Description of complete mitochondrial genome of the black-veined white,Aporia crataegi (Lepidoptera: Papilionoidea), and comparison to papilionoid species

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 used to rescue such endangered species is to launch a re-introduction program after a proper amount of genetic information is analyzed from donor and donee populations. In this study, we sequenced the complete mitochondrial genome (mitogenome) of A. crataegi to accumulate genetic information for subsequent population studies and to further understand the mitogenome evolution in true butterflies, Papilionoidea. The 15,140-bp long A. crataegi mitogenome has typical sets of 37 genes and is the smallest among the true butterfly species, with overall slightly smaller size genes and regions throughout the genome. The A/T content of the genome (81.3%) is the highest in Pieridae, where A. crataegi belongs, but lower than that of the lycaenid species (81.7%–82.7%). Unlike the diversified or modified usage of an anticodon for tRNA^Ser(AGN), the species of Pieridae including A. crataegi all contain GCT that has been hypothesized as being ancestral for Lepidoptera. A total of 111 bp of non-coding sequences are interspersed in 13 regions, ranging in size from 1–49 bp. Among these sequences, 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.     

Complete nucleotide sequence and organization of the mitochondrial genome of eri-silkworm,Samia cynthia ricini (Lepidoptera: Saturniidae)

Samia cynthia ricini is a commercial silk-producing insect that is now reared year-round in Korea, with the expectation of being utilized for diverse purposes. In this report, we present the complete mitochondrial genome (mitogenome) of S. c. ricini. The 15,384-bp long S. cynthia ricini mitogenome was amplified into 26 short fragments using three long overlapping fragments using primers designed from reported lepidopteran mitogenome sequences. The genome comprises 37 genes (13 protein-coding genes, two rRNA genes, and 22 tRNA genes), and one large non-coding region termed the A + T-rich region. The A/T content of the third codon position was 91.7%, which was 18.8% and 21.6% higher than those of first and second codon positions, respectively. The high A/T content in the genome is reflected in codon usage, accounting for 39.5% of A/T-composed codons (TTA, ATT, TTT, and ATA). Unlike a previous report on the start codon for the COI gene, the S. c. ricini COI gene commences with a typical ATT codon. A total of 221 bp of non-coding sequences are dispersed in 17 regions, ranging in size from 1 to 54 bp, which comprise 1.4% of the total genome. One of the non-coding sequence located between tRNA^Gln and ND2 (54 bp) has 77% sequence homology with the 5′-sequence of the neighboring ND2 gene, suggesting partial duplication of the sequence during evolution. The 361-bp long A + T-rich region contains an 18 bp-long poly-T stretch, ATAGA motif, ATTTA element, microsatellite-like A/T sequence, poly-A stretch and one tRNA-like sequence, as typically found in Lepidoptera including Bombycoidea.     

Complete nucleotide sequence and organization of the mitogenome of the red-spotted apollo butterfly, Parnassius bremeri (Lepidoptera: Papilionidae) and comparison with other lepidopteran insects

The 15,389-bp long complete mitogenome of the endangered red-spotted apollo butterfly, Parnassius bremeri (Lepidoptera: Papilionidae) was determined in this study. The start codon for the COI gene in insects has been extensively discussed, and has long remained a matter of some controversy. Herein, we propose that the CGA (arginine) sequence functions as the start codon for the COI gene in lepidopteran insects, on the basis of complete mitogenome sequences of lepidopteran insects, including P. bremeri, as well as additional sequences of the COI start region from a diverse taxonomic range of lepidopteran species (a total of 53 species from 15 families). In our extensive search for a tRNA-like structure in the A+T-rich region, one tRNA^Trp-like sequence and one tRNA^Leu (UUR)-like sequence were detected in the P. bremeri A+T-rich region, and one or more tRNA-like structures were detected in the A+T-rich region of the majority of other sequenced lepidopteran insects, thereby indicating that such features occur frequently in the lepidopteran mitogenomes. Phylogenetic analysis using the concatenated 13 amino acid sequences and nucleotide sequences of PCGs of the four macrolepidopteran superfamilies together with the Tortricoidea and Pyraloidea resulted in the successful recovery of a monophyly of Papilionoidea and a monophyly of Bombycoidea. However, the Geometroidea were unexpectedly identified as a sister group of the Bombycoidea, rather than the Papilionoidea.     

