The blackish cicada, Cryptotympana atrata Fabricius, 1775 (Hemiptera: Cicadidae) was originally distributed mainly in the southernmost remote island, Jeju and rarely throughout low lands in South Korea, but has been explosively increased at the urban areas, where annual temperature is higher. In this study, we sequenced a partial mitochondrial COI from a total of 171 individuals collected throughout 12 localities in South Korea. The haplotype found with the highest frequency in Jeju island shares only with two inland localities in southern region with a low frequency, whereas the haplotype found with the highest frequency throughout inland localities was not found in Jeju island. These results showed that Jeju population, southern region, and other inland populations form somewhat different genetic groups.

The onion thrips, Thrips tabaci, is a serious global pest attacking many agricultural crops such as onion and Welsh onion. The thrips, assumed to originate in the Mediterranean region, has been reported for a long time in South Korea. According to worldwide molecular works, the species composes of three genetic lineages (LI, L2 and T) which related to reproductive mode (arrhenotoky vs. thelotoky). To understand the genetic diversity of T. tabaci in South Korea, we investigated genetic lineage and haplotype composition, using about 80 mitochondrial COI gene sequences (369bp) along with foreign sequences from GenBank and BOLD. The COI gene analysis shows that both of thelotokous L1 and arrhenotokous L2 population distribute in South Korea. Among 97 COI-haplotypes worldwide, only six haplotypes are found and thelotokous H1 dominantly distributes.

담배가루이는 경제적으로 매우 중요한 농업 해충들 중의 하나이며, 전세계적으로 40개 이상의 종들로 구성된 종복합군(species complex) 으로 알려져 있다. 본 연구에서는 담배가루이 종복합군의 유전적 변이와 구성하는 종들의 수를 550개의 COI 염기서열들을 바탕으로 재평가하였 다. 담배가루이의 유전적 변이는 0% - 27.8%이며(평균 11.1%), 이는 담배가루이 종복합군이 서로 다른 속들 혹은 아과들에 속하는 다양한 종들 로 구성되어 있음을 나타낸다. 217개 COI 염기서열들을 바탕으로 분석된 계통수는 담배가루이 종복합군이 잠재적인 신종(Java)을 포함한 43개 종들로 구성되어 있고, 이 가운데 9종(Australia, Asia II 1, Asia II 6, Asia II 7, Asia II 10, Mediterranean, New world, New world 2, Sub Saharan Africa 1)의 종내 유전적 변이는 기존의 종구분 한계인 4.0%가 담배가루이 종복합군의 종들을 구분하는데 적합하며, 높은 종내 유전변이를 보이 는 종들은 은밀종과 관련이 있을 것으로 판단된다.

