The Complete Mitochondrial Genome of Leucoptera malifoliella Costa (Lepidoptera: Lyonetiidae)

Abstract

The mitochondrial genome (mitogenome) of Leucoptera malifoliella (=L. scitella) (Lepidoptera: Lyonetiidae) was sequenced. The size was 15,646 bp with gene content and order the same as those of other lepidopterans. The nucleotide composition of L. malifoliella mitogenome is highly A+T biased (82.57%), ranked just below Coreana raphaelis (82.66%) (Lepidoptera: Lycaenidae). All protein-coding genes (PCGs) start with the typical ATN codon except for the cox1 gene, which uses CGA as the initiation codon. Nine PCGs have the common stop codon TAA, four PCGs have the common stop codon T as incomplete stop codons, and nad4l and nad6 have TAG as the stop codon. Cloverleaf secondary structures were inferred for 22 tRNA genes, but trnS1(AGN) was found to lack the DHU stem. The secondary structure of rrnL and rrnS is generally similar to other lepidopterans but with some minor differences. The A+T-rich region includes the motif ATAGA, but the poly (T) stretch is replaced by a stem-loop structure, which may have a similar function to the poly (T) stretch. Finally, there are three long repeat (154 bp) sequences followed by one short repeat (56 bp) with four (TA)_n intervals, and a 10-bp poly-A is present upstream of trnM. Phylogenetic analysis shows that the position of Yponomeutoidea, as represented by L. malifoliella, is the same as traditional classifications. Yponomeutoidea is the sister to the other lepidopteran superfamilies covered in the present study.

Introduction

M itogenomes are maternally inherited, with a covalently closed double-stranded DNA structure, a relatively stable arrangement of genes, and are broadly applied in phylogenetic reconstruction, phylogeography, and molecular evolution (Zhang et al., 1995; Nardi et al., 2003; Arunkumar et al., 2006). Animal mitogenomes are generally 14–20 kb in length, including 37 genes—13 being protein-coding genes (PCGs), 22 transfer RNAs (tRNA), and 2 genes for ribosomal RNA (rRNA). They also contain an A+T-rich noncoding area that regulates transcription and replication of the mitogenome (Boore, 1999; Taanman, 1999) and influences the length of mitogenome (Wolstenholme, 1992). As sequencing technology develops, there is a rapid growth of mitogenome data in GenBank. To date, there have been more than 30 Lepidoptera species sequenced in their entirety or to near completion (Coates et al., 2005; Kim et al., 2006; Lee et al., 2006; Cha et al., 2007; Cameron and Whiting, 2008; Hong et al., 2008; Liu et al., 2008; Pan et al., 2008; Salvato et al., 2008; Hong et al., 2009; Jiang et al., 2009; Kim MI et al., 2009; Hu et al., 2010; Li et al., 2010; Liao et al., 2010; Zhao et al., 2010, Kim MJ et al., 2010; Margam et al., 2011).

The Lepidoptera includes moths and butterflies, with more than 160,000 described species distributed worldwide in 124 families (Kristensen et al., 2007). Leucoptera malifoliella Costa belongs to Lyonetiidae (Lepidoptera). Its junior synonym is Leucoptera scitella Zeller (Mey, 1994). Lyonetiidae is a microlepidopteran family with more than 500 described species. These moths are small and slender, with a very narrow forewing and a wingspan, which rarely exceeds 1 cm. Their larvae are generally leaf miners. L. malifoliella damage apples, pears, and other plants, and withering fruit tree leaves. Recent studies focused on the sex pheromone for prevention and control (Francke et al., 1987; Koutinkova et al., 1999), but few studies have been carried out on their mitogenome, and there is a lack of mitogenome data.

In this study, we described the complete mitogenome of L. malifoliella, the first sequenced specie of Lyonetiidae, and compared its features with other available lepidopteran mitogenomes. Finally, the phylogenetic relationships among the lepidopteran superfamilies were reconstructed using complete mitochondrial genomes.

Materials and Methods

DNA sample extraction

Larvae were collected from an orchard in Beijing, China, L. malifoliella were identified according to Mey (1994), and raised in laboratory. The hatched moths were collected, preserved in 100% ethanol, and stored at −20°C. Total DNA was extracted and isolated from single specimens using the DNeasy Tissue kit (QIAGEN) according to the manufacturer's instructions.

