We carried out DNA barcoding of five Korean Lymantria species to establish identification references library for quarantine inspection. Total of 118 samples including 34 samples obtained through quarantine inspection, two from USDA, and one collected from Philiphine were used for this study. And 30 sequences of 10 species from GenBank of NCBI were used as reference sequences. In a result of DNA barcoding of the Korean Lymantria species, sequence divergence of 148 DNA barcodes ranged from null to 17.0%, intraspecific divergence from null to 1.0%, and interspecific divergence from 5.1 to 17.0%. In NJ tree, L. dispar contained three clusters, which were identified as L. dispar asiatica, L. albescens, and L. xylina, respectively. L. xylina was collected through quarantine inspection on a foreign merchant ship in Yeosu port, and L. albescens was obtained by pheromone trap on L. dispar installed in Busan port. And L. monacha known as single species in Korea was revealed as species complex with three species, L. monacha, L. minomonis, and L. sugii. In subspecies level, L. dispar dispar (EGM) built single cluster, but L. d. asiatica (AGM) and L. d. japonica showed as multiple cluster. Therefore, DNA barcoding lead to rapid and accurate identification in species level, but in subspecies level, only a taxon showing geographically far distance was discriminated from the others. And the results could provide a taxonomic outline of the Korean Lymantria fauna and might be used as identification reference for Lymantria species in quarantine inspection.

The mitochondrial COI gene has proven successful for identifying the lepidopteran species. In addition, this gene has been applied to recognize cryptic species and confirm a polyphagous species as one species. However, it has been also reported misidentification in the COI gene. As a result, some researchers have suggested the use of additional genes in species identification, especially, nuclear genes. In this study, we observed that a nuclear EF1agene is better than the mitochondrial COI gene in recognizing Stathmopoda species. For example, among the all species, 47 individuals of nine species, a large intraspecific pairwise difference were detected, up to 15.0% in universal barcoding regions of the COI gene, 22.0% in other part of the COI gene; however, 2.0% in EF1a. In contrast, Stathmopoda sp2 and S. commoda were separated into several clusters in each different COI regions, and some individuals of S. auriferella and S. commoda, were closer to each other separating from the cluster of the same species. But, we get clear results from the EF1a, Stathmopoda species well clustered and could be distinguished.

Standardized short DNA sequences of the genome provide a DNA barcode for identifying species. Assembling DNA barcode data of regional fauna could provide a new identifying system and it will aid regional and global taxonomic studies and other applied biology. Recently, partial cytochrome c oxidase I (COI) sequences, around 670-bp region of the mitochondrial gene, has been qualified as a good DNA barcode for the identification of insect species. The superfamily Nepoidea includes two families-Belostomatidae and Nepidae. And it contains about 377 species in 23 genera worldwide. In Korea, each family is recognized 4 species 3 genera, respectively. As a part of the Korean insect DNA barcode project, we conducted preliminary DNA barcode study on Korean Nepoidea to provide useful molecular data for aiding accurate identification. This study also tested the effectiveness of a COI barcode in discriminating Korean Nepoidea species. So far, 43 individuals of 8 species belonging to two families were analyzed. We used a 658-bp fragment of COI gene was sequenced. Pairwise sequence divergences were calculated using a Kimura 2-parameter model and constructed neighbor-joining tree to calculate generic differences among species and within species.

Many species of the genus Bradysia larvae are known as agriculture pest, because they are feed on stem or root of agriculture plants. Even though the damages are made by larval stages, the immature stages are hardly identified morphologically. The partial sequences of mitochondrial cytochrome c oxidase subunit I (COI) gene are constructed for 25 species of Bradysia with the identified voucher specimens. Comparing these data, Bradysia procera, and B. sp.1 (tilicola Group) could be identified from the larvae specimens by molecular COI universal region. Bradysia atracornea, B. nomica, and B. difformis are also confirmed from the larval samples in greenhouse crops, chinese cabbage, onion, and scallion etc. The mean Kimura two-parameter (K2P) interspecific divergence of genus Bradysia was 16.78%, and mean K2P intraspecific divergence was 0.52%. DNA