한국 및 그 인근에 서식하는 매미나방의 개체군 구조 분석을 수행하였다. 한국 및 러시아의 33개 지역 중 샘플이 채집된 32개 지역 960 개체를 대상으로 COI gene 서열을 분석하였으며, 이 중 서열이 확보된 906개체(n=20~30)를 이용하여 개체군 구조를 분석하였다. 그 결과, 전체 개체에 대한 haplotype diversity는 0.6774±0.0144으로 나타났는데, 33번 지역(러시아)이 0.3034 ± 0.1041로 가장 낮았고, 25번 지역이 0.8128±0.0648로 가장 높았다. 전체 개체에 대한 nucleotide diversity는 0.016701±0.012517로 나타났는데, 2번 지역이 0.005875±0.006619로 가장 낮았고, 25번 지역이 0.027003±0.018376으로 가장 높았다. Fst pairwise distance를 이용한 개체군 구조 분석 결과, 러시아를 비롯한 한국 내 분포하는 매미나방은 2개의 계통이 존재하는 것으로 나타났으며, 이에 대한 F-statistic value는 계통 간(Fct)에는 0.27066 (P=0.00000), 각 계통 내의 개체군 간(Fsc)에는 0.01174 (P=0.00489), 전체 개체군 간(Fst)에는 0.27922 (P=0.00000)으로 나타나 각 계통 간의 유전적 격리가 있음을 확인할 수 있었다. Median joining network에서도 한국의 남부지역에서만 나타나는 haplotype이 확인되었다. 이로 볼 때, 한국 내로의 매미나방 개체군의 형성은 크게 2가지 경로로 이루어진 것으로 사료되었다. mismatch analysis를 통해 각 계통에 대한 demographic history를 추론하였다. 그 결과, 러시아를 비롯한 한국 중부 지역의 개체군은 약 35,000년 전(34,782 (18826~54652) generation time)에 sudden expansion이 있었던 것으로 나타났으며, 지리적 거리와 유전적 거리의 상관관계를 나타내는 mentel's test에서도 지리적 거리가 증가함에 따라 유전적 거리도 증가하는 것으로 나타났다. 남부 지역 개체군은 약 48,000년 전(47,826 (35,478~66,652) generation time)에 확산이 일어난 것으로 나타났다. 다만, 남부지역 개체군의 경우, mentel's test에서 지리적 거리와 유전적 거리의 상관관계가 반비례하는 것으로 나타났으며, 남부지역에만 나타나는 haplotype은 NCBI GenBank Blast search 결과, 한국 내에만 분포하는 유전자형으로 나타나 유전적 병목현상이 발생했을 가능성이 있을 것으로 추론되었다. 그러나 이에 대한 분석은 추후 Mitochondrial control region이나 Microsatellite loci의 분석을 통해 확증할 예정이다. 결과적으로 한국 및 인근 지역의 매미나방 개체군 구조 분석을 통해 지역 개체군간의 관계를 밝힘과 분산에 대한 가설을 제공함으로서 유사한 양상을 나타내는 곤충 종에 대한 한국 내에서의 개체군 형성 모델을 제시할 수 있을 것으로 사료된다.

The mitochondrial COI gene has often been utilized as a molecular marker for species identification. However, it has sometimes caused misidentification for some pairs of closely related species. For detecting complementary barcoding loci, we first screened candidate genes by calculating genetic distances within and between species based on 542 sequences collected from the Genbank by using aphids of the Eriosomatini as an example. Of eight genes analyzed, we selected the ATP6 and ATP8 genes, which exhibited lower intraspecific and higher interspecific genetic divergences than did the COI gene. Secondly, we tested the usefulness of these genes by calculating genetic distances between all the combinations of 44 individuals of 23 eriosomatine species for each of the ATP6, ATP8, and COI genes. In the ATP8 gene, the average intraspecific divergence was lowest (0.6%) and the average interspecific divergence was highest (14.7%). The ATP8 gene evolved more rapidly than did the COI gene if genetic divergence between individuals was sufficiently large, whereas it evolved more slowly than did COI if genetic divergence was less than a threshold (1% in COI distance). As a result, species with intraspecific variation in COI and ATP6 exhibited no genetic variation in ATP8. The pattern of genetic divergence in ATP8 well accorded with the pattern of species delimitation in the present taxonomic system. Thus, we conclude that the use of the ATP8 gene in DNA barcoding could improve the accuracy of species identification in the Eriosomatini and possibly other insect groups.

The phylogenetic relationships of species and genera in the subfamily Nymphalinae from Myanmar were inferred using mtDNA sequence data from 608 bp of cytochrome oxidase subunit I (COI). A total of 20 species in 10 genera were sequenced and used to construct phylogenetic trees. The base composition of COI sequences was 38.1% T, 15.6% C, 31.6% A, 14.7% G, revealing strong AT bias (69.7%). The sequence distance of 20 species of Nymphalinae ranged from 1.5% to 15.5%. The transition of nucleotide substitution was more common than transversion. The transition between T and C were higher than transition between A and G, and the transversion between A and T was the highest amongst other types of transversion. The phylogenetic trees were constructed using the neighbor-joining (NJ) and maximum likelihood (ML) methods and showed almost identical topologies. The results indicated that the tribes Junoniini and Nymphalini (sensu Wahlberg et al., 2005) formed monophyletic groups but Kallimini was not monophyletic group. Rhinoplapa polynice formed sister group to Junoniini clade with moderate support in both trees. The relationship of species in Junoniini was ((Junonia + Yoma) + Hypolimnas) and the relationship in Nymphalini was (Symbrenthia + (Vanessa + (Kaniska + Polygonia))). The clustering results were almost identical to current morphological classification.