The complete mitochondrial genome of the mantid shrimp Oratosquilla oratoria (Crustacea: Malacostraca: Stomatopoda): Novel non-coding regions features and phylogenetic implications of the Stomatopoda

The complete mitochondrial (mt) genome sequence of Oratosquilla oratoria (Crustacea: Malacostraca: Stomatopoda) was determined; a circular molecule of 15,783 bp in length. The gene content and arrangement are consistent with the pancrustacean ground pattern. The mt control region of O. oratoria is characterized by no GA-block near the 3′ end and different position of [TA(A)]n-blocks compared with other reported Stomatopoda species. The sequence of the second hairpin structure is relative conserved which suggests this region may be a synapomorphic character for the Stomatopoda. In addition, a relative large intergenic spacer (101 bp) with higher A + T content than that in control region was identified between the tRNA^Glu and tRNA^Phe genes. Phylogenetic analyses based on the current dataset of complete mt genomes strongly support the Stomatopoda is closely related to Euphausiacea. They in turn cluster with Penaeoidea and Caridea clades while other decapods form a separate group, which rejects the monophyly of Decapoda. This challenges the suitability of Stomatopoda as an outgroup of Decapoda in phylogenetic analyses. The basal position of Stomatopoda within Eumalacostraca according to the morphological characters is also questioned.     

Complete mtDNA of Meretrix lusoria (Bivalvia: Veneridae) reveals the presence of an atp8 gene, length variation and heteroplasmy in the control region

The complete nucleotide sequence of the mitochondrial genome of the clam Meretrix lusoria (Bivalvia: Veneridae) was determined. It comprises 20,268 base pairs (bp) and contains 13 protein-coding genes, including ATPase subunit 8 (atp8), two ribosomal RNAs, 22 transfer RNAs, and a non-coding control region. The atp8 encodes a protein of 39 amino acids. All genes are encoded on the same strand. A putative control region (CR or D-loop) was identified in the major non-coding region (NCR) between the tRNA^Gly and tRNA^Gln. A 1087 bp tandem repeat fragment was identified that comprises nearly 11 copies of a 101 bp motif and accounts for approximately 41% of the NCR. The 101 bp tandem repeat motif of the NCR can be folded into a stem–loop secondary structure. Samples of eight individuals from Hainan and Fujian provinces were collected and their NCR regions were successfully amplified and sequenced. The data revealed a highly polymorphic VNTR (variable number of tandem repeats) associated with high levels of heteroplasmy in the D-loop region. The size of the CR ranged from 1942 to 3354 bp depending upon the copy number of the repeat sequence.     

The complete mitogenome of Bombyx mori strain Dazao (Lepidoptera: Bombycidae) and comparison with other lepidopteran insects

The complete mitochondrial genome (mitogenome) of Bombyx mori strain Dazao (Lepidoptera: Bombycidae) was determined to be 15,653 bp, including 13 protein-coding genes (PCGs), two rRNA genes, 22 tRNA genes and a A + T-rich region. It has the typical gene organization and order of mitogenomes from lepidopteran insects. The AT skew of this mitogenome was slightly positive and the nucleotide composition was also biased toward A + T nucleotides (81.31%). All PCGs were initiated by ATN codons, except for cytochrome c oxidase subunit 1 (cox1) gene which was initiated by CGA. The cox1 and cox2 genes had incomplete stop codons consisting of just a T. All the tRNA genes displayed a typical clover-leaf structure of mitochondrial tRNA. The A + T-rich region of the mitogenome was 495 bp in length and consisted of several features common to the lepidopteras. Phylogenetic analysis showed that the B. mori Dazao was close to Bombycidae.     