DNA barcoding is a strong species identification tool for all animal taxa, and can easily be conducted when materials are under DNA friendly conditions. In contract, a full-length (659 bp) sequencing has been limited for the degraded DNAs extracted from old museum specimens. The initial challenges to retrieve the authentic DNA fragments from old museum specimens were attempted by obtaining short sequences (<300 bp) with the cloning process after PCR, making it both expensive and time-consuming. In this study, we employed a modified method to analyze the full-length DNA barcoding regions in 31~52 year-old butterfly specimens (301 dried specimens of 39 species) using direct sequencing after PCR with two different methods: 1) the successful PCR rates of 0 to 5.6% using four universal primer sets were too low to obtain authentic sequences and the cross-contamination was detected in almost all successful amplicons; 2) the success rates of PCR using specie-specific overlapping primer sets were distinctly high, reaching up to 75% with 98% authentic and 2% non-specific sequences. Thus, the result showed the method that using species-specific primer set per species yields the most effective success rates of both PCR and sequencing from degraded DNA without incorrect sequences.

We tested the identification ability of DNA barcodes comparing with morphological data using the Korean butterflies. The 921 samples (4.6 samples per species) for 202 resident Korean species except migratory species were used. The obtained samples were morphologically identified based on wing patterns. In a result, genetic divergence to the nearest-neighbouring taxon varied from 0 to 28.2%, with an average of 13.4 per cent. The neighbour joining (NJ) tree profile showed that sequence data for 185 of the 202 species formed distinct barcode clusters. Thus, our results indicated that 91.6 percent of the species were possible to allow the reliable identification using DNA barcoding. The rest 17 species (8.4%) consist of following four cases: clustering separated from each species by less than 1% branch length (two species pairs), paraphyletic clustering (two species pairs and one triple species pair), polyphyletic clustering with sharing barcodes (three species pairs), and clustering separated from existing species by the deep branch divergence (four clusters). However, it was not easy to interpret these ambiguous cases only using our current taxonomic evidences. Therefore, we are performing integrative taxonomy on these cases using other additional evidences such as examination on male genitalia and analysis of other gene regions.

In DNA barcoding, the DNA degradation of old museum specimens has been limited full-length (658bp) sequencing. The challenges associated with the retrieval and authentication of degraded DNA extracts from fossil and old museum specimens were principally limited to analyze the relatively short sequences (<300 bp). Furthermore, almost protocols in other to analyzed the degraded DNA contained the cloning process after PCR causing the time-consuming and the rising costs. To overcome these problematic circumstances, we tried a modified method to analyze full-length of DNA barcoding region in 30~60 year-old butterfly specimens (225 samples in 28 species), using direct sequencing after PCR with species-specific overlapping primer sets per each species. As a result, all of 28 species have been successfully analyzed, although 178 samples (79%) are completely generated barcoding sequences ranged from 640 to 658 bp and 47 samples (21%) are partially sequenced ranged from 100 to 500 bp. Thus, the result showed that the direct PCR sequencing using the overlapping primer sets per species appears to have great potential efficiency for analysis of degraded DNA without incorrect sequences.

DNA barcode (mitochondrial COI) has been widely attempted for species identification of many animal groups including aphids. In this study, we newly found a DNA barcoding problem in a case study of the grain aphid, Sitobion avenae. Unexpectedly, five S. avenae individuals showed considerable differences of, on average, 32.6% in the DNA sequences from other conspecific individuals. BLAST search revealed that the five sequences are similar to those of aphid parasitoids such as Aphidius, Ephedrus, and Praon spp. (Hymenoptera: Braconidae). Based on these results, we concluded that the universal primers used in aphid DNA barcodes can amplify barcode sequences from parasitoid species within host aphids.

