DNA Barcoding of Two Gonioctena Species (Coleoptera: Chrysomelidae) Described from the Korean Peninsula

The genus Hexatoma Latreille, 1809 is a large group of aquatic crane flies, with almost 600 species worldwide. The largest subgenus is Eriocera Macquart, 1838, which includes all nine species known from the Korean Peninsula. Molecular methods were used to associate Hexatoma larvae with their putative adult species from South Korea. Mitochondrial Cytochrome c Oxidase Subunit I (COI) gene fragment sequences (DNA barcodes) of recently collected adults of H. (E.) gifuensis, H. (E.) ilwola, H. (E.) pernigrina, and H. (E.) pianigra were compared with twelve sequences of Hexatoma larvae. The larvae of H. (E.) pernigrina, H. (E.) pianigra, and H. (E.) gifuensis were associated with their putative adults. The larvae of H. (E.) gifuensis and H. (E.) pianigra and the larvae and pupae of H. (E.) pernigrina are described and illustrated. The larvae of two species not associated with any adult are described, and their COI gene fragment sequences (DNA barcodes) are presented. This paper presents the morphological characteristics suitable for distinguishing larval species. A key for the identification of larvae of the genus Hexatoma on the Korean Peninsula has been compiled. H (E.) sachalinensis is recorded from the Korean Peninsula for the first time. Our study is the first contribution to the Hexatoma larvae taxonomy using phylogenetic analysis based on mitochondrial COI fragment (DNA barcode) and one of the first attempts to reveal phylogenetic relationships between Hexatoma species using molecular markers.

We collected one new species and one new record in the genus Mesogastrura Bonet, 1930 (family Hypogastruridae) from three caves with different origins in the Korean Peninsula; Mesogastrura seotalensis sp. nov. and Mesogastrura ojcoviensis (Stach, 1919). The genus Mesogastrura Bonet, 1930 is newly recorded from the Korean Peninsula. Mesogastrura seotalensis sp. nov. shows various body color (light brown or much less and light purple), 3 + 3 eyes and unguis with three inner teeth. On the other hand, Mesogastrura ojcoviensis (Stach, 1919) is characterized by white body color, 2 + 2 eyes and unguis without inner tooth (rarely with 1). These species were found in piles of bat's guano inside of caves, so they are considered as troglophilous and guanophilous species. Partial DNA sequences of mitochondrial cytochrome c oxidase subunit I (COI) gene were used as DNA barcodes to clarify the species delimitation.

DNA barcoding has been widely used in species identification and biodiversity research. A short fragment of the mitochondrial cytochrome c oxidase subunit I (COI) sequence serves as a DNA bio-barcode. We collected DNA barcodes, based on COI sequences from 156 species (529 sequences) of fish, insects, and shellfish. We present results on phylogenetic relationships to assess biodiversity the in the Korean peninsula. Average GC% contents of the 68 fish species (46.9%), the 59 shellfish species (38.0%), and the 29 insect species (33.2%) are reported. Using the Kimura 2 parameter in all possible pairwise comparisons, the average interspecific distances were compared with the average intraspecific distances in fish (3.22 vs. 0.41), insects (2.06 vs. 0.25), and shellfish (3.58 vs. 0.14). Our results confirm that distance-based DNA barcoding provides sufficient information to identify and delineate fish, insect, and shellfish species by means of all possible pairwise comparisons. These results also confirm that the development of an effective molecular barcode identification system is possible. All DNA barcode sequences collected from our study will be useful for the interpretation of species-level identification and community-level patterns in fish, insects, and shellfish in Korea, although at the species level, the rate of correct identification in a diversified environment might be low.

Twelve species in total are recognized for the subgenus Lasioglossum (Dialictus) Robertson, 1902 from Japan, the Korean Peninsula and Taiwan. Lasioglossum (Dialictus) alishanense sp. nov. and L. (D.) taiwanense sp. nov. from Taiwan, are described as new. Four species are recorded from the following localities for the first time: L. (D.) miyabei Murao, Ebmer & Tadauchi, 2006 from the Korean Peninsula (South Korea); L. (D.) ellipticeps (Blüthgen, 1923), L. (D.) virideglaucum Ebmer & Sakagami, 1994 and L. (D.) viridellum (Cockerell, 1931) from South Korea. Lasioglossum (Dialictus) atroglaucum (Strand, 1913) is redescribed, L. (D.) ellipticeps, L. (D.) problematicum (Blüthgen, 1923), L. (D.) virideglaucum, and L. (D.) viridellum are supplementary described. A key to species is provided. Bionomical data, such as flight and flower records or habitat, are reported for some species. The distributions of all species are mapped. DNA barcodes of L. (D.) miyabei, L. (D.) pseudannulipes (Blüthgen, 1925), and L. (D.) virideglaucum are provided

DNA barcode (mitochondrial COI) sequences are provided for species identification of aphids from the Korean Peninsula. Most (98%) of the 154 species had distinct COI sequences (average 0.05% intraspecific pairwise divergence) relative to the degree of sequence divergence among species (average value 5.84%). For species in common with other regions, barcodes for Korean samples fell near or within known levels of variation. Based on these results, we conclude that DNA barcodes can provide an effective tool for identifying aphid species in such applications as pest management, monitoring and plant quarantine.