Primer design, polymerase chain reaction, and sequencing

Short fragment amplifications were performed using the universal polymerase chain reaction (PCR) primers from Simon et al. (1994). The degenerate and specific primer pairs were designed based on the known mitochondrial sequences in Lepidoptera, or designed by Primer5.0 software on the fragments that we previously sequenced (Table 1). All the primers were synthesized by Shanghai Sangon Biotechnology Co., Ltd. (Beijing, China). For fragments of length less than 2 kb, PCR conditions were as follows: 95°C for 5 min; 34 cycles of 94°C for 30 s; 50°C–55°C (depending on primer combinations), 1–3 min (depending on putative length of the fragments) at 68°C; and a final extension step of 72°C for 10 min. For fragments of length more than 2 kb, PCR conditions were as follows: 92°C for 2 min; 40 cycles of 92°C for 30 s, 50°C–55°C for 30 s (depending on primer combinations), 60°C for 12 min; and a final extension step of 60°C for 20 min.

Table 1.

Region, Primers, and Sequences for Polymerase Chain Reactions in This Study

Region	Upstream primer	Upstream primer sequence (5′–3′)	Downstream primer	Downstream primer sequence (5′–3′)	Size (bp)
trnQ-coxl	Gln-J-518^a	ATTAAGGCAATTATGGGAGCT	Cl-N-1391^a	AATCTGAGTATCGACGTGGTA	2000
trnQ-nad2	Glnl0486^b	TAAACTATATCTAATAATATCAAAAATTATTGTG	ND2-N-784^a	TTTAATCCTCCGATAGCTCCAAT	600
nad2	Gln-J-171^a	ATTTACTTAGATTTATCCCCC	C1-N-1031^a	GAAGGGGGGAGAAGTCAGAAT	1200
cox1	LCO1490^a	GGTCAACAAATCATAAAGATATTGG	HCO2198^a	TAAACTTCAGGGTGACCAAAAAATCA	650
cox1-cox2	C1-J-2183^a	CAACATTTATTTTGATTTTTTGG	C2-N-3494^a	GGTAAAACTACTCGATTATCAAC	1300
trnL2-trnK	Leu-J-3029^c	CTAATATGGCAGACTATATGTAATGGA	Lysl4111Re^a	GACCATTACTTGCTTTCAGTCATCTAATG	750
cox2-nad5	C2-J-2085^a	TAGATAATCGCATTGTTTTACCCT	N5-N-2826^a	TGCTCTTCAGGGATATTTTGTTTA	5000
nad5-cob	N5-J-532^a	ATGAGCAACTGAAGAATAAGC	Cytb-N-989^a	ATAATAGGGATGGAAAGGAAT	2500
Cob	CB-J-10933^c	TATGTTTTTCCTTGAGGACAAATATC	CB-N-11367^c	TAACTCCTCCTAATTTATTGGGA	460
cob-nad1	Cytb-J-625^a	TCGACCTGTAGAAGAACCCTA	N1-N-57^a	CAAATTATGCTTTATTAGGAGGT	730
cob-rrnL	CB-5971^b	CAAACAGGATCTAATAACCCTTTAGG	LR-N-12866^d	ACATGATCTGAGTTCAAACCGG	1800
rrnL-rrnS	Lr-J-2275^a	AACTCTATAGGGTCTTCTCGT	Sr-N-3095^a	GAGATACTGGAAAGTGTTTCT	1100
rrnS	SR-J-14233^C	GAAAGCGACGGGCAATATG	SR-N-14588^d	AAACTAGGATTAGATACCCTATTAT	350
rrnS-nad2	Sr-J-6512^a	TCTCTACTTTGTTACGACTTA	Gln-N-193^a	AAGGGGGATAAATCTAAGTAA	1100

Primers newly designed for this genome.

Primers from Lee et al. (2006).

Primers from Zhao et al. (2010).

Primers from Simon et al. (1994).

