The spotted-wing drosophila Drosophila suzukii (Diptera: Drosophilidae) is an Asian species introduced into North America and Europe. It damages a wide variety of thin-skinned fruits. We sequenced the complete mitochondrial genome (mitogenome) of D. suzukii to better understand the mitogenomic characteristics of this species and understand phylogentic relationships of Drosophila. The 16,230-bp complete mitogenome of the species consists of a typical set of genes, including 13 protein-coding genes (PCGs), two rRNA genes, and 22 tRNA genes, and one major non-coding A+T-rich region, with an arrangement typical of insects. Twelve PCGs began with the typical ATN codon, whereas the COI began with TCG, which has been designated as the start codon for other Drosophila species. The 1,525-bp A+T-rich region is the second longest in Drosophila species for which the whole mitogenome has been sequenced, after D. melanogaster. Phylogenetic analysis with the 13 PCGs of the Drosophila species using Bayesian Inference and Maximum likelihood methods both placed D. suzukii at the basal lineage of the previously defined Melanogaster group, with a strong support.

The Gelechioidea is the second most species-rich group of Lepidoptera, but only limited number of mitochondrial genome (mitogenome) sequences is available. Thus, we sequenced the complete mitogenome of a gelechioid Hieromantis kurokoi (Lepidoptera: Stathmopodidae) to use the data for future study for the higher phylogeny of Ditrysia in Lepidoptera. The arrangement of the genome was identical to typical one found in Ditrysia (trnM-trnI-trnQ) (underline for inverted gene). The COI began with CGA, which has been designated as the start codon for majority of lepidopteran species, whereas other protein-coding genes (PCGs) began with the typical ATN codon. The 360-bp long A+T-rich region harbored the conserved sequence blocks Phylogenetic analysis using the 13 PCGs both by Bayesian inference (BI) and Maximum-likelihood (ML) methods indicated that H. kurokoi belonging to the family Stathmopodidae grouped together with within-familial species Atrijuglans hetaohei with the highest nodal support (BI, 1.0; ML, 100%).

In this study, we tested the effect of a range of insect orders including Trichoptera as outgroups for lepidopteran phylogeny. Phylogenetic analyses performed with four different partitioning schemes using the maximum-likelihood method provided four different topologies (T1-T4) and topological test most supported T1 topology. When the means of first principle component for nucleotide frequency between A/T and G/C of PCGs was considered Trichoptera, Diptera, Coleoptera, and Orthoptera tended to result in T1 topology more frequently in the given ingroup taxa and outgroups tested. This result contradicts to the general view that the sister taxon might be the best outgroup. The T1 topology was largely consistent with a recent large molecular dataset-based lepidopteran phylogeney, presenting the relationships ((((((((((Noctuoidea + Geometroidea) + (Bombycoidea + Lasiocampoidea)) + Drepanoidea) + Mimallonoidea) + Pyraloidea) + Gelechioidea) + Papilionoidea) + Tortricoidea) + (Gracillaroidea + Yponomeutoidea)) + Hepialoidea).

We sequenced the complete mitochondrial (mt) genome of Camponotus atrox (Hymenoptera: Formicidae) that is distributed only in Korea. This genome is 16,540 bp in size, contains typical sets of genes (13 protein-coding genes, 22 tRNAs, and two rRNAs). The C. atrox A+T-rich region is the longest in the sequenced ants as 1,402 bp and is comprised of an identical tandem repeat consisting of six 100-bp copies and one 96-bp copy. A total of 315 bp of intergenic-spacer sequences were spread over 23 regions. An attempt to align spacer sequences in ants turned out that alignment was mostly feasible among congeneric species, with a substantial sequence divergence, indicating the potential of these sequences as congeneric molecular markers. The A/T content in first and second codon positions of PCGs are similar in ants including C. atrox (73.9 vs. 72.3% on average). Estimation of degree of genetic divergence (e.g. non-synonymous substitution rate) with an increased taxon sampling among hymenopteran superfamilies indicated the presence of different rates of divergence between the suborders Symphyta and Apocrita as has previously been reported. The C. atrox mt genome has a unique gene arrangement, trnI-trnM-trnQ at the A+T-rich region and ND2 junction (underline for inverted gene), possibly originated from tandem duplication of trnM-trnI, resulting in trnM-trnI-trnM-trnI-trnQ and loss of first trnM and second trnI, resulting in trnI-trnM-trnQ.