2000년도 이후로 많은 분자 연구 논문들이 육각강에서 진행되었으며, 그 결과 방대한 양의 미토콘드리아 유전자 염기서열이 생산되었다. 본 연구에서는 2000년도부터 2009년까지 육각강에 보고된 COI 염기서열과 이들을 활용한 분자 연구 논문들을 분석하기 위하여, 58,323개의 COI 염기서열들을 바탕으로 보고된 488개의 분자 연구 논문들을 26개 목들과 사용된 COI 염기서열들의 5', 3', 중간 위치에 따라 재분류하였다. 세 지역의 COI 염기서열을 이용한 연구 논문의 수는 26개 목들에 따라 매우 다양하였다. 하지만, 7개 목들은 특정 지역의 COI 염기서열들이 선호되었음을 나타내었다. 예를 들어, 파리목과 메뚜기목에서는 5' 지역의 COI 염기서열을 사용하는 논문의 수가 가장 높았으며, 이에 반해 딱정벌레, 이목, 잠자리목, 더듬이벌레목, 대벌레목에서는 3' 지역의 COI 염기서열을 사용하는 논문의 수가 가장 높았다. 2000년 이전에 보고된 84개의 분자 연구 논문들과의 비교를 통해, 2000년부터 2009년까지 딱정벌레목, 파리목, 이목, 대벌레목에서는 1999년 이전에 각 목들에서 주로 사용되었던 COI 염기서열의 특정 지역과 같은 지역을 사용하는 경향성을 보여주었다. 본 연구는 육각강에서 2000년에서 2009년까지 보고되었던 분자 연구 논문 뿐만 아니라 이들 논문에서 사용된 COI 염기서열에 대한 전체적인 경향을 이해하는데 유용한 정보를 제공한다.

The bumblebee, Bombus ignitus (Hymenoptera: Apidae), is a valuable natural resource that is widely utilized for greenhouse pollination in South Korea. Understanding the magnitude of genetic diversity and geographic relationships is of fundamental importance for long term preservation and utilization. As a first step, we sequenced a partial COI gene of mitochondrial DNA (mtDNA) corresponding to the “DNA barcode” region and the complete internal transcribed spacer 2 (ITS2) of nuclear ribosomal DNA from 88 individuals collected in nine South Korean localities. The complete ITS2 sequences were longest among known insects, ranging in size from 2,034 bp ~ 2,052 bp, harboring two duplicated 112-bp long repeats. The 658-bp long mtDNA sequences provided only six haplotypes with a maximum sequence divergence of 0.61% (4 bp), whereas the ITS sequences provided 84 sequence types with a maximum sequence divergence of 1.02% (21 sites). The combination of the current COI data with those of published data suggest that the B. ignitus in South Korea and China are genetically a large group, but those in Japan can be roughly separated into another group. Overall, a very high per generation migration ratio, a very low level of genetic fixation, and no discernable hierarchical population were found to exist among the South Korean populations of B. ignitus, which suggests panmixia. This finding is consistent with our understanding of the dispersal capability of the species.

The leaf beetle, Chrysolina aurichalcea (Coleoptera: Chysomelidae), is a pest damaging plants of Compositae. In order to understand the genetic diversity and geographic variation we sequenced a portion of mitochondrial COI gene (658 bp) and complete nuclear internal transcribed spacer 2 (ITS2) of the species collected from seven Korean localities. A total of 17 haplotypes (CACOI01 ~ CACOI17), with the maximum sequence divergence of 3.04% (20 bp) were obtained from COI gene sequence, whereas 16 sequence types (ITS2CA01 ~ ITS2CA16), with the maximum sequence divergence of 2.013% (9 bp) were obtained from ITS2, indicating substantially larger sequence divergence in COI gene sequence. Phylogenetically, the COI gene provided two haplotype groups with a high nodal support (≥ 87%), whereas ITS2 provided one sequence type group with a high nodal support (≥ 92%). The result of COI gene may suggest the presence of historical biogeographic barriers that bolster genetic subdivision in the species. Different grouping pattern between COI gene and ITS2 sequences were interpreted in terms of recent dispersal, reflected in the ITS2 sequence. Finally, finding of unique haplotypes and sequence types only from Beakryeng-Islet population was interpreted as an intact remnant of ancient polymorphism. As more samples are analyzed using further hyper-variable marker, further fruitful inference on the geographic contour of the species might be available.

