Reconstructing intraordinal relationships in Lepidoptera using mitochondrial genome data with the description of two newly sequenced lycaenids, Spindasis takanonis and Protantigius superans (Lepidoptera: Lycaenidae)

Lepidoptera is one of the largest insect orders, but the phylogenetic relationships within this order, have yet to be adequately described. Among these unresolved relationships include those regarding the monophyly of the Macrolepidoptera and interfamilial relationships of the true butterflies superfamily Papilionoidea. We present two new mitochondrial genomes (mitogenomes) belonging to the butterfly family Lycaenidae to explore the phylogenetic relationships existing among lepidopteran superfamilies and true butterfly families from a mitogenome perspective, and to evaluate the characteristics of the lepidopteran mitogenomes. Our consensus phylogeny of the Lepidoptera largely supported the superfamilial relationships (((((Bombycoidea + Geometroidea) + Noctuoidea) + Pyraloidea) + Papilionoidea) + Tortricoidea), signifying a lack of support for a traditionally defined Macrolepidoptera. The familial relationships of the true butterflies concordantly recovered the previously proposed phylogenetic hypothesis (((Lycaenidae + Nymphalidae) + Pieridae) + Papilionidae). The test for the effect of optimization schemes (exclusion and inclusion of third codon position of PCGs and two rRNA genes, with and without partitions) on the resolution and relationships within the Lepidoptera have demonstrated that the majority of analyses did not substantially alter the relevant topology and node support, possibly as the result of relatively strong signal in mitogenomes for intraordinal relationships in Lepidoptera.     

武铠蛱蝶线粒体基因组全序列测定和分析

【目的】了解闪蛱蝶亚科属间及种间的分子系统进化关系。【方法】采用PCR步移法对武铠蛱蝶 Chitoria ulupi 线粒体基因组全序列进行了测定和分析。基于线粒体基因组13个蛋白质编码基因的核苷酸序列构建了38种鳞翅目昆虫的系统发育树。【结果】分析结果表明,武铠蛱蝶线粒体基因组全长15 279 bp,包括13个蛋白质编码基因、22个tRNA基因、2个rRNA基因和一段长度为391 bp的A+T富含区,基因排列顺序与其他已知近缘种昆虫相同。武铠蛱蝶线粒体基因组中存在很高的A+T含量（79.9％）。13个蛋白质编码基因中,COII以TTG作为起始密码子,COI以CGA作为起始密码子外,其余均为昆虫典型的起始密码子ATN。COII和ND4基因使用了不完全终止密码子T,其余基因均以典型的TAA为终止密码子。在所测得的22个tRNA基因中,除tRNA^Ser(AGN)缺少DHU臂外,其余tRNA均能形成典型的三叶草结构。与其他多数鳞翅目昆虫一样,武铠蛱蝶的A+T富含区中有一段由ATAGAA引导的保守的多聚T结构,长度为21 bp,并散布着一些长短不一的串联重复单元。系统发育树结果显示,总科级别的系统发育关系为：卷蛾总科＋（凤蝶总科＋（螟蛾总科＋(夜蛾总科＋蚕蛾总科＋尺蛾总科)））;在蛱蝶科物种中,武铠蛱蝶与猫蛱蝶Timelaea maculate 亲缘关系最近。【结论】基于分子标记构建的鳞翅目昆虫系统发育关系与传统的形态学分类结果基本一致。     

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 Paracymoriza prodigalis (Leech, 1889) (Lepidoptera), with a preliminary phylogenetic analysis of Pyraloidea

The complete mitochondrial genome sequence is determined for Paracymoriza prodigalis (Leech, 1889). The 15,326 bp circular molecule possesses a gene organization and order identical to other sequenced Pyraloidea mitochondrial genomes. All tRNAs have the typical clover-leaf structure except for tRNA^Ser(AGN), which lacks the dihydrouridine (DHU) arm. The A+T-rich region of 343 bp includes the features common to the Lepidoptera, including the ‘ATAGA’ followed by an 19-bp poly-T stretch, but the tandem repeat sequences often appearing in available insects are not found. Phylogenetic relationships of eight subfamilies of 14 Pyraloidea species were constructed based on 13 PCGs of mitochondrial genomes using Bayesian inference (BI) and maximum likelihood (ML) methods. These phylogenies of the subfamilies within Pyraloidea accord well with morphological phylogenetic analysis except for the position of Schoenobiinae.     