The entire mitogenome of L. malifoliella was amplified in 14 fragments. For most fragments, we used 2× Taq PCR MasterMix (Tiangen Biotech Co., Ltd., Beijing, China) in the amplification; for fragments longer than 2 kb (cox2-nad5 and nad5-cob) and with higher AT contents (A+T-rich region), amplification used Takara LA Taq (Takara Co., Dalian, China). All amplifications were performed on an Eppendorf Mastercycler and Mastercycler gradient in 50-μL reaction volumes. The reaction volume of 2× Taq PCR MasterMix contains 22 μL sterilized distilled water, 25 μL 2× Master Mix, 1 μL of each primer (10 μM), and 1 μL of DNA template; the one of Takara LA Taq consists of 26.5 μL of sterilized distilled water, 5 μL of 10× LA PCR Buffer II (Takara), 5 μL of 25 mM MgCl₂, 8 μL of dNTPs Mixture, 2 μL of each primer (10 μM), 1 μL of DNA template, and 0.5 μL (1.25 U) of TaKaRa LA Taq polymerase (Takara).

The PCR products were detected via electrophoresis in 1% agarose gel, purified using the 3S Spin PCR Product Purification Kit, and sequenced directly with ABI-377 automatic DNA sequencer. All fragments were sequenced from both strands. Short amplified products were sequenced directly by internal primers, long amplified products were sequenced completely by primer walking, but the rrnS-nad2 regions were sequenced after cloning. The purified PCR products were ligated to the pEASY-T3 Cloning Vector (Beijing TransGen Biotech Co., Ltd., Beijing, China), and then sequenced by M13-F and M13-R primers and walking. Sequencing was performed using ABI BigDye ver 3.1 dye terminator sequencing technology and run on ABI PRISM 3730×1 capillary sequencers.

Analysis and annotation

Sequence annotation was performed using the DNAStar package (DNAStar Inc. Madison, WI). The tRNA genes were identified using the tRNAscan-SE v.1.21 software (Lowe and Eddy, 1997). The putative tRNAs were then confirmed by sequence alignment with other insects of lepidoptera using the Bioedit (Hall, 1999). Secondary structure was inferred using DNA-SIS 2.5 (Hitachi Engineering, Tokyo, Japan). The trnS1(AGN) secondary structure was developed as proposed by Steinberg and Cedergren (1994). rrnL and rrnS secondary structures were drawn by XRNA (developed by B. Weiser and available at http://rna.ucsc.edu/rnacenter/xrna/xrna.html). Helix numbering follows the convention established at the CRW site (Cannone et al., 2002) and Grapholita molesta rRNA secondary structure (Gong et al., 2011), with minor modification. The stem-loop structure of A+T-rich region was determined by the Mfold Web Server (Zuker, 2003; http://mfold.rna.albany.edu/?q=mfold). PCGs and rRNAs were identified by similarity to other lepidopterans. The nucleotide sequences of PCGs were translated based on the invertebrate mtDNA genetic code. Nucleotide composition and codon usage were calculated using MEGA5.0 (Tamura et al., 2011).

Phylogenetic analysis

To infer the phylogenetic relationships of lepidopterans, other available complete mitogenomes in Lepidoptera were obtained from GenBank. Bactrocera oleae (NC_005333) (Nardi et al., 2003) and Anopheles gambiae (NC_002084) (Beard et al., 1993) were used as outgroups. The alignment of the amino acid sequences and nucleotide sequences of each of the 13 mitochondrial PCGs was performed with MUSCLE (Edgar, 2004) using default settings, and concatenated into an amino acid (3,872 sites in length) and nucleotide (11,616 sites in length) matrix. The concatenated set of amino acid sequences and nucleotide sequences were used in phylogenetic analyses, using Bayesian Inference (BI) and Maximum Likelihood (ML) methods. Substitution model selection was conducted via a comparison of Akaike Information Criterion scores (Akaike, 1974), calculated using the programs ProTest ver. 1.4 (Abascal et al., 2005) for amino acid sequence alignment and Modeltest ver. 3. 7 (Posada and Crandall, 1998) for nucleotide sequence alignment. The MtRev (Adachi and Hasegama, 1996)+I+G model and GTR (Lanave et al., 1984)+I+G model were chosen as the best-fitting model for amino acid sequences and nucleotide sequences, respectively, for BI analyses and ML analyses. The BI analysis was conducted using MrBayes 3.1 (Huelsenbeck and Ronquist, 2001) with four independent Markov chains run for 1,000,000 metropolis-coupled MCMC generations, with tree sampling every 100 generations and a burn-in of 2000 trees. The ML analysis was performed using RAxML (Stamatakis, 2006) with 1000 bootstrap replicates.