노랑부리백로(Egretta eulophotes)는 천연기념물 제 361 호이며, 멸종위기 Ⅰ급으로 등록 되어 있다. 국제적 희귀종 으로 IUCN의 Redlist에 취약종(Vulnerable: VU)이며, 야생 에서 2,500개체 미만만이 남은 것으로 알려져 있다. 국내에 서 노랑부리백로 집단번식지는 1987년 신도에서 최초 발견 되어 천연기념물 360호로 지정받았고, 1991년 약 400여 둥 지가 관찰되었으나, 이후 인위적 방해요인으로 인접 섬으로 이동하여 신도에는 현재 번식하지 않는다. 최근 노랑부리백 로의 번식이 확인된 곳은 천연기념물로 지정되어 보호받는 영광군 칠산도(제 389호), 연평도, 구지도, 서만도, 황서도, 보령의 목도 등이 있다. 노랑부리백로는 서해안 무인도서에 서 집단으로 번식하고 있고, 전 세계 최대 번식집단을 형성 하고 있다. 한편, 노랑부리백로와 같이 무인도서에 서식하는 종은 외 부 위협 요인에 쉽게 취약하거나 교란될 수 있고, 괭이갈매 기와 같이 무인도서내 집단번식의 확산은 서식지 감소로 이어져 멸종위기로 이어지고 있다. 이러한 점 때문에 종의 개체군 구조, 유전적 다양성, 소수개체군의 유전자 관리에 대한 분자생물학적 연구 방법은 노랑부리백로와 같이 무인 도서에서 집단으로 번식하는 멸종위기종의 보전에 중요한 정보와 기초자료를 제공할 수 있다. 본 논문은 노랑부리백로 서식지의 개체군 조사와 더불어 NGS(Next Generation Sequencing)의 빠른 분석기법을 이 용하여 노랑부리백로의 미토콘드리아 유전체 분석과 계통 분류를 수행하고 근연종과의 비교를 통해 종 다양성 및 개 체군 보전을 위한 기초자료로 이용하고자 한다. 노랑부리백로 번식 및 서식현황을 파악하기 위해 문화재 청의 허가를 받고, 번식 및 서식 피해를 최소화하면서 번식 지내 노랑부리백로 43개체의 혈액, 조직(사체), 알껍질 등에 서 유전자원을 확보하였다. 이후 DNA 추출 후 QC테스트 한 총 18개체의 유전자원 샘플로 2개의 primer set을 제작하 고 PCR 증폭을 진행한 후 100bp 이상 조각으로 해독하여, 약 1.2Gb의 미토콘드리아 유전체 데이터를 생산 및 분석하 였다. 노랑부리백로의 번식현황 조사결과 전남 영광군의 칠산 도, 납대기섬, 서만도, 황서도, 보령의 목도(나무섬) 등에서 번식이 확인되었다. 하지만 기존 번식지이었던 신도, 구지 도, 비도, 예도 등에서는 번식을 확인할 수 없었다. 최근 10년간 노랑부리백로의 둥지 수 변화를 보면 2004년 674개 의 둥지에서 2006년 461개, 2008년 524개의 둥지로 감소하 였다가 2010년 696개로 증가한 후, 본 조사인 2013년에는 367개로 감소하였는데, 이는 구지도의 번식 집단이 사라졌 고, 서만도와 황서도의 개체군이 감소한 결과로 판단된다. NGS로 생산된 데이터를 기 보고된 노랑부리백로의 reference sequence에 BWA 프로그램을 이용하여 align을 수행하였으며, samtools를 이용하여 각 샘플별 미토콘드리 아 서열을 생산하였다. 