The superfamily Tephritoidea is a large group of acalyptrate flies including over 7,300 described species arranged in nine families. All the tephritoid families excluding Richardiidae are represented in Korea, but their taxonomy has not been well studied. For example, a total of 80 species of the family Tephritidae are currently known in Korea, but our personal data shows that there actually are at least 150 species. Presence of highly variable or cryptic species has been hampering taxonomic progress involving this group of flies. Some genera such as Trypeta, Campiglossa, Oxyna, and Tephritis are good examples of taxonomically difficult taxa. We find that DNA barcoding is extremely helpful to clarify such taxonomic problems. We are currently trying to bacode multiple samples for every single Korean tephritoid species. A total of 271 specimens representing 185 species have been barcoded so far. Neighbor-Joining analysis of our preliminary data shows many interesting findings that are potentially useful to resolve long standing taxonomic problems. The followings are some of our findings through the DNA barcoding analyses: 1) multiple samples for each included species were almost always clustered together showing utility for specific identification; 2) multiple species per each genus were mostly clustered together; 3) clarification of male and female association of two closely resembling new Acidiella species; 4) strongly support synonymy of Sinacidia and Chetostoma; 5) possible existence of two cryptic species within Campiglossa defasciata; 6) possible synonymy of Dioxyna and Campiglossa; 7) possible synonymy of Herina zojae and H. hennigi; and 8) discrimination of two closely resembling Chaetostomella species.

DNA barcoding uses a 650 bp segment of the mitochondrial cytochrome c oxidase I (COI) gene as the basis for an identification system for members of the animal kingdom and some other groups of eukaryotes. PCR amplification of the barcode region is a key step in the analytical chain, but it sometimes fails because of a lack of homology between the standard primer sets and target DNA. Two forward PCR primers were developed following analysis of all known arthropod mitochondrial genome arrangements and sequence alignment of the tRNA-W gene which was usually located within 200 bp upstream of the COI gene. These two primers were combined with a standard reverse primer (LepR1) to produce a cocktail which generated a barcode amplicon from 125 of 141 species that included representatives of 121 different families of Hexapoda. High quality sequences were recovered from 79% of the species including groups, such as scale insects, that invariably fail to amplify with standard primers. The current results show that primers designed to bind to highly conserved gene regions upstream of COI will aid the amplification of this gene region in species where standard primers fail and provide valuable information to design a primer for problem groups.

지난 10여 년간의 한국산 병대벌레과에 대한 분류학적 연구를 통하여 연노랑목가는병대벌레(Asiopodabrus fragiliformis)는 체색이나 앞가슴등판의 형태에 의해 2개의 무리로 뚜렷하게 구분되었지만, 수컷 개체의 부족으로 생식기에서는 명확한 차이를 충분히 확인할 수 없어서 형태 변이폭이 큰 종으로만 인식해 왔다. 최근 한국산 병대벌레과의 전종에 대한 DNA barcoding 분석에서 9개체의 연노랑목가는병대벌레가 각각 5개체와 4개체로 나누어져 2개의 단일 집단으로 구분되어 새로운 은밀종이 있을 가능성이 제시되었다. 특히, 두 집단 사이에서는 3.9-5.2%의 큰 서열 차이를 보였고, 이들 각각의 집단 내 서열 차이를 보면 기존 연노랑목가는병대벌레집단은 0.0-2.0%, 신종 추정집단은 0.3-2.0%으로 차이를 나타내었다. 따라서 두 집단 모두 2.0%의 divergence threshold를 보여주면서 뚜렷한 barcoding gap이 형성됨을 확인하였다. 이를 근거로 각 집단의 형태 형질을 재검토하였으며, 그 결과로 신종 추정집단에서는 수컷 생식기에서 화살모양의 ventral process가 개체마다 동일하게 나타남을 확인하였다. 반면에 기존 연노랑목가는 병대벌레집단에서는 기부에서 두껍다가 점점 가늘어지는 형태의 ventral process를 가짐을 볼 수 있었다. 결과적으로 과거 형태적 분류로서 연노랑목가는병대벌레의 개체변이는 체색과 앞가슴등판의 형태적 차이를 가진 2종으로 구분될 수 있었다. 따라서 신종추정집단을 기존 연노랑목가는병대벌레에서 독립된 Asiopodabrus sp. nov.로 명명하여 신종으로 보고하고자 한다.