Results and Discussion

Genome structure and organization

The L. malifoliella mitogenome is a circular molecule of 15,646 bp in length, deposited in GenBank under accession number JN790955. The L. malifoliella mitogenome showed the typical metazoan gene content, containing 13 PCGs, 2 rRNAs, 22 tRNAs, and noncoding regions. The gene order in L. malifoliella is A+T-rich region-trnM-trnI-trnQ, whereas the ancestral gene order for the Lepidoptera is A+T-rich region-trnI-trnQ-trnM (Junqueira et al., 2004). This placement of trnM may be a molecular feature exclusive to lepidopteran mitogenomes (Cameron and Whiting, 2008).

The L. malifoliella mitogenome is biased toward A+T (82.57%) with the value falling into lepidopteran range of 77.84% (Ochrogaster lunifer, Salvato et al., 2008) to 82.66% (Coreana raphaelis, Kim et al., 2006). The A+T content was 80.24% in PCGs, 85.49%, in rrnL genes, 87.14% in rrns genes, and 95.36% in the A+T-rich region. These values were also high in other lepidopterans reported (Table 2).

Table 2.

Characteristics of the Lepidopteran Mitogenomes

									A+T-rich region
				PCG A+T content (%)	rmL A+T content (%) 16S		rmS A+T content (%) 12S		A+T content(%)
Superfamily	Species	Size (bp)	A+T content (%)	A+T (%)	Size (bp)	A+T (%)	Size (bp)	A+T (%)	Size (bp)	A+T (%)	GenBank accession no.
Yponomeutoidea			82.57	80.24	1351	85.49	770	87.14	733	95.36	JN790955
	Corcyra cephalonica	15273	80.43	78.96	1355	82.95	778	85.86	351	96.58	HQ897685
	Chilo suppressalis	15395	80.67	78.9	1383	84.24	788	86.17	348	95.4	JF339041
Pyraloidea	Diatræa saccharalis	15490	80.02	77.9	1412	84.77	781	85.53	335	94.43	FJ240227
	Ostrinia nubilalis	14535	80.17	79.16	1339	84.91	434	82.03			AF442957
	Ostrinia furnacalis	14536	80.37	79.42	1339	84.99	435	82.76			AF467260
	Antheraea pernyi	15566	80.16	78.53	1369	83.86	775	84.13	552	90.4	AY242996
	Antheraea yamamai	15338	80.29	78.94	1380	83.99	776	84.41	334	89.52	EU726630
	Bombyx mandarina	15928	81.68	79.64	1377	84.75	783	85.95	747	95.91	AB070263
Bombycoidea	Bombyx mori	15643	81.32	79.57	1375	84.36	783	85.57	499	95.39	AF149768
	Eriogyna pyretorum	15327	80.82	79.41	1338	84.6	778	84.45	358	92.18	FJ685653
	Manduca sexta	15516	81.79	80.3	1391	85.26	777	85.71	324	95.37	EU286785
	Saturnia boisduvalii	15360	80.63	79.15	1391	84.76	774	84.11	330	91.52	EF622227
Geometroidea	Phthonandria atrilineata	15499	81.02	79.1	1400	85.71	803	86.03	457	98.25	EU569764
Tortricoidea	A doxophyes honmai	15680	80.39	78.48	1387	83.56	779	85.37	490	94.29	DQ073916
	Grapholita molesta	15717	80.87	78.89	1377	84.75	772	85.36	771	95.85	HQ392511
	Spilonota lechriaspis	15368	81.19	79.72	1382	85.17	778	86.25	441	92.74	HM204705
	Helicoverpa armigera	15347	80.97	79.43	1395	84.73	794	85.89	328	95.12	GU188273
	Hyphantria cunea	15481	80.39	78.95	1426	84.99	808	84.53	357	94.96	GU592049
Noctuoidea	Lymantria dispar	15569	79.88	77.84	1351	84.23	799	85.23	435	96.09	FJ617240
	Ochrogaster lunifer	15593	77.84	75.73	1351	81.5	806	83.25	319	93.42	AM946601
	Sesamia inferens	15413	80.24	78.62	1385	83.39	784	85.33	311	95.82	JN039362
	Acraea issoria	15245	79.76	78.11	1331	83.85	788	83.76	430	96.05	GQ376195
	Apatura metis	15236	80.44	78.96	1333	84.47	779	84.85	394	92.89	JF801742
	Artogeia melete	15140	79.78	78.52	1319	83.47	777	85.46	351	89.17	EU597124
	Pieris rapae	15157	79.74	78.28	1320	84.01	764	84.95	393	91.61	GQ398376
	Calinaga davidis	15267	80.45	78.94	1337	83.84	773	85.9	389	92.03	HQ658143
Papilionoidea	Coreana raphaelis	15314	82.66	81.51	1330	85.26	777	85.84	375	94.13	DQ 102703
	Hipparchia autonoe	15489	79.09	76.91	1335	83.67	775	85.29	678	94.54	GQ868707
	Papilio maraho	16094	80.5	78.17	1333	83.72	779	85.49	1270	94.62	FJ810212
	Parnassius bremeri	15389	81.27	80.18	1344	83.93	773	85.12	504	93.65	FJ871125
	Sasakia charonda	15244	79.87	78.22	1323	84.35	775	85.03	380	91.84	AP011824
	Sasakia charonda kuriyamaensis	15222	79.89	78.3	1311	84.21	775	85.03	380	91.84	AP011825
	Teinopalpus aureus	15242	79.81	78.31	1320	82.42	781	85.66	395	93.16	HM563681