Description of new mitochondrial genomes (Spodoptera litura, Noctuoidea and Cnaphalocrocis medinalis, Pyraloidea) and phylogenetic reconstruction of Lepidoptera with the comment on optimization schemes

We newly sequenced mitochondrial genomes of Spodoptera litura and Cnaphalocrocis medinalis belonging to Lepidoptera to obtain further insight into mitochondrial genome evolution in this group and investigated the influence of optimal strategies on phylogenetic reconstruction of Lepidoptera. Estimation of p-distances of each mitochondrial gene for available taxonomic levels has shown the highest value in ND6, whereas the lowest values in COI and COII at the nucleotide level, suggesting different utility of each gene for different hierarchical group when individual genes are utilized for phylogenetic analysis. Phylogenetic analyses mainly yielded the relationships (((((Bombycoidea + Geometroidea) + Noctuoidea) + Pyraloidea) + Papilionoidea) + Tortricoidea), evidencing the polyphyly of Macrolepidoptera. The Noctuoidea concordantly recovered the familial relationships (((Arctiidae + Lymantriidae) + Noctuidae) + Notodontidae). The tests of optimality strategies, such as exclusion of third codon positions, inclusion of rRNA and tRNA genes, data partitioning, RY recoding approach, and recoding nucleotides into amino acids suggested that the majority of the strategies did not substantially alter phylogenetic topologies or nodal supports, except for the sister relationship between Lycaenidae and Pieridae only in the amino acid dataset, which was in contrast to the sister relationship between Lycaenidae and Nymphalidae in Papilionoidea in the remaining datasets.     

The complete mitogenome sequence of the Japanese oak silkmoth, <Emphasis Type="Italic">Antheraea yamamai</Emphasis> (Lepidoptera: Saturniidae)

The 15,338-bp long complete mitochondrial genome (mitogenome) of the Japanese oak silkmoth, Antheraea yamamai (Lepidoptera: Saturniidae) was determined. This genome has a gene arrangement identical to those of all other sequenced lepidopteran insects, but differs from the most common type, as the result of the movement of tRNA^Met to a position 5′-upstream of tRNA^Ile. No typical start codon of the A. yamamai COI gene is available. Instead, a tetranucleotide, TTAG, which is found at the beginning context of all sequenced lepidopteran insects was tentatively designated as the start codon for A. yamamai COI gene. Three of the 13 protein-coding genes (PCGs) harbor the incomplete termination codon, T or TA. All tRNAs formed stable stem-and-loop structures, with the exception of tRNA^Ser(AGN), the DHU arm of which formed a simple loop as has been observed in many other metazoan mt tRNA^Ser(AGN). The 334-bp long A + T-rich region is noteworthy in that it harbors tRNA-like structures, as has also been seen in the A + T-rich regions of other insect mitogenomes. Phylogenetic analyses of the available species of Bombycoidea, Pyraloidea, and Tortricidea bolstered the current morphology-based hypothesis that Bombycoidea and Pyraloidea are monophyletic (Obtectomera). As has been previously suggested, Bombycidae (Bombyx mori and B. mandarina) and Saturniidae (A. yamamai and Caligula boisduvalii) formed a reciprocal monophyletic group.     

The first mitochondrial genome for Brahmin moths (Brahmaeidae) and implications for the higher phylogeny of Bombycoidea

Bombycoidea comprises 10 families and 4723 species, and the phylogenetic relationships among families are still in debate. In this study, we have determined the complete mitochondrial genome (mitogenome) of Brahmaea porphyria. The 15,429-bp mitogenome contains a common set of 37 mitochondrial genes including 13 protein-coding genes, 22 transfer RNA genes (tRNAs), two ribosomal RNA genes (rRNAs) and an inferred control region, and shares the conserved gene rearrangement (trnM-trnI-trnQ) in most ditrysian mitogenomes. Moreover, we analysed the secondary structure for all the tRNA genes of B. porphyria and the preference of codon usage in the PCGs of B. porphyria. The putative 373-bp control region (CR) possesses three types of conserved elements, including ATAGA, Ploy-T stretch, and microsatellite-like elements. A phylogenetic analysis among available Bombycoidea mitogenomes using the concatenated 37 mitochondrial genes appears to support the hypothesis of (Sphingidae+Bombycidae)+Saturniidae and the relatively basal phylogenetic position of Brahmaeidae within Bombycoidea.     