전체 미토콘드리아 서열을 비교해본 결과, 총 87개의 위치에서 다형성(polymorphism)이 발견되 었으며, 대부분이 D-loop(Control region)에 모여 있는 것을 확인할 수 있었다. 또한 기 보고된 중국의 노랑부리백로 데이터와 비교한 결과, 새로운 두 개의 Haplotype이 한국 노랑부리백로에 있음을 확인하였다. 미토콘드리아의 유전 자 다양성을 측정하기 위해 기 보고된 중국의 노랑부리백로 데이터의 미토콘드리아 서열 중 D-loop 영역의 일부를 DnaSP 프로그램을 이용하여 비교하였다. Haplotype/Nucleotide diversity를 계산한 결과 각각 0.956, 0.00908을 얻었고, 중국의 노랑부리백로(Haplotype diversity: 0.920, Nucleotide diversity: 0.00878)에 비해 높게 나타났다. Tajima's D test를 통해 한국에 번식하는 노랑부리백로의 미토콘드리아 control region에 선택압 (selection pressure)이 없다는 결과도 확인할 수 있었다.한국과 중국에 서식하는 노랑부리백로의 지리적 변이를 확인하기 위하여, Arlequin을 통해 AMOVA (Analysis of Molecular Variance)를 진행하였으며, Haplotype frequency를 이용하였을 때 약 2.27 %의 variation 차이가 있음을 확인할 수 있었으며, Genetic distance를 이용하였을 때는 유의한 값 을 얻을 수 없었다. 또한 번식지별로 개체군간 차이가 있는 지 확인해보았으나, 샘플의 수가 적어 통계적으로 유의한 값이 도출되지 않았다. 노랑부리백로의 번식지별 개체군간 의 비교와 새롭게 발견된 Haplotype의 분석을 위하여 RAxML을 이용하여 계통도 분석을 한 결과, 특별하게 번식 지별로 클러스터링(clustering) 되지 않아 번식지별 차이가 없었다. 한편, KHap1, KHap2는 이번 연구에서 새롭게 발견된 Haplotype으로서 기 보고된 중국의 노랑부리백로 Haplotype 과 클러스터링이 되는 것을 확인하였다. 이는 국내 번식하는 노랑부리백로 집단이 중국에서 번식하는 집단과 유전적 교 류가 있다고 판단되며, 번식시기보다는 월동지인 동남아시 아 등지에서 모여 집단 월동할 가능성이 있다. 향후, 노랑부 리백로 mtDNA와 STR genotyping의 분석을 추가적으로 수 행하여 한국에서 번식하는 노랑부리백로의 유전자 다양도 와 계통분석을 심층적으로 수행할 필요가 있다.