Protein-coding genes

The initial and termination codons of 13 PCGs are shown in Table 3. Twelve PCGs start with a typical ATN codon (ATT for nad2, nad3, nad5, atp8; ATA for cox2, nad6, nad1; ATG for atp6, cox3, nad4, nad4l, cob). The exception is the cox1 gene, which uses CGA as the start codon. Seven PCGs have the common stop codon TAA, nad4l and nad6 have the common stop codon TAG, and four PCGs have the codon T as incomplete stop codons, which was also found in other animal mitochondrial genes (Clary and Wolstenholme, 1985).

Table 3.

Summary of Mitogenome of Leucoptera malifoliella

Gene	Direction	Location	Size (bp)	Anticodon	Start codon	Stop codon
trnM	F	1–66	66	CAT
trn1	F	68–139	72	GAT
trnQ	R	137–205	69	TTG
Spacer 1	N/A	206–248	43
nad2	F	249–1257	1009		ATT	T
trnW	F	1258–1325	68	TCA
trnC	R	1318–1392	75	GCA
trnY	R	1395–1459	65	GTA
cox1	F	1464–2994	1531		CGA	T
trnL2(UUR)	F	2995–3062	68	TAA
cox2	F	3063–3744	682		ATA	T
trnK	F	3745–3815	71	TTT
trnD	F	3818–3883	66	GTC
atp8	F	3884–4045	162		ATT	TAA
atp6	F	4039–4716	678		ATG	TAA
cox3	F	4720–5508	789		ATG	TAA
trnG	F	5515–5581	67	TCC
nad3	F	5582–5935	354		ATT	TAA
Spacer2		5936–5954	19
trnA	F	5955–6017	63	TGC
trnR	F	6017–6083	67	TCG
trnN	F	6083–6151	69	GTT
trnS1(AGN)	F	6150–6211	68	GCT
trnE	F	6214–6280	67	TTC
trnF	R	6279–6343	65	GAA
nad5	R	6344–8069	1726		ATT	T
Spacer3	N/A	8070–8090	21
trnH	R	8091–8157	67	GTG
nad4	R	8157–9497	1341		ATG	TAA
nad4L	R	9503–9781	279		ATG	TAG
trnT	F	9793–9859	67	TGT
trnP	R	9860–9924	65	TGG
nad6	F	9927–10448	522		ATA	TAG
cob	F	10460–11614	1155		ATG	TAA
Spacer4	N/A	11615–11628	14
trnS2(UCN)	F	11629–11695	67	TGA
Spacer5	N/A	11696–11714	19
nad1	R	11715–12650	936		ATA	TAA
trnL1(CUN)	R	12651–12720	70	TAG
rrnL	R	12721–14071	1351
trnV	R	14072–14143	72	TAC
rrnS	R	14144–14913	770
A+T-rich region		14914–15646	733

The putative start codons of PCGs in the L. malifoliella mitogenome are ATN, except for the CGA start codon of the cox1 gene. The start codon of the cox1 gene is controversial in many studies. The putative codon CGA is common across