The Complete Mitochondrial Genome of the Beet Webworm,Spoladea recurvalis (Lepidoptera: Crambidae) and Its Phylogenetic Implications

The complete mitochondrial genome (mitogenome) of the beet webworm, Spoladea recurvalis has been sequenced. The circular genome is 15,273 bp in size, encoding 13 protein-coding genes (PCGs), two rRNA genes, and 22 tRNA genes and containing a control region with gene order and orientation identical to that of other ditrysian lepidopteran mitogenomes. The nucleotide composition of the mitogenome shows a high A+T content of 80.9%, and the AT skewness is slightly negative (-0.023). All PCGs start with the typical ATN codons, except for COX1, which may start with the CGA codon. Nine of 13 PCGs have the common stop codon TAA; however, COX1, COX2 and ND5 utilize the T nucleotide and ND4 utilizes TA nucleotides as incomplete termination codons. All tRNAs genes are folded into the typical cloverleaf structure of mitochondrial tRNAs, except for the tRNA^Ser(AGY) gene, in which the DHU arm fails to form a stable stem-loop structure. A total of 157 bp intergenic spacers are scattered in 17 regions. The overlapping sequences are 42 bp in total and found in eight different locations. The 329 bp AT-rich region is comprised of non-repetitive sequences, including the motif ATAG, which is followed by a 14 bp poly-T stretch, a (AT₁₁ microsatellite-like repeat, which is adjacent to the motif ATTTA, and a 9 bp poly-A, which is immediately upstream from the tRNA^Met gene. Phylogenetic analysis, based on 13 PCGs and 13 PCGs+2 rRNAs using Bayesian inference and Maximum likelihood methods, show that the classification position of Pyraloidea is inconsistent with the traditional classification. Hesperioidea is placed within the Papilionoidea rather than as a sister group to it. The Pyraloidea is placed within the Macrolepidoptera with other superfamilies instead of the Papilionoidea.     

The complete mitochondrial genome of Biston panterinaria (Lepidoptera: Geometridae), with phylogenetic utility of mitochondrial genome in the Lepidoptera

The complete mitochondrial genome (mitogenome) of the Chinese pistacia looper Biston panterinaria was sequenced and annotated (15,517 bp). It contains the typical 37 genes of animal mitogenomes and a high A + T content (79.5%). All protein coding genes (PCGs) use standard ATN initiation codons except for cytochrome c oxidase 1 (COX1) with CGA. Eleven PCGs use a common stop codon of TAA or TAG, whereas COX2 and NADH dehydrogenase 4 (ND4) use a single T. All transfer RNA (tRNA) genes have the typical clover-leaf structure with the exception of tRNA^Ser(AGN). We reconstructed a preliminary mitochondrial phylogeny of six ditrysian superfamilies and performed comparative analyses of inference methods (Bayesian Inference (BI), Maximum Likelihood (ML), and Maximum Parsimony (MP)), dataset compositions (including and excluding 3rd codon positions), and alignment methods (Muscle, Clustal W, and MAFFT). Our analyses indicated that inference methods and dataset compositions more significantly affected the phylogenetic results than alignment methods. BI analysis consistently revealed uncontroversial relationships with all dataset compositions. By contrast, ML analysis failed to reconstruct stable phylogeny at two nodes, whereas MP analysis had more difficulties in the tree resolution and nodal support. Distinct from most previous studies, our analyses revealed that Geometroidea had a closer lineage relationship with Bombycoidea than Noctuoidea. Similar to previous molecular studies, our analyses revealed that Hesperiidae were nested in the Papilionoidea clade, providing further evidence to the previous concept that Papilionoidea was paraphyletic, and none of the butterflies were associated with the Macroheterocera.     