The mulberry white caterpillar, Rondotia menciana, belongs to the lepidopteran family Bombycidae, in which the domestic silkworm, Bombyx mori is included. In this study, we describe the complete mitochondrial genome (mitogenome) sequences of the species in terms of general genomic features and characteristic features found in the A+T-rich region. The 15,364-bp long genome consisted of a typical set of genes (13 protein-coding genes, two rRNA genes and 22 tRNA genes) and one major non-coding A+T-rich region, with the typical arrangement found in Lepidoptera. Twelve of the 13 PCGs start with typical ATN codons, except for the COI, which begins with CGA. Twelve of the 13 PCGs have complete stop codon, except for COII, which ends up with a single T. The 360-bp long A+T-rich region harbored the conserved sequence blocks that are typically found in lepidopteran insects. Additionally, the A+T-rich region of R. menciana contained one tRNAMet-like structure, which has a proper anticodon and secondary structure.

We report the complete mitochondrial genome (mitogenome) of Apodemia mormo, which belongs to the lepidopteran family Riodinidae. The 15,262-bp long complete genome is comprised of 13 protein-coding genes, two rRNA genes, 22 tRNA genes, and one major non-coding A+T-rich region, with the arrangement typically found in majority of Lepidoptera. The genes of A. mormo are interleaved with a total of 168 bp, which are spread over 16 regions and overlap in a total of 58 bp at eight locations. All tRNAs of the A. mormo mitogenome formed typical cloverleaf structure, except for tRNASer(AGN), which formed the truncated dihydrouridine arm. COI gene started with CGA, instead of canonical ATN as seen in other Lepidoptera. The 349-bp long A+T-rich region harbored the conserved sequence blocks, such as ATAGA motif, poly-T stretch, the conserved ATTTA sequence, and microsatellite A/T repeat that are typically found in Lepidoptera, but absent for tRNA-like pseudogene.

Up to now the complete mitochondrial genome (mitogenome) sequences of only three species of clitellate have been available. We have determined the complete mitogenome sequences of the elusive Burmese giant earthworm Tonoscolex birmanicus (Clitellata: Megascolecidae), which is endemic to Myanmar. The 15,170-bp long genome contains the 37 genes typical of metazoan mitogenomes [13 protein-coding genes (PCG), two rRNA genes and 22 tRNA genes] and one major non-coding region. All of the 37 genes are transcribed from the same DNA strand. The arrangement of the T. birmanicus mitogenome is identical to that of two within-ordinal species Lumbricus terrestris and Perionyx excavates. All 13 PCGs start with the ATG. For the stop codon, only six PCGs end with the TAA, whereas the remaining ones ends with the incomplete stop codon, T. Genes overlap in a total of 14 bp in five locations, and harbor a total of 16 bp of intergenic spacer sequences in nine locations.

Glyphodes quadrimaculalis (Lepidoptera: Crambidae) feed on a root tuber of Cynanchum wilfordii (Asclepiadaceae) that is one of the most famous traditional medicines in Korea. The genus Glyphodes includes ~130 species distributed worldwide, so the complete mitochondrial genome (mitogenome) would be helpful for bio-identification, biogeographic studies, and multigene-based phylogeny. The 15,255-bp long G. quadrimaculalis genome comprises 37 typical genes and one large non-coding region, with the typical arrangement found in Lepidoptera. Of the 13 protein coding genes (PCGs), 12 begin with typical start codons found in insect mitochondrial PCGs, but the COI gene starts with atypical CGA. One of the noteworthy features of the genome includes the presence of a 51-bp long non-coding space sequence located between tRNAGln and ND2 that reveals high sequence homology (71.4%) to the neighboring ND2 gene, indicating the origin of the region by partial duplication of the ND2 gene.

We present the nearly complete mitogenome sequences of the garden chafer, Polyphylla laticollis manchurica, which is listed as an endangered species in Korea. The P. l. manchurica mitogenome, which includes unfinished whole A+T-rich region and a partial srRNA was 14,473-bp long, possessing typical sets of genes (13 PCGs, 22 tRNA genes, and 2 rRNA genes). Gene arrangement of the P. l. manchurica mitogenome was identical to the common one found in the majority of insects. The 5 bp-long motif sequence (TAGTA) that has been suggested to be the possible binding site for the transcription termination peptide for the major-strand was also found in the P. l. manchurica mitogenome between tRNASer(UCN) and ND1. As has been previously determined, the high A/T content was unanimously observed in P. l. manchurica in terms of A/T bias in the third codon position (73.5%) compared with the first (66.4%) and second codon position (66.2%), and a high frequency of A/T-containing codons (a total of 28.22% for TTA, ATT, TTT, and ATA). The PCGs encoded in major-strands are slightly T-skewed, whereas those of the minor-strand are A-skewed, indicating strand asymmetry in nucleotide composition in the Coleoptera including P. l. manchurica.

We sequenced 17,329 bp of mitochondrial genome (mitogenome) of the black dwarf honey bee, Apis andreniformis (Hymenoptera: Apidae), that lacks ~200 bp of the A+T-rich region for the completion of the genomic sequence. The gene arrangement of A. andreniformis mitogenome is identical to that of A. cerana. However, the genome contains 5 additional tRNALeu(CUN) located 4 copies between tRNAMet and tRNAGln, and 1 copy between tRNAGln and tRNAAla, along with the typical sets of genes (13 protein-coding genes, 22 tRNAs, and 2 rRNAs) including regular tRNALeu(CUN) and the A+T-rich region (at least 923 bp). Only 1 copy of tRNALeu(CUN) differed by 1 bp from other 4 copies of tRNALeu(CUN). Each additional tRNALeu(CUN) is followed by nearly identical 68-bp long repeat sequence (95.6% identity). All 13 protein coding genes have typical start codons found in insect mitochondrial PCGs (2 ATA, 9 ATT, and 2 ATG).