The Scarites aterrimus (Coleoptera: Carabidae), is one of the carabid beetles dwelling exclusively on coastal sandy dunes. Habitat deterioration and equivalent activity have greatly concerned population declines in several species dwelling on the coastal sandy dunes. As a first step to establish long-term conservation strategy, we investigated the nation-wide magnitude and nature of genetic diversity of the species. As a first step, we sequenced a portion of mitochondrial COI gene, corresponding to "DNA Barcode" region (658 bp) from a total of 24 S. aterrimus individuals collected over nine sandy dunes belonging to four Korean provinces. The sequence analysis evidenced moderate to low magnitude of sequence diversity compared with other insect species distributed in Korean peninsula (0.152% to 0.912%). The presence of closely related haplotypes and relatively high gene flow estimate collectively suggest that there had been no historical barriers that bolster genetic subdivision. Population decline was postulated on the basis of several missing haplotypes that are well found in the species with a large population size. This interpretation is consistent with field observation of small population size in the coastal sandy dune habitats. The highest genetic diversity estimates were found in the coastal sand dune population of Seogwipo, Jeju Island, justifying a prior attention to the population, in order to sustain overall genetic diversity of the species. Further scrutinized study might be required for further robust conclusion.

Discovery of a highly variable bug killing fly species, Clytiomya continua (Diptera: Tachinidae: Phasiinae), in Korea with discussion on its systematic status

Using reverse taxonomy and morphological analyses, this study describes a new species belonging to the C. flavicans species complex in the Korean Peninsula, Chaoborus pseudoflavicans Bang & Shin sp. nov. Descriptions of the new species from larvae to adults are provided, and the key to the C. flavicans species complex is updated accordingly. DNA barcodes (COI partial sequences) are shown to be sufficient for molecular identification in the C. flavicans species complex. Finally, the taxonomic accounts of all species in the C. flavicans complex are completely resolved for the first time.

*Department of Biological and Environmental Science, Dongguk University, Seoul 10326, Korea. **Department of Herb Crop Resources, NIHHS, RDA, Eumseong 27709, Korea.ABSTRACTBackground : Plants belonging to 5 species of the genus Eleutherococcus are currently distributed in the Korean peninsula. Thetraditional medicine ‘Ogapi’, derived from Eleutherococcus sessiliflorus and other related species, and ‘Gasiogapi’, derived fromEleutherococcus senticosus, are frequently mixed up and marketed. Therefore, accurated identification of their origins in urgentlyrequired. Methods and Results : Candidate genes from nuclear ribosomal DNA (nrDNA) and chloroplast DNA (cpDNA) of Eleutherococ-cus plants were analyzed. Whereas the nrDNA-internal transcribed spacer (ITS) regions were useful in elucidating the phylogeneticrelationships among the plants, the cpDNA regions were not as effective. Therefore, a combined analysis with nrDNA-ITS was per-formed. Various combinations of nrDNA and matK were effective for discriminating among the plants. However, the matK andrpoC1 combination was ineffective for discriminating among some species. Based on these results, it was found that OG1, OG4,OG5, OG7, GS1, GS2, and GS3 were derived from E. sessiliflorus . In particular, it was confirmed that GS1, GS2, and GS3 were notderived from E. senticosus. However, more samples need to be analyzed because identification of the origins of OG2, OG3, OG6and GS4 was not possible.Conclusion : The ITS2, ITS5a, and matK combination was the most effective in identifying the phylogenetic relationship amongEleutherococcus plants and traditional medicines based on Eleutherococcus.Key Words : Eleutherococcus, Chloroplast DNA, DNA Barcoding, Internal Transcribed Spacers, Nuclear Ribosomal DNA