The complete nucleotide sequence and gene organization of the mitochondrial genome of the oriental mole cricket, Gryllotalpa orientalis (Orthoptera: Gryllotalpidae)

The complete nucleotide sequences of the mitochondrial genome (mitogenome) of the oriental mole cricket, Gryllotalpa orientalis (Orthoptera: Gryllotalpidae), were determined. The 15,521-bp-long G. orientalis mitogenome contains typical gene complement, base composition, and codon usage found in metazoan mitogenomes. The G. orientalis mitogenome contains the third lowest A+T content (70.5%) among the complete insects mt genome sequences. The initiation codon for the G. orientalis COI gene appears to be ATG, instead of the tetranucleotides, which have been postulated to act as initiation codon for Locusta migratoria and some lepidopteran COI genes. The initiation codon for ND2 appears to be GTG, which is rare, but has been designated as an initiator of Tricholepidion gertschi ND2. All anticodons of G. orientalis tRNAs were identical to Drosophila yakuba and L. migratoria. The tRNA(Ser)(AGN) could not form a stable stem loop structure in the DHU arm as shown in many other insect tRNA(Ser)(AGN). Phylogenetic analysis of nucleotide sequence information from all mt genes supported a monophyletic Diptera, a monophyletic Lepidoptera, a monophyletic Coleoptera, a monophyletic Mecopterida (Diptera+Lepidoptera), and a monophyletic Endopterygota (Diptera+Lepidoptera+Coleoptera), suggesting that the complete insect mitogenome sequence has a resolving power to the diversification events within Endopterygota. However, the relationships of ancient insect orders were unstable, indicating the limited use of mitogenome information at deeper phylogenetic depth.     

Phylogenetic and genomic analysis of the complete mitochondrial DNA sequence of the spotted asparagus beetle Crioceris duodecimpunctata

We report the complete mitochondrial DNA sequence of the spotted asparagus beetle, Crioceris duodecimpunctata. The genome complement, gene order, and nucleotide composition of this beetle's mitochondrial genome were found to be typical of those reported for other insects. Unusual features of this genome include the substitution of UCU for GCU as the anticodon for tRNA(Ser), an unusual TpsiC loop for the tRNA(Ile) gene, and the identification of a putative ATT start codon for cox1. The utility of complete mitochondrial genome data for phylogenetic inference of the insect orders was tested, and compared to that of cox1 and combined mitochondrial ribosomal DNA sequences. 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 orders Diptera, Lepidoptera, and Coleoptera were consistently recovered. Monophyly of the Holometabola was also observed in some (though not all) analyses. The accumulation of complete mitochondrial sequences from a broader array of insect orders holds the promise of clarifying the early diversification of insects.     

Characterization of the complete mitochondrial genome of Tryporyza incertulas, in comparison with seven other Pyraloidea moths

The complete 15,223-bp mitochondrial genome (mitogenome) of Tryporyza incertulas (Walker) (Lepidoptera: Pyraloidea: Crambidae) was determined, characterized and compared with seven other species of superfamily Pyraloidea. The order of 37 genes was typical of insect mitochondrial DNA sequences described to date. Compared with other moths of Pyraloidea, the A + T biased (77.0%) of T. incertulas was the lowest. Eleven protein-coding genes (PCGs) utilized the standard ATN, but cox1 used CGA and nad4 used AAT as the initiation codons. Ten protein-coding genes had the common stop codon TAA, except nad3 having TAG as the stop codon, and cox2, nad4 using T, TA as the incomplete stop codons, respectively. All of the tRNA genes had typical cloverleaf secondary structures except trnS1(AGN), in which the dihydrouridine (DHU) arm did not form a stable stem-loop structure. There was a spacer between trnQ and nad2, which was common in Lepidoptera moths. A 6-bp motif ‘ATACTA’ between trnS2(UCN) and nad1, a 7-bp motif “AGC(T)CTTA” between trnW and trnC and a 6-bp motif “ATGATA” of overlapping region between atp8 and atp6 were found in Pyraloidea moths. The A + T-rich region contained an ‘ATAGT(A)’-like motif followed by a poly-T stretch. In addition, two potential stem-loop structures, a duplicated 19-bp repeat element, and two microsatellites ‘(TA)₁₂’ and ‘(TA)₉’ were observed in the A + T-rich region of T. incertulas mitogenome. Finally, the phylogenetic relationships of Pyraloidea species were constructed based on amino acid sequences of 13 PCGs of mitogenomes using Bayesian inference (BI) and maximum likelihood (ML) methods. These molecular-based phylogenies supported the morphological classification on relationships within Pyraloidea species.     