Lepidoptera is one of the largest insect orders, but the phylogenetic relationships within this order, have yet to be completely described. One of the unresolved relationships includes the monophyly of Papilionoidea in relationship with the monotypic superfamily Hesperioidea. We newly sequenced five hesperid mitochondrial genomes (mitogenomes), representing four subfamilies: Pyrginae (Daimio tethys and Lobocla bifasciatus), Coeliadinae (Choaspes benjaminii), and Hesperiinae (Potanthus flavus), and Heteropterinae (Carterocephalus silvicola). Along with these newly sequenced hesperid genomes phylogenetic analysis was conducted with all available lepidopteran mitogenomes including three reported species of Hesperiidae that consisted of ~70 species in ten lepidopteran superfamilies. The test for the effect of optimization schemes, such as exclusion and inclusion of third codon position of 13 PCGs, other genes (22 tRNAs and two rRNAs), and with and without partitions also was performed. Majority of datasets consistently placed the monophyletic Hesperiidae the sister to ((Pieridae + Lycaenidae) + Nymphalidae), placing another true butterfly family Papilionidae as the basal lineage of this group, presenting the relationships (Papilionidae + (Hesperiidae + ((Pieridae + Lycaenidae) + Nymphalidae))). Consistent to previous result, Pyraloidea was placed as the sister to ((Bombycoidea + Geometroidea) + Noctuoidea), placing the Macrolepidoptera as non-monophyletic group.

The larch hawk moth, Sphinx morio, belongs to the lepidopteran family Sphingidae that has long been studied as a family of model insects in a diverse field. In this study, we describe the complete mitochondrial genome (mitogenome) sequences of the species in terms of general genomic features and characteristic short repetitive sequences found in the A+T-rich region. The 15,299-bp long genome consisted of a typical set of genes (13 protein-coding genes, two rRNA genes and 22 tRNA genes) and one major non-coding A+T-rich region, with the typical arrangement found in Lepidoptera. The 316-bp long A+T-rich region located between srRNA and tRNAMet harbored the conserved sequence blocks that are typically found in lepidopteran insects. Additionally, the A+T-rich region of S. morio contained three characteristic repeat sequences that are rarely found in Lepidoptera: two identical 12-bp repeat, three identical 5-bp long tandem repeat, and six nearly identical 5~6 bp long repeat sequences.

In the present study, the 17,694-bp long complete mitochondrial genome (mitogenome) of the dwarf honey bee, Apis florea (Hymenoptera: Apidae), is described with an emphasis on the noteworthy triplicated tRNAser(AGN) region and an extraordinary long A+T-rich region with repeat regions. The gene arrangement of A. florea mitogenome is identical to that of A. mellifera, but has triplicated tRNASer(AGN), each of which contains the precedent 44 bp-long and following another 64 bp-long repeats plus one complete first repeat abutting to tRNAMet. A total of 1,610-bp long two repeat regions in 1,987 bp-long A+T-rich region is composed of nearly identical 141 ~ 219-bp long five tandem repeats and 50 ~ 52-bp long 12 tandem repeats that are encompassed by three non-repeat sequences. One of the common interpretations for such repeat sequence is slipped-strand mispairing and unequal crossing-over events during DNA replication.