오색나비(Apatura ilia)와 황오색나비(Apatura metis)는 과거로부터 동일종, 또는 동일종의 아종관계, 별종 등으로 분류학적 위치에 대한 논란이 있어왔다. 이 두 종은 주로 날개 무늬로 구분되는데, 오색나비와 황오색나비의 중간적 양상을 나타내는 경우가 빈번하여 생물학적 종을 식별하는데 혼란이 있어왔다. 이에 따라서 일부 전문가는 두 종의 공유 분포 지역에서는 잡종이 형성될 수 있다는 주장을 제시하였다. 또한 최근 아마추어 나비전문가에 의하여 사육된 황오색나비는 2세대에서 2가지 형이 모두 나타났다는 보고도 있었다. 따라서 형태형질을 바탕으로 동정된 표본을 근거로 DNA barcoding을 이용하여 오색나비와 황오색나비의 생물학적 실체를 검증해 보고자 하였다. 오색나비류의 전세계 분포지 중, 우선 중국, 극동러시아, 한국, 일본에서 확보된 극동아시아산 오색나비류 총 60 개체들을 대상으로 시도하였다. 그 결과, 오색나비 집단과 황오색나비 집단의 유전적 차이는 개체에 따라 0.0%에서 1.0%까지 밖에는 차이가 나지 않는 것으로 확인되었으며, NJ tree에서도 두 집단이 각각 별도의 묶음으로 구분되지 않았다. 특히, 형태적으로 오색나비로 동정된 북방계 개체들의 일부가 단계통 묶음을 형성하는 듯한 양상을 보이나, 그의 나머지 개체들과 남부집단 그리고 황오색나비의 전 개체들은 모두 하나의 섞긴 묶음을 형성하였다. 또한 두 묶음과는 0.5% 정도의 극히 미미한 서열 차이만 보였고, 특정 지역 집단으로 구성된 무리도 존재하지 않았다. 따라서 기존의 오색나비와 황오색나비의 극동아시아산 개체들은 오색나비 한 종이며, 황오색나비로 알려진 개체들은 오색나비의 변이체로 진단하는 것이 분류학적 당위성을 가질 것으로 판단되었다.

Recently, hundreds thousands of CO1 barcodes have been produced in many insect groups however, relatively little is known in Hemiptera. In this study, we newly determine 1173 sequences for 374 species distributed among 190 genera and 33 families collected mostly from North America. Average age of the specimen was 12.2 years and about 70% of the specimens produce barcode over 500 bp among examined 1700 specimens. Con-generic divergency calculated by K2P distance was 9.99 which was 11fold higher than the mean intraspecific variation. A steady increase of genetic variation through increasing taxonomic levels was also observed. We found several taxon groups with deep divergency (over 2.0 K2P distance) which were morphologically identified as a same species. Some mixed clusters with several species were also found and suggested more detailed study for these clusters through both morphological and molecular biological method.

DNA barcode is known to be successfully applied in identification on the members of Insecta. In recent studies, however, it was known that the DNA approach may fail in several taxa with following cases: (1) very recent speciation and hybridization, (2) recent diverged groups with complex gene histories, (3) the spread of maternally transmitted bacteria, (4) adding more than one geographical race and at least one congener, (5) different levels of dispersal. In this study, we taxonomically review on the Korean Hatchiana using the morphological data and DNA barcodes. In morphology, they are distinct from each other by the characteristics of body coloration, eye, pronotum, scutellum, and aedeagus. In molecular data, however, the interspecific sequence distance ranged from 0.0-3.4%. This result is caused by H. glochidiatus, of which the sequence divergence is 0.2-2.8% in H. rosinae, 0.8-2.6% in H. baekripoensis, and 0.0-3.3% in H. jirisanensis. Also, H. glochidiatus produces mixed-clusters with H. rosinae and H. jirisanensis in NJ phenogram. Through this presentation, therefore, we discuss on why the four Korean Hatchiana species distinct by morphological characters produce mixed-clusters in DNA barcoding.