The complete mitochondrial genome of the butterfly <Emphasis Type="Italic">Apatura metis</Emphasis> (Lepidoptera: Nymphalidae)

As an important pest in the Slender Leaved Willow (Salix alba), Apatura metis is called Freyer’s purple emperor, and its mitochondrial genome is 15,236 bp long. The encoded genes for 22 tRNA genes, two ribosomal RNA (rrnL and rrnS) genes, and 13 protein-coding genes (PCGs), and a control region in the A. metis mitochondria are highly homologous to other lepidopteran species. The mitochondrial genome of A. metis is biased toward a high A + T content (A + T = 80.5%). All protein-coding genes, except for COI begins with the CGA codon as observed in other lepidopterans, start with a typical ATN initiation codon. All tRNAs show the classic clover-leaf structure, except that the dihydrouridine (DHU) arm of tRNA ^Ser(AGN) forms a simple loop. The A. metis A + T-rich region contains some conserved structures including a structure combining the motif ‘ATAGA’ and 19 bp poly (T) stretch, which is similar to those found in other lepidopteran mitogenomes. The phylogenetic analyses of lepidopterans based on mitogenomes sequences demonstrate that each of the six superfamilies is monophyletic, and the relationship among them is (((Noctuoidea + (Geometroidea + Bombycoidea)) + Pyraloidea) + Papilionoidea) + Tortricoidea. In Papilionoidea group, our conclusion argues that ((Lycaenidae + Pieridae) + Nymphalidae) + Papilionidae.     

The complete mitochondrial genomes of two ghost moths, Thitarodes renzhiensis and Thitarodes yunnanensis: the ancestral gene arrangement in Lepidoptera

ABSTRACT: BACKGROUND: Lepidoptera encompasses more than 160,000 described species that have been classified into 45-48 superfamilies. The previously determined Lepidoptera mitochondrial genomes (mitogenomes) are limited to six superfamilies of the most derived lepidopteran lineage Ditrysia. Compared with the ancestral insect gene order, these mitogenomes all contain a tRNA rearrangement. To gain new insights into Lepidoptera mitogenome evolution, we sequenced the mitogenomes of two ghost moths that belong to primitive lepidopteran lineages and conducted a comparative mitogenomic analysis across Lepidoptera. RESULTS: The mitogenomes of Thitarodes renzhiensis and T. yunnanensis are 16,173 bp and 15,814 bp long with an A+T content of 81.28% and 82.33%, respectively. Different tandem repeats in the A+T-rich region mainly account for the size difference between the two mitogenomes. Both mitogenomes include 13 protein-coding genes, 22 transfer RNA genes, and 2 ribosomal RNA genes. The 1,584-bp sequence from rrnS to nad2 was also determined for Thitarodes sp.QL, which has no repetitive sequence in the A+T-rich region. All three Thitarodes species possess the ancestral gene order with trnI-trnQ-trnM located between the A+T-rich region and nad2, which is different from the gene order trnM-trnI-trnQ in all previously sequenced Lepidoptera species. The formerly identified conserved elements of Lepidoptera mitogenomes (i.e. the motif 'ATAGA' and poly-T stretch in the A+T-rich region and the long intergenic spacer upstream of nad2) are absent in the Thitarodes mitogenomes. The phylogenetic analysis supports that Hepialoidea, represented by T. renzhiensis and T. yunnanensis, occupies a basal position in the currently sampled seven superfamilies. The relationships of the other six superfamilies are (((((Bombycoidea + Geometroidea) + Noctuoidea) + Pyraloidea) + Papilionoidea) + Tortricoidea). CONCLUSION: The mitogenomes of T. renzhiensis and T. yunnanensis exhibit unusual features compared with the previously determined Lepidoptera mitogenomes. Their ancestral gene order indicates that the tRNA rearrangement event occurred after Lepidoptera diverged from other holometabolous insect orders. Phylogenetic analysis based on mitogenome sequences is a power tool for addressing phylogenetic relationships among major Lepidoptera superfamilies. Characterization of the two ghost moth mitogenomes has enriched our knowledge of Lepidoptera mitogenomes and contributed to our understanding of the mechanisms underlying mitogenome evolution, especially gene rearrangements.     