The mitochondrial genome of the butterfly Papilio xuthus (Lepidoptera: Papilionidae) and related phylogenetic analyses

The nearly complete mitochondrial genome of the butterfly Papilio xuthus (Lepidoptera: Papilionidae) was sequenced for its nucleotide sequence of 13,964 bp. The genome has a typical gene order identical to other lepidopteran species. All tRNAs showed same stable canonical clover-leaf structure as those of other insects, except for tRNA^Ser (AGN), in which the dihydrouracil arm (DHU arm) could not form stable stem–loop structure. Anomalous initiation codons have been observed for the cox1 gene, where the ATTACG hexa-nucleotide was believed to be involved in the initiation signaling. Twelve mitochondrial protein-coding gene sequence data were used to infer the phylogenetic relationships among the insect orders. Even though the number of insect orders represented by complete mitochondrial genomes is still limited, several well-established relationships are evident in the phylogenetic analysis of the complete sequences. Monophyly of the Homometabola was not supported in this paper. Phylogenetic analyses of the available species of Bombycoidea, Pyraloidea, Papilionoidea and Tortricidea bolstered the current morphology-based hypothesis that Bombycoidea, Pyraloidea and Papilionoidea are monophyletic (Obtectomera). Bombycoidea (Bombyx mandarina and Antheraea pernyi) and Papilionoidea (P. xuthus and Coreana raphaelis) formed a sister group.     

Characterisation of the complete mitochondrial genome of Helice wuana (Grapsoidea: Varunidae) and comparison with other Brachyuran crabs

The mitochondrial genome (mitogenome) provides important information for phylogenetic analysis and understanding evolutionary origins. Herein, we sequenced, annotated, and characterised the mitogenome of the crab Helice wuana to better understand its molecular evolution and phylogeny. The 16,359 bp mitogenome includes 13 protein-coding genes (PCGs), 22 transfer RNA (tRNA) genes, two ribosomal RNA (rRNA) genes and one control region. The genome composition is highly A + T biased 68.42%, and exhibits a negative AT–skew (? 0.036) and GC–skew (? 0.269) among Brachyura crabs. Gene rearrangements were detected, as was tandem duplication followed by random loss, which explains the translocation of mitochondrial genes. Phylogenetic analysis showed that H. wuana and H. tientsinensis clustered on one branch with high nodal support values. These results confirm that the placement of H. wuana within the Varunidae family of Thoracotrematan crabs. This study will provided a better understanding for gene rearrangements and crab evolution in the further.     

The complete mitochondrial genome of the fall webworm,Hyphantria cunea (Lepidoptera: Arctiidae)

The complete mitochondrial genome (mitogenome) of the fall webworm, Hyphantria cunea (Lepidoptera: Arctiidae) was determined. The genome is a circular molecule 15 481 bp long. It presents a typical gene organization and order for completely sequenced lepidopteran mitogenomes, but differs from the insect ancestral type for the placement of tRNA^Met. The nucleotide composition of the genome is also highly A + T biased, accounting for 80.38%, with a slightly positive AT skewness (0.010), indicating the occurrence of more As than Ts, as found in the Noctuoidea species. All protein-coding genes (PCGs) are initiated by ATN codons, except for COI, which is tentatively designated by the CGA codon as observed in other lepidopterans. Four of 13 PCGs harbor the incomplete termination codon, T or TA. All tRNAs have a typical clover-leaf structure of mitochondrial tRNAs, except for tRNA^Ser(AGN), the DHU arm of which could not form a stable stem-loop structure. The intergenic spacer sequence between tRNA^Ser(AGN) and ND1 also contains the ATACTAA motif, which is conserved across the Lepidoptera order. The H. cunea A+T-rich region of 357 bp is comprised of non-repetitive sequences, but harbors several features common to the Lepidoptera insects, including the motif ATAGA followed by an 18 bp poly-T stretch, a microsatellite-like (AT)₈ element preceded by the ATTTA motif, an 11 bp poly-A present immediately upstream tRNA^Met. The phylogenetic analyses support the view that the H. cunea is closerly related to the Lymantria dispar than Ochrogaster lunifer, and support the hypothesis that Noctuoidea (H. cunea, L. dispar, and O. lunifer) and Geometroidea (Phthonandria atrilineata) are monophyletic. However, in the phylogenetic trees based on mitogenome sequences among the lepidopteran superfamilies, Papillonoidea (Artogeia melete, Acraea issoria, and Coreana raphaelis) joined basally within the monophyly of Lepidoptera, which is different to the traditional classification.     