Gene arrangement in the mitochondrial genome (mitogenome) has been regarded as an important evolutionary event that is useful as a phylogenetic signal. The mountainous duskywing, Erynnis montanus, belongs to a lepidopteran family Hesperiidae. We sequenced 15,530-bp long complete mitogenome of the species. The genome has the typical gene content of animals (13 protein-coding genes, two rRNA genes, 22 tRNA genes, and one major non-coding A+T-rich region). Further, E. montanus mitogenome also contained a high A/T content in the whole genome (81.7%) and the CGA (arginine) as the start codon for the COI gene, as typical in lepidopteran mitogenome. However, unlike other lepidopteran species, including two sequenced skippers, the E. montanus mitogenome has a unique arrangement tRNASer-tRNAAsn, instead of the tRNAAsn-tRNASer found unanimously in other lepidopteran species, providing a new gene arrangement in Lepidoptera. Such rearrangement probably was likely caused by duplication of gene block tRNASer-tRNAAsn and subsequent random loss of tRNAAsn in the first copy and tRNASer in the second copy, resulting in the arrangement tRNASer-tRNAAsn. Considering current phylogenetic relationships among available lepidopteran groups in connection with lepidopteran gene arrangement the new gene arrangement found in E. montanus seems to be apomorphy, requiring cautious interpretation as a phylogenetic signal.

The black-veined white, Aporia crataegi (Lepidoptera: Papilionoidea), is nearly extinct in South Korea, although substantial numbers of dried specimens are available. One of the common practices for such species is to launch re-introduction program after proper amount of genetic information are analyzed from donor and donee populations. In this study, we sequenced complete mitochondrial genome (mitogenome) of A. crataegi to design species-specific primers for subsequent population works and to further understand the mitogenome evolution in lepodiopteran Papilionoidea. The 15,140-bp long A. crataegi mitogenome that has typical sets of 37 genes is smallest among true butterfly species with overall slightly smaller size in genes and regions throughout the genome. Arrangement of the genome is identical to those of other lepidopteran mitogenomes, in which tRNA cluster located between the A+T-rich region and ND2 gene is translocated into tRNAMet, tRNAIle, and tRNAGln from ancestral arrangement, tRNAIle, and tRNAGln, tRNAMet. The A/T content of the genome at 81.3% is the highest in Pieridae, but lower than that of lycaenid species (81.7% ~ 82.7%) The high A/T content in the genome is also reflected in codon usage, accounting for 41.69% of A/T-composed codons (TTA, ATT, TTT, and ATA). Unlikely the diversified or modified usage of anticodon for tRNASer(AGN) the species of Pieridae including A. crataegi all unanimously have GCT that has been hypothesized as ancestral for Lepidoptera. A total of 111 bp of non-coding sequences are dispersed in 13 regions, ranging in size from 1–49 bp. Among them relatively longer ones (≥ 16 bp) all have relatively higher sequence identity to other regions of the genome, suggesting partial duplication of the sequences during A. crataegi evolution. As has been reported in some species of Lepidoptera, the A. crataegi A+T-region also has typically found conserved sequences (e.g., poly-T stretch, ATAGA motif, ATTTA element, microsatellite-like A/T sequence, and poly-A stretch) and one tRNA-like sequence, and this feature was commonly found in true butterfly species.

The complete mitogenome (20,456 bp) of Challia fletcheri (Dermaptera: Pygidicranidae) as the first dermapteran insect is the longest among sequenced insects. The genome contained typical gene sets, but harbored the largest TRU among Exopterygota and Palaeoptera. The AT- and GC-skewness showed more Ts and Gs encoded on the major strand, whereas more As and Cs on the minor strand, presenting a reversal to the general pattern found in most insect mitogenomes. This pattern was explained in terms of inversion of replication origin. The gene arrangement of C. fletcheri genome is unique in insects and differs from the ancestral type found in insects by a series of gene translocations and/or inversions. We hypothesize that the markedly different gene arrangement is probably due to some unique organism-level properties, which allow relaxed selection against mitochondrial gene rearrangement. All phylogenetic analyses consistently placed Orthoptera as the sister to the group composed of a monophyletic Isoptera + Mantodea + Blattodea and a monophyletic Grylloblattodea + Mantophasmatodea + Phasmatodea, and placed Dermaptera as the sister to Plecoptera, leaving them as the most basal lineage of Polyneoptera.