Complete mitochondrial genomes of five skippers (Lepidoptera: Hesperiidae) and phylogenetic reconstruction of Lepidoptera

We sequenced mitogenomes of five skippers (family Hesperiidae, Lepidoptera) to obtain further insight into the characteristics of butterfly mitogenomes and performed phylogenetic reconstruction using all available gene sequences (PCGs, rRNAs, and tRNAs) from 85 species (20 families in eight superfamilies). The general genomic features found in the butterflies also were found in the five skippers: a high A + T composition (79.3%–80.9%), dominant usage of TAA stop codon, similar skewness pattern in both strands, consistently length intergenic spacer sequence between tRNA^Gln and ND2 (64–87 bp), conserved ATACTAA motif between tRNA^{Ser (UCN)} and ND1, and characteristic features of the A + T-rich region (the ATAGA motif, varying length of poly-T stretch, and poly-A stretch). The start codon for COI was CGA in four skippers as typical, but Lobocla bifasciatus evidently possessed canonical ATG as start codon. All species had the ancestral arrangement tRNA^Asn/tRNA^{Ser (AGN)}, instead of the rearrangement tRNA^{Ser (AGN)}/tRNA^Asn, found in another skipper species (Erynnis). Phylogenetic analyses using all available genes (PCGs, rRNAS, and tRNAs) yielded the consensus superfamilial relationships ((((((Bombycoidea + Noctuoidea + Geometroidea) + Pyraloidea) + Papilionoidea) + Tortricoidea) + Yponomeutoidea) + Hepialoidea), confirming the validity of Macroheterocera (Bombycoidea, Noctuoidea, and Geometroidea in this study) and its sister relationship to Pyraloidea. Within Rhopalocera (butterflies and skippers) the familial relationships (Papilionidae + (Hesperiidae + (Pieridae + ((Lycaenidae + Riodinidae) + Nymphalidae)))) were strongly supported in all analyses (0.98–1 by BI and 96–100 by ML methods), rendering invalid the superfamily status for Hesperioidea. On the other hand, current mitogenome-based phylogeny did not find consistent superfamilial relationships among Noctuoidea, Geometroidea, and Bombycoidea and the familial relationships within Bombycoidea between analyses, requiring further taxon sampling in future studies.     

Complete mitochondrial genome of Saturnia jonasii (Lepidoptera: Saturniidae): Genomic comparisons and phylogenetic inference among Bombycoidea

The complete mitochondrial genome (mitogenome) of Saturnia jonasii (Lepidoptera: Saturniidae) was sequenced and compared to those of 19 other bombycoid species. Furthermore, the mitogenome sequences were used to infer phylogenetic relationships among bombycoid species. The 15,261-bp Saturnia jonasii mitogenome contained the typical sets of genes and gene arrangements found in majority of Lepidoptera. All Bombycoidea species, including Saturnia jonasii, have a 15–33-bp spacer sequence at the trnS₂-ND1 junction. The phylogenetic reconstruction of bombycoid species consistently and strongly supported monophylies of the families, Saturniidae, Bombycidae, and Sphingidae, based on Bayesian inference (BI) and maximum-likelihood (ML) methods. Among these families, the Bombycidae and Sphingidae species consistently showed a sister relationship, regardless of data partitions; the BI method strongly supported this relationship, whereas it was moderately supported using the ML method.     

The mitochondrial genome of the firefly, Pyrocoelia rufa: complete DNA sequence, genome organization, and phylogenetic analysis with other insects

The complete nucleotide sequences of the mt genome from the firefly, Pyrococelia rufa (Coeleoptera: Lampyridae) was determined. The circular genome is 17,739-bp long, and contains a typical gene complement, order, and arrangement identical to Drosophila yacuba. The presence of 1,724-bp long intergenic spacer in the P. rufa mt genome is unique. The putative initiation codon for ND1 gene appears to be TTG, instead of frequently found ATN. All tRNAs showed stable canonical clover-leaf structure of other mt tRNAs, except for tRNA(Ser) (AGN), DHU arm of which could not form stable stem-loop structure. Phylogenetic analysis among insect orders confirmed a monophyletic Endopterygota, a monophyletic Mecopterida, a monophyletic Diptera, a monophyletic Lepidoptera, and a monophyletic Coleoptera, suggesting that the complete insect mt genome sequence has a resolving power in the diversification events within Endopterygota. However, internal relationships among three coleopteran species are not clear, and the inclusion of some insect orders (i.e., apterygotan T. gertschi) in the analysis provided inconsistent results compared to other molecular studies.     