菜粉蝶线粒体基因组的全序列测定和分析

目前关于蝶类线粒体基因组全序列及其分子进化的研究还不多见。本研究通过长PCR和引物步移法对菜粉蝶Pieris rapae Linnaeus线粒体基因组全序列进行了测定和初步分析。结果表明：菜粉蝶线粒体基因组全长15 157 bp, 包含13个蛋白编码基因、22个tRNA和2个rRNA基因以及1个非编码的控制区域, 它们的长度分别是11 196 bp, 1 474 bp, 2 093 bp和393 bp。37个基因的位置与已报道的其他蝶类基本一致, 共有10对基因间存在总共59 bp的重叠, 重叠碱基数在1~35 bp之间; 基因间隔序列共计13处120 bp, 间隔长度1~46 bp不等, 最大的基因间隔46 bp, 位于tRNA^Ile和tRNA^Gln基因之间。另外, 基于13个蛋白质编码基因的氨基酸序列, 重建了基于蛋白质编码基因序列数据的11种代表性蝶类的NJ和MP系统树。结果表明:凤蝶类(包括凤蝶和绢蝶)为一大支系, 粉蝶类、 灰蝶类与蛱蝶类(包括蛱蝶、 珍蝶)构成另一大支系。结果不支持粉蝶科与凤蝶科（包括凤蝶类和绢蝶类）构成单系群, 却显示粉蝶科、 灰蝶科和蛱蝶科的组合为单系群。     

Complete mitochondrial genome of Odontobutis haifengensis (Perciformes,Odontobutiae): A unique rearrangement of tRNAs and additional non-coding regions identified in the genus Odontobutis

Herein, the complete mitochondrial genome of Odontobutis haifengensis was sequenced for the first time. The O. haifengensis mitogenome was 17,016 bp in length and included 13 protein-coding genes, 22 transfer RNAs (tRNAs), 2 ribosomal RNAs (rRNAs), and a control region (CR). The genome organization, base composition, codon usage, and gene rearrangement was similar to other Odontobutis species. Furthermore, a tRNA gene rearrangement within the SLH cluster was found to be identical to other Odontobutis species. Moreover, the gene order and the positions of additional intergenic non-coding regions suggests that the observed unique gene rearrangement resulted from a tandem duplication and random loss of large-scale gene regions. Additionally, phylogenetic analysis showed that Odontobutis species form a monophyletic clade due to the conserved mitochondrial gene rearrangement. This study provides useful information that aids in a better understanding of mitogenomic diversity and evolutionary patterns of Odontobutidae species.     

The complete mitochondrial genome of the Ailanthus silkmoth, Samia cynthia cynthia (Lepidoptera: Saturniidae)

The complete mitochondrial genome (mitogenome) of the Ailanthus silkmoth, Samia cynthia cynthia (Lepidoptera: Saturniidae) was determined. The circular genome is 15,345 bp long, and presents a typical gene organization and order for sequenced mitogenomes of Bombycidea species. The nucleotide composition of the genome is highly A+T biased, accounting for 79.86%. The AT skew of the genome is slightly negative, indicating the occurrence of more Ts than As, as found in other Saturniidae species. All protein-coding genes (PCGs) are initiated by ATN codons, except for COI and COII, which are tentatively designated by CGA and GTG, respectively, as observed in other insects. Four of 13 PCGs, including COI, COII, ATP6, and ND3, harbor the incomplete termination codons, T or TA. With an exception for tRNA^Ser(AGN), all other tRNAs can form a typical clover-leaf structure of mitochondrial tRNA. The 359 bp A+T-rich region of S. c. cynthia contains non-repetitive sequences, but harbors several features common to the Bombycidea insects, including the motif ATAGA followed by a poly-T stretch of 19 bp, a microsatellite-like (AT)₇ element preceded by the ATTTA motif, and a poly-A element upstream tRNA^Met. The phylogenetic analyses support the morphology-based current hypothesis that Bombycidae and Saturniidae are monophyletic. Our result confirms that Saturniini and Attacini form a reciprocal monophyletic group within Saturniidae.     