The leaf beetle, Chrysolina aurichalcea (Coleoptera: Chysomelidae), is a pest damaging plants of Compositae. In order to understand the genetic diversity and geographic variation of the species we sequenced a portion of mitochondrial COI gene (658 bp) and complete nuclear internal transcribed spacer 2 (ITS2) collected from seven Korean localities. A total of 18 haplotypes (BARCA01 ~ BARCA18), with the maximum sequence divergence of 3.04% (20 bp) were obtained from COI gene sequence, whereas 17 sequence types (ITS2CA01 ~ ITS2CA17), with the maximum sequence divergence of 2.013% (9 bp) were obtained from ITS2, indicating substantially larger sequence divergence in mitochondrial gene sequence. Phylogenetically, the mitochondrial DNA has shown several haplotypes formed independent groups with substantially high node support (≥ 90%), whereas no such grouping was evidenced for ITS2, indicating different behaviors of the two molecules. Such difference may reflect a diverse dynamics of the species such as biogeographic history, mating behaviors, and also possibly different mode of inheritance of the two molecules, but requires further scrutinized examination of the dataset. In terms of population genetic perspective, overall no population subdivision was detected from both molecules, except for locality 7 (Eocheong islet) from mitochondrial DNA. As more scrutinized analysis is performed, further fruitful inference on the geographic contour of the species might be available.

The seven-spotted lady beetle, Coccinella septempunctata (Coleoptera: Coccinellidae), known also as the seven-spot ladybird, is natural enemy for aphids and has a broad ecological range, living almost anywhere there are aphids for it to eat. In order to understand the genetic diversity and geographic variation of the species we sequenced a portion of mitochondrial COI gene (658 bp) and complete nuclear internal transcribed spacer 2 (ITS2) collected from nine Korean localities. A total of 21 haplotypes (CSCOI01 ~ CSCOI21), with the maximum sequence divergence of 4.56% (30 bp) were obtained from COI gene sequence (from 78 individuals), whereas 65 sequence types (CSITS201 ~ CSITS265), with the maximum sequence divergence of 2.06% (11 positions) were obtained from ITS2 (from 79 individuals), indicating substantially larger sequence divergence in mitochondrial gene sequence. Both COI gene and ITS2 shows the distribution pattern that only a few haplotypes or sequence types are widely distributed, whereas majority of them are highly restricted in one geographic location, even represented as a single individual. Unlikely the ITS2 sequence types the mitochondrial COI haplotypes evidenced the presence of two main phylogenetic groups, reciprocally monophyletic to each other. Geographically, these two groups are spread in all localities surveyed. Considering both COI gene and ITS2 sequence together, current our data may suggest the presence of ancestral polymorphism, rather than on-going speciation, but more scrutinized analysis will be performed soon. Due partially by the presence of both COI groups in all surveyed localities, the genetic diversity estimates of all localities are similar from the perspective of COI gene, but ITS data showed extremely lower genetic diversity of one islet locality, Anmyeon-do (locality 2; 0.002530 vs. 0.008054 ~ 0.012060). Analysis of gene flew estimates between localities indicates that most populations are highly interconnected to each other. However, one islet locality, Anmyeon-do (locality) has shown statistically significant distance from the remaining localities on the basis of only ITS2 data (FST = 0.19 ~ 0.34), requiring scrutinized phylogeographic inference on this population with expanded sampling. As more scrutinized analysis is performed, further fruitful inference on the geographic contour of the species might be available.