최근에 급속도로 확산되고 있는 DNA 바코드 연구는 새로운 종의 발견이나 상위분류군의 판단에도 결정적인 단서를 제공해 줄 수 있다. 즉 표준화된 DNA 부분을 많은 종에 걸쳐 비교함으로서 종을 정확하게 동정할 수 있다는 것이다. 2004 년에 발표된 한 연구에 따르면, 중남미에 분포하는 팔랑나비과의 1종으로 기록되어 있던 분류군에서 무려 10종의 근연종들을 DNA 바코드 분석을 통하여 구분한 바 있다. 이들은 성충시기에는 큰 차이를 보이지 않으나 유충의 형태 및 숙주에서 상탕한 종간 변이를 갖고 있는 것이 확인되었다. 이외에도 성적인 이형을 보이는 암수의 연계, 유충과 성충의 연계에 관련된 연구들도 상당수 발표되고 있다. 과실파리과는 많은 해충들을 포함하여 전 세계적으로 4,000종 이상이 기록되어 있는 농업적으로 매우 중요한 분류군이다. 현재 약 300종의 과실파리가 유실수의 작물을 공격하는 해충들로 알려져 있으며 많은 종들이 동남아시아와 남아메리카, 오스트레일리아 등 열대 및 아열대 지방에서 다양한 과실에 극심한 피해를 주고 있다. 많은 과실파리과 곤충들이 세계 각국에서 과채류의 해충으로서 원산지로부터 다른 지역으로 도입되어 문제를 일으키고 있으므로, 겸역상으로 매우 중요한 분류군이라 할 수 있다. 한국의 국립식물검 역소에서도 과실파리 42종을 금지해충으로 5종을 관리해충으로 선정하여 검역에 힘쓰고 있다. 과실파리의 정확한 동정을 위해서는 일반적으로 잘 제작된 성충의 표본이 필요하다. 그러나 검역을 통하여 입수되는 표본들은 대부분 유충태이며, 이들을 정확히 동정하는 것은 거의 불가능하다. 드물게 사육을 통하여 성충을 우화시켰다 하더라도 이들을 동정하기 위해서는 분류전문가의 도움이 필요 한 경우가 많다. 그러나 국제적으로 과실파리 분류학자들의 수가 한정되어 있어 동정을 의뢰하여 도움을 받기가 쉽지 않다. 따라서 DNA 바코딩 작업을 통하여 주요 해충분류군을 포함하는 DNA 데이터베이스를 조성할 수 있다면 간단한 DNA 염기서열 분석작업을 통하여 검역현장에서 분류학자의 도움 없이 정확한 동정을 실시할 수 있는 기반을 마련할 수 있을 것이다.

Background : Gentinae Macrophyllae Radix is one of the traditional medicines originated from the roots of multiple plants, Gentiana macrophylla Pall., Gentiana straminea Maxim., Gentiana crassicaulis Duthie ex Burkill and Gentiana dahurica Fisch., in Gentianaceae. Multi-origin traditional medicine usually has adulteration problem based on the morphological similarity and/or misunderstanding the species. Therefore, accurate and reliable identification criteria to ensure drug safety and quality is necessary. Methods and Results : We collected four original species of Gentinae Macrophyllae Radix from plantations and markets in China and Korea. DNA barcoding with four barcoding markers (Internal Transcribed Spacer (ITS), rbcL, trnL intron, trnL-F intergenic sapcer) was performed. Intra-specific variation was observed in ITS nucleotide sequence however, successfully distinct four original species based on the nucleotide discrepancy while trnL intron has no difference. trnL-F intergenic spacer has two transitions(T→C and A→G) sites only in G. crassicaulis and rbcL shows one transition(C→T) site in G. dahurica and G. macrophylla. Phylogenetic relationship analysis of the Gentinae Macrophyllae Radix revealed two major clades – clade I including three groups, G. macrophylla, G. straminea and G. dahurica, and clade II including G. crassicaulis. This aspects was shown more clear with multi-region combined analysis. Conclusion : DNA barcoding will be accurate and powerful criteria for the analysis the origin of Gentinae Macrophyllae Radix. However, single region analysis might be deficient such as trnL intron, rbcL and trnL-F intergenic spacer results in this research. Multi-region combined analysis based on the multiregional DNA barcode markers will be overcome the disadvantage and also increase the precision.