Complete mitochondrial genome of Coelomactra antiquata (Mollusca: Bivalvia): The first representative from the family Mactridae with novel gene order and unusual tandem repeats

The complete mitochondrial genome plays an important role in the accurate inference of phylogenetic relationships among metazoans. Mactridae, also known as trough shells or duck clams, is an important family of marine bivalve clams in the order Veneroida. Here we present the complete mitochondrial genome sequence of the Xishishe Coelomactra antiquata (Mollusca: Bivalvia), which is the first representative from the family Mactridae. The mitochondrial genome of C. antiquata is of 17,384 bp in length, and encodes 35 genes, including 12 protein-coding, 21 transfer RNA, and 2 ribosomal RNA genes. Compared with the typical gene content of animal mitochondrial genomes, atp8 and tRNAS₂ are missing. Gene order of the mitochondrial genome of C. antiquata is unique compared with others from Veneroida. In the mitochondrial genome of the C. antiquata, a total of 2189 bp of non-coding nucleotides are scattered among 26 non-coding regions. The largest non-coding region contains one section of tandem repeats (99 bp × 11), which is the second largest tandem repeats found in the mitochondrial genomes from Veneroida. The phylogenetic trees based on mitochondrial genomes support the monophyly of Veneridae and Lucinidae, and the relationship at the family level: ((Veneridae + Mactridae) + (Cardiidae + Solecurtidae)) + Lucinidae. The phylogenetic result is consistent with the morphological classification. Meanwhile, bootstrap values are very high (BP = 94–100), suggesting that the evolutionary relationship based on mitochondrial genomes is very reliable.     

Structure,DNA sequence variation and phylogenetic implications of the mitochondrial control region in horseshoe bats

There have been few studies of the structural and evolutionary characteristics of the mitochondrial control region (CR) in rhinolophids, yet this could have important consequences for the interpretation of phylogenetic relationships within this group. Here we sequenced and analyzed the CR of 37 individuals from 12 Rhinolophus species, including 2 species from GenBank. The length of the CR ranged from 1335 to 1514 bp, and the base composition was very similar among species. The CR of horseshoe bats, like that of other mammals, could be subdivided into a central conserved domain (CD) and two flanking variable domains, extended termination associated sequences (ETAS), and conserved sequence blocks (CSB). Besides the common conserved blocks (ETAS1, ETAS2, F-B boxes, CSB1, CSB2 and CSB3) found in 3 domains, an ETAS2-like and a CSB1-like element were also detected in the ETAS and CSB domains, respectively, in all individuals. Notwithstanding a short tandem repeat (11 or 13 bp) between CSB1 and CSB2 in all specimens, the base composition, copy number and arrays are all variable. A long tandem repeat (79 bp) was only identified in the ETAS domain in one individual of R. pusillus. Phylogenetic reconstructions based on the CR sequences indicated that the molecular phylogenetic relationships among some Rhinolophus species were inconsistent with the results of phenetic analyses, but similar to phylogenetic constructions using cytochrome b. An unidentified species R. sp and 3 species from the philippinensis-group that were clearly morphologically different comprised a monophyletic group, which could have resulted from morphological independent evolution.     