Anoplophora glabripennis (Motschulsky 1853) (Coleoptera: Cerambycidae), the Asian Longhorned Beetle, is native to temperate and subtropical areas of China and the Korean peninsula. Due to its wide range of host plants, it is considered among the most economically important invasive plant pests. The morphological identification of A. glabripennis larvae can be confirmed by DNA barcoding, but obtaining the specimens from infested trees can be a demanding and challenging task. Therefore, non-invasive diagnostic tools based on DNA extracted from frass samples can be of key importance in phytosanitary surveys. In this study, an in silico generated real-time quantitative PCR test was developed for the detection of A. glabripennis DNA from frass material, which is naturally extruded from larval tunnels through cracks in the bark. Specificity was confirmed against a wide range of other wood-boring insect species frequently encountered during phytosanitary surveys and inclusivity was demonstrated for different populations of A. glabripennis from all main European outbreak areas. The test proved sensitive and reliable in detecting A. glabripennis DNA extracted from woody frass material of Acer saccharinum and Aesculus hippocastanum at least up to the 100-fold dilution. Furthermore, the test allowed the molecular identification of any life stage of the insect, including eggs and young larvae, whose morphological identification is impossible or very challenging. This study provides a reliable and sensitive molecular tool to detect A. glabripennis DNA in woody frass material, thus allowing a non-invasive sampling approach.

Copepods, small aquatic crustaceans, are the most abundant metazoan zooplankton and outnumber every other group of multicellular animals on earth. In spite of ecological and biological importance in aquatic environment, their morphological plasticity, originated from their various lifestyles and their incomparable capacity to adapt to a variety of environments, has made the identification of species challenging, even for expert taxonomists. Molecular approaches to species identification have allowed rapid detection, discrimination, and identification of cryptic or sibling species based on DNA sequence data. We examined sequence variation of a partial mitochondrial cytochrome C oxidase I gene (COI) from 133 copepod individuals collected from the Korean Peninsula, in order to identify and discriminate 94 copepod species covering six copepod orders of Calanoida, Cyclopoida, Harpacticoida, Monstrilloida, Poecilostomatoida and Siphonostomatoida. The results showed that there exists a clear gap with ca. 20 fold difference between the averages of within-specific sequence divergence (2.42%) and that of between-specific sequence divergence (42.79%) in COI, suggesting the plausible utility of this gene in delimitating copepod species. The results showed, with the COI barcoding data among 94 copepod species, that a copepod species could be distinguished from the others very clearly, only with four exceptions as followings: Mesocyclops dissimilis–Mesocyclops pehpeiensis (0.26% K2P distance in percent) and Oithona davisae–Oithona similis (1.1%) in Cyclopoida, Ostrincola japonica–Pseudomyicola spinosus (1.5%) in Poecilostomatoida, and Hatschekia japonica–Caligus quadratus (5.2%) in Siphonostomatoida. Thus, it strongly indicated that COI may be a useful tool in identifying various copepod species and make an initial progress toward the construction of a comprehensive DNA barcode database for copepods inhabiting the Korean Peninsula.

Recent molecular approaches toward insect taxonomy are revealing cryptic diversity within biting midge genus Culicoides Latreille, 1809. A new biting midge species was collected from highly restricted area of Dokdo Island, East Sea of the Republic of Korea, and was identified as Culicoides dokdoensis, sp. nov., based on morphological characters and DNA barcoding. The specimens collected from the island were previously known as Culicoides circumscriptus Kieffer, 1918, a cosmopolitan blood‐feeder, due to its morphological similarity. However, the specimens from Dokdo Island can be distinguished from other congeners by the following characters: more curved arms in male aedeagus, wing spot pattern, sensory pit depth, and absence of a spermatheca neck in female. In addition, mitochondrial cytochrome c oxidase I sequence exhibited substantial genetic difference compared to C. circumscriptus populations from the Korean Peninsula, sufficiently supporting the genetic differentiation between the two species. The discovery of this cryptic species highlights the necessity of integrated taxonomy for Culicoides.

Chironomid larvae are among the dominant benthic macroinvertebrates in all types of water systems in South Korea. They may pass through pipes in rivers (raw water) and occur in drinking water, thus creating public health issues. However, little is known about the larval stages of chironomids in large South Korean rivers. Therefore, we examined larval–adult associations in chironomids inhabiting major rivers used as water sources. The larvae were collected in 2015 and 2016 from nine locations along the four largest rivers in South Korea using a Ponar grab. Cytochrome oxidase subunit I (COI) sequences were generated from the larval specimens, and the species were identified by comparing these sequences to those in a newly constructed DNA barcode library of Chironomidae in South Korea. The samples from the four rivers yielded 61 mitochondrial COI sequences belonging to 18 species, including Hydrobaenus kondoi Saether, 1989, which was reported for the first time in the Korean Peninsula. Further, morphological identification of the larvae was conducted, and a pictorial taxonomic key to Chironomidae species in large rivers in South Korea was developed to facilitate freshwater biomonitoring research. Finally, an action flow chart was created for the rapid identification of chironomid larvae in infested drinking water or water purification facilities.

Mitochondrial DNA sequence variation of the bumblebee, Bombus ardens (Hymenoptera: Apidae)

The second introduction of a non-native Embioptera to Korea via ornamental plants: Evaluating DNA barcoding to prepare for a global inflow of unfamiliar taxa