The pear psylla, Cacopsylla pyricola Foerster (Homoptera: Psyllidae), is a serious insect pest of commercial pear crops. The species, which resides on pear trees throughout its life cycle, is rapidly spreading in some regions of the world. Given the life cycle, it is unclear how such a rapid spread has been facilitated. Presently, the population genetic structure of the species including genetic diversity and gene flow was studied to understand the nature of dispersal and field ecology of the species. Pear psylla was collected from several pear orchards in Korea. The 658-bp region of mitochondrial COI gene and the 716-bp long complete internal transcribed spacer 2 (ITS2) of the nuclear ribosomal DNA were sequenced. Unlikely other previously studied insect pests, the COI-based genetic diversity of the pear psylla was extremely low (maximum sequence divergence of 0.15%). This finding allowed us to conclude that the species may have been introduced in Korea relatively recently, possibly with the phenomenon of genetic bottlenecks. ITS2 sequence-based analyses of phylogeny, population differentiation, gene flow, and hierarchical population structure all concordantly suggested that the pear psylla populations in Korea are neither genetically isolated nor hampered for gene flow. These genetic data are concordant with the dispersal of an overwintering winterform morph outside the non-pear habitat in the fall and the possibility of subsequently longer distant dispersal.

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.

The bumblebee, Bombus ignitus (Hymenoptera: Apidae), is a valuable natural resource that is one of the most notably utilized for greenhouse pollination in Korea. In order to understand the nature of genetic relationships, gene flow, and population structure of the species we sequenced a partial COI gene of mitochondrial DNA (mtDNA) corresponding to “animal barcode” region and the complete internal transcribed spacer 2 (ITS2) of the nuclear ribosomal DNA (nrDNA) collected from Korean localities. Although the 658-bp long mtDNA sequence provided only six haplotypes with the maximum sequence divergence of 0.61% (4 bp), the ITS sequences provided 84 sequence types with the maximum sequence divergence of 1.02% (21 sites), confirming better applicability of the ITS sequences to the study of intraspecific variation. The complete ITS2 sequences of B. ignitus were shown to be longest among known insects, ranging in size from 2,034 bp ~ 2,052 bp, harboring two duplicated repeats. Overall, a very high per generation migration ratio, a very low level of genetic fixation, and no discernable hierarchical population/ population group were noted to exist among populations of B. ignitus on the basis of both molecules, thus suggesting that the B. ignitus populations on the Korean peninsula are panmictic, which is consistent with our understanding of the dispersal capability of the species

In the family Sciaridae, only 3 species have been recorded in Korea, as the pests on various crops and mushrooms in glasshouse (Bradysia difformis Frey, 1948, Bradysia procera (Winnertz, 1868), Lycoriella ingenua (Dufour, 1839)), even though Sciaridae is one of the species-rich families in the order Diptera. There are plenty of species unknown in Korea which are very hard to identify by morphological characters in the larval, pupal stages and also female adults as well. Therefore, using the sequences of cytochrome oxidase I gene (COI) from adult male, we tested the utilities of DNA barcode to identify the species of sciarid flies.

The bumblebee, Bombus ignitus (Hymenoptera: Apidae), is a valuable natural resource that is one of the most notably utilized for greenhouse pollination in Korea. In order to understand the nature of genetic relationships, gene flow, and population structure of the species we sequenced a partial COI gene of mitochondrial DNA (mtDNA) corresponding to “animal barcode” region and the complete internal transcribed spacer 2 (ITS2) of the nuclear ribosomal DNA (nrDNA) with the individuals collected from Korean localities. Although the 658-bp long mtDNA sequence provided only six haplotypes with the maximum sequence divergence of 0.456% (3 bp, from 91 individuals), but the ITS sequences provided 33 sequence types with the maximum sequence divergence of 0.78% (16 bp, from 35 individuals), confirming better applicability of the ITS sequences to the study of intraspecific variation. The ITS2 sequences of B. ignitus were shown to be the longest among known insects, ranging in size from 2,034 bp ~ 2,045 bp and harbored two duplicated repeats, indicating unusual structure of B. ignitus ITS2 sequences compared with other insect ITS sequences. Overall, a very high per generation migration ratio, a very low level of genetic fixation, and no discernable hierarchical population/population group were noted to exist among populations of B. ignitus on the basis of both molecules, thus suggesting that the B. ignitus populations on the Korean peninsula are panmictic, which is consistent with our understanding of the dispersal capability.