Barcoding old Korean lepidopteran specimens using newly designed specific primer pairs

Currently, DNA barcodes are often required to be analyzed using old museum specimens when they are the only available specimens for rare or endangered species, or even type series. In this study, using eight universal primers and newly designed 315 species-specific primers, we tried to recover full-length barcode sequences from 45 dried specimens of 36 butterfly species collected between 1959 and 1980 in Korea. The eight universal primers failed entirely in the PCR amplification and sequencing of all the specimens. On the other hand, 284 primer pairs consisting of the 315 primers, targeting fragments of 71–417 bp, amplified various lengths of barcode sequences from all specimens. The fragments were successfully combined to generate the barcode sequences ranging from 444 bp to 658 bp. Notably, of the 284 primer pairs, 26 primer pairs designed for Limenitis camilla, Argynnis niobe, and Brenthis daphne successfully amplified the barcode sequences of congeneric species, Limenitis doerriesi, Argynnis nerippe, and Brenthis ino, suggesting that the species-specific primers can be available for analyzing barcode sequences of closely related species. Our study reveals that the newly designed species-specific primers will be effective in acquiring COI sequences from old butterfly specimens.     

Complete mitochondrial genome of Periplaneta brunnea (Blattodea: Blattidae) and phylogenetic analyses within Blattodea

The complete mitochondrial genome (mitogenome) of Periplaneta brunnea was sequenced in this study and used to reconstruct the phylogenetic relationship of Blattodea. The circular mitogenome was 15,604?bp long and exhibited typical gene organization and order, consistent with other sequenced Periplaneta mitogenomes. The initiation codon of the P. brunnea COX1 gene was unusual in that no typical ATN or TTG start codon was found. The two longest intergenic spacer sequences found in the P. brunnea mitogenome were 21 and 17?bp long. Twenty-one base spacer had a 4?bp motif (TATT) between tRNA-Glu and tRNA-Met that conservatively displayed in 9 sequenced blattarian mitogenomes. The second spacer was between tRNA-Ser (UCN) and NAD1 containing a 7?bp motif (WACTTAA) that was highly conserved in 14 blattarian mitogenomes. The control region showed a relatively fixed motif present in 6 Blattidae mitogenomes, with a big stem-loop structure. Phylogenetic analyses were conducted using site-homogeneous models based on 13 protein-coding genes (PCGs) and two RNA genes. The trees derived from Bayesian inference and maximum likelihood analyses and recovered a relatively stable relationship among major lineages except for the position of Polyphagidae and inter-family relationships of Blaberidae. Analyses supported the monophyly of Blattidae, Blaberidae, Blattellidae, Polyphagidae, Dictyoptera, and the paraphyly of Blattaria. We also found Mantodea was the sister clade to (Blattaria?+?Isoptera), being the basal position of Dictyoptera in all topologies. Meanwhile, our results also consistently supported that Isoptera should be clustered with Blattaria of Blattodea.     

Complete mitochondrial genome sequence of the yellow-spotted long-horned beetle <Emphasis Type="Italic">Psacothea hilaris</Emphasis> (Coleoptera: Cerambycidae) and phylogenetic analysis among coleopteran insects

We have determined the complete mitochondrial genome of the yellow-spotted long horned beetle, Psacothea hilaris (Coleoptera: Cerambycidae), an endangered insect species in Korea. The 15,856-bp long P. hilaris mitogenome harbors gene content typical of the animal mitogenome and a gene arrangement identical to the most common type found in insect mitogenomes. As with all other sequenced coleopteran species, the 5-bp long TAGTA motif was also detected in the intergenic space sequence located between tRNA^Ser(UCN) and ND1 of P. hilaris. The 1,190-bp long non-coding A+T-rich region harbors an unusual series of seven identical repeat sequences of 57-bp in length and several stretches of sequences with the potential to form stem-and-loop structures. Furthermore, it contains one tRNA^Arg-like sequence and one tRNA^Lys-like sequence. Phylogenetic analysis among available coleopteran mitogenomes using the concatenated amino acid sequences of PCGs appear to support the sister group relationship of the suborder Polyphaga to all remaining suborders, including Adephaga, Myxophaga, and Archostemata. Among the two available infraorders in Polyphaga, a monophyletic Cucujiformia was confirmed, with the placement of Cleroidea as the basal lineage for Cucujiformia. On the other hand, the infraorder Elateriformia was not identified as monophyletic, thereby indicating that Scirtoidea and Buprestoidea are the basal lineages for Cucujiformia and the remaining Elateriformia.     