Background : Ixeris dentata is the perennial herbaceous plant in the Ixeris genera within the Compositae family. The whole plant has been used traditionally as herbal medicine. It is widely distributed in South Korea and the genetic difference among the plants harvested from different regions may differ due to the disparity in cultivation climate. Therefore, this research was performed to discriminate the I. dentata that are collected from four locations in South Korea based on sequence analysis of nrDNA-ITS region. Methods and Results : Genomic DNA was extracted from I. dentata obtained from Goesan, Dangjin, Yangpyeong, and Chuncheon. I. stonolifera was included for a comparison of genetic distance with I. dentata. PCR amplification was performed by using an universal barcode nrDNA-ITS primer for DNA barcoding. After sequencing, the data was aligned using ClustalW multiple alignment tool in BioEdit version 7.2.5 software. SNP and phylogenetic analysis were conducted with the MEGA7 program. Phylogenetic analysis was presented with Neighbor-joining method using the K2P model. Statistical significance are evaluated using bootstrap (1,000 replicates). PCR products were amplified with about 800bp length for the ITS1-4 sequences of all samples. Nineteen SNPs were detected within a 578bp fragment of the aligned sequences. The mean GC content was 51.88% for ITS1-4 sequences of them. The interspecific genetic distance between I. dentata and I. stonolifera was 0.029%. The highest region-specific distance was confirmed to 0.010% between the plants from Dangjin with Goesan and Chuncheon group. Meanwhile, the mean intraspecific distance among the plants from Yangpyeong was 0.002%. Conclusion : In the phylogenetic analysis, the plants from Goesan and Chuncheon were placed within the same clade, while the plants from Dangjin formed independent clade. On the other hand, the plants from Yangpyeong were not grouped into one clade followed by the intraspecies variation. In conclusion, the data from the research will be useful for the regional identification of Dangjin, even if it is required to perform some additional researches.

Background : Correct identification of Panax species is important to ensure food quality, safety, authenticity and health for consumers. This paper describes a high resolution melting (HRM) analysis based method using internal transcribed spacer (ITS) and 5.8S ribosomal DNA barcoding regions as target (Bar-HRM) to obtain barcoding information for the major Panax species and to identify the origin of ginseng plant. Methods and Results : A PCR-based approach, Bar-HRM was developed to discriminate among Panax species. In this study, the ITS1, ITS2, and 5.8S rDNA genes were targeted for testing, since these have been identified as suitable genes for use in the identification of Panax species. The HRM analysis generated cluster patterns that were specific and sensitive enough to detect small sequence differences among the tested Panax species. Conclusion : The results of this study show that the HRM curve analysis of the ITS regions and 5.8S rDNA sequences is a simple, quick, and reproducible method. It can simultaneously identify three Panax species and screen for variants. Thus, ITS1HRM and 5.8SHRM primer sets can be used to distinguish among Panax species.

The plant family chrysanthemum is known for its medicinal, ornamental, and economic purposes. Owing to its economic and biological significance to the difficult identification based on morphological characters, it is useful to develop DNA barcodes. DNA sequence data enable not only the inference of phylogenetic relationships but also provide an efficient method for species-level identifications under terms DNA barcoding or DNA taxonomy. The purpose of this study is to evaluate the utility of DNA barcoding in discriminating Chrysanthemum species. Four cpDNA regions (matK, rpoC, rpoB, trnH-psbA) and one nuclear (ITS) marker have sequenced from 28 specimens of 11 species from 4 genera of Chrysanthemum which were collected from 5 provinces in Korea. Comparisons of within and between species levels of sequence divergence showed that genetic variation between species exceeds variation within species.