The first complete mitochondrial genome from Bostrychus genus (Bostrychus sinensis) and partitioned Bayesian analysis of Eleotridae fish phylogeny

To understand the phylogenetic position of Bostrychus sinensis in Eleotridae and the phylogenetic relationships of the family, we determined the nucleotide sequence of the mitochondrial (mt) genome of Bostrychus sinensis. It is the first complete mitochondrial genome sequence of Bostrychus genus. The entire mtDNA sequence was 16508 bp in length with a standard set of 13 protein-coding genes, 22 transfer RNA genes (tRNAs), two ribosomal RNA genes (rRNAs) and a noncoding control region. The mitochondrial genome of B. sinensis had common features with those of other bony fishes with respect to gene arrangement, base composition, and tRNA structures. Phylogenetic hypotheses within Eleotridae fish have been controversial at the genus level. We used the mitochondrial cytochrome b (cytb) gene sequence to examine phylogenetic relationships of Eleotridae by using partitioned Bayesian method. When the specific models and parameter estimates were presumed for partitioning the total data, the harmonic mean –lnL was improved. The phylogenetic analysis supported the monophyly of Hypseleotris and Gobiomorphs. In addition, the Bostrychus were most closely related to Ophiocara, and the Philypnodon is also the sister to Microphlypnus, based on the current datasets. Further, extensive taxonomic sampling and more molecular information are needed to confirm the phylogenetic relationships in Eleotridae.     

Identifying two moth species (Lepidoptera: Ditrysia) from Saudi Arabia using mitochondrial 16S rRNA sequences

The mitochondrial genetic markers are considered useful tools for discrimination between more closely related lepidopteran taxa. Therefore, the present study aimed to investigate the role of mitochondrial (mt) 16 s rRNA gene in the determination of the taxonomic position for two moth species within Ditrysia clade. Maximum likelihood analysis has indicated a well-supported dendrogram based on the Tamura-Nei model for the recovered lepidopterans. The mt 16 s rRNA query sequences from 24 species within seven families were analyzed. This analysis and bootstrap confidence revealed two major clades representing Glossata suborder within Lepidoptera, with a close relationship of Noctuoidea + (Pyraloidea (Hesperioidea + Papilionoidea)). The subfamily Heliothinae forming a sister group with Risobinae (Noctinae + Hadeninae). In addition, there is a clear observation about the close relation between Phycitinae + Galleriinae within Pyraloidea and Cyrestinae + Limenitidinae within Papilionoidea. The present study supported that the Helicoverpa and Meroptera species are the first accounts of these genera inhabiting Saudi Arabia.     

Characterization of the complete mitochondrial genome of the giant silkworm moth, Eriogyna pyretorum (Lepidoptera: Saturniidae)

The complete mitochondrial genome (mitogenome) of Eriogyna pyretorum (Lepidoptera: Saturniidae) was determined as being composed of 15,327 base pairs (bp), including 13 protein-coding genes (PCGs), 2 rRNA genes, 22 tRNA genes, and a control region. The arrangement of the PCGs is the same as that found in the other sequenced lepidopteran. The AT skewness for the E. pyretorum mitogenome is slightly negative (-0.031), indicating the occurrence of more Ts than As. The nucleotide composition of the E. pyretorum mitogenome is also biased toward A + T nucleotides (80.82%). All PCGs are initiated by ATN codons, except for cytochrome c oxidase subunit 1 and 2 (cox1 and cox2). Two of the 13 PCGs harbor the incomplete termination codon by T. All tRNA genes have a typical clover-leaf structure of mitochondrial tRNA, with the exception of trnS1(AGN) and trnS2(UCN). Phylogenetic analysis among the available lepidopteran species supports the current morphology-based hypothesis that Bombycoidea, Geometroidea, Notodontidea, Papilionoidea and Pyraloidea are monophyletic. As has been previously suggested, Bombycidae (Bombyx mori and Bombyx mandarina), Sphingoidae (Manduca sexta) and Saturniidae (Antheraea pernyi, Antheraea yamamai, E. pyretorum and Caligula boisduvalii) formed a group.