of all species and both sexes of adults collected in Korea are presented. Most females are illustrated or described for the first time. Elevation range, period of activity, habitat information, general distribution and a distribution map in Korean Peninsula is given for each species.

Genetic structure and demographic history of Lymantria dispar (Linnaeus, 1758) (Lepidoptera: Erebidae) in its area of origin and adjacent areas.

We analyzed the population genetic structure and demographic history of 20 Lymantria dispar populations from Far East Asia using microsatellite loci and mitochondrial genes. In the microsatellite analysis, the genetic distances based on pairwise FST values ranged from 0.0087 to 0.1171. A NeighborNet network based on pairwise FST genetic distances showed that the 20 regional populations were divided into five groups. Bayesian clustering analysis (K = 3) demonstrated the same groupings. The populations in the Korean Peninsula and adjacent regions, in particular, showed a mixed genetic pattern. In the mitochondrial genetic analysis based on 98 haplotypes, the median-joining network exhibited a star shape that was focused on three high-frequency haplotypes (Haplotype 1: central Korea and adjacent regions, Group 1; Haplotype 37: southern Korea, Group 2; and Haplotype 90: Hokkaido area, Group 3) connected by low-frequency haplotypes. The mismatch distribution dividing the three groups was unimodal. In the neutral test, Tajima's D and Fu's FS tests were negative. We can thus infer that the Far East Asian populations of L. dispar underwent a sudden population expansion. Based on the age expansion parameter, the expansion time was inferred to be approximately 53,652 years before present (ybp) for Group 1, approximately 65,043 ybp for Group 2, and approximately 76,086 ybp for Group 3. We propose that the mixed genetic pattern of the inland populations of Far East Asia is due to these expansions and that the inland populations of the region should be treated as valid subspecies that are distinguishable from other subspecies by genetic traits.