Secondary structural modeling of the second internal transcribed spacer (ITS2) from Pfiesteria-like dinoflagellates (Dinophyceae)

Pfiesteria piscicida is a harmful bloom-forming alga that has received a great deal of attention due to its potential association with large fish kills and neurological problems in humans. Since the discovery of Pfiesteria, several other Pfiesteria-like dinoflagellates (PLDs) have also been identified. Genetic identification and phylogenetic relationships among the PLDs commonly utilize sequence data from the genes and spacers of the ribosomal DNA (rDNA) operon. Of these, the internal transcribed spacers (ITSs) have been previously shown to fold into secondary structures that are critical for proper ribosomal processing. In this study, we modeled the secondary structure of the second internal transcribed spacer (ITS2) from 16 PLDs (as well as an outgroup taxon) using phylogenetic comparative methods and minimum free energy. The secondary structural models predicted for these dinoflagellates consisted of four paired helices separated by five unpaired regions, consistent with those reported from many eukaryotes. All of the structures were highly stable (ΔG = ?66.1 to ?122.3 kcal·mol at 37 °C) and several structural characters were found to be conserved either across the PLDs or were specific to monophyletic subgroups, strengthening previously inferred phylogenetic relationships among taxa. Additionally, an 18 bp motif was identified in the PLDs whose position corresponds to a ribosomal processing site described from other eukaryotes. Potential applications of these ITS2 secondary structures include utility in strain and species identification, phylogenetic inference and serving as a tool for identifying and excluding rDNA pseudogenes when assessing biodiversity within the PLDs.     

Identification of butterfly based on their shapes when viewed from different angles using an artificial neural network

Identification of butterfly species is essential because they are directly associated with crop plants used for human and animal consumption. However, the widely used reliable methods for butterfly identification are not efficient due to complicated butterfly shapes. We previously developed a novel shape recognition method that uses branch length similarity (BLS) entropy, which is a simple branching network consisting of a single node and branches. The method has been successfully applied to recognize battle tanks and characterize human faces with different emotions. In the present study, we used the BLS entropy profile (an assemble of BLS entropies) as an input feature in a feed-forward back-propagation artificial neural network to identify butterfly species according to their shapes when viewed from different angles (for vertically adjustable angle, θ = ± 10°, ± 20°, …, ± 60° and for horizontally adjustable angle, φ = ± 10°, ± 20°, …, ± 60°). In the field, butterfly images are generally captured obliquely by camera due to butterfly alignment and viewer positioning, which generates various shapes for a given specimen. To generate different shapes of a butterfly when viewed from different angles, we projected the shapes captured from top-view to a plane rotated through angles θ and φ. Projected shapes with differing θ and φ values were used as training data for the neural network and other shapes were used as test data. Experimental results showed that our method successfully identified various butterfly shapes. In addition, we briefly discuss extension of the method to identify more complicated images of different butterfly species.     

ITS2 sequence variations among members of Diachasmimorpha longicaudata complex (Hymenoptera: Braconidae) in Thailand

The parasitoid Diachasmimorpha longicaudata complex in Thailand contains at least 3 cryptic species informally designated as species D. longicaudata A, B and BB. DNA sequence data of nuclear ITS2 (second internal transcribed spacer) were used to characterize members of this D. longicaudata complex. The polymerase chain reaction (PCR) amplicon of ITS2 region of D. longicaudata B (≈ 650 bp) clearly differentiated this species from A and BB (amplicon of ≈ 590 bp). Sequence alignment of individual parasitoids revealed that low intraspecies differences ranged from 0.457 to 3.991%, but interspecies differences ranged from 7.566 to 12.989%. Phylogenetic trees constructed using Neighbor-Joining (NJ) and Maximum Parsimony (MP) methods, taking the parasitoid Psyttalia concolor complex as an outgroup, revealed that D. longicaudata A, B and BB formed a monophyletic group, with species A and BB being more closely related than species B. ITS2 characterization of D. longicaudata complex has revealed an interesting divergence of the three cryptic sibling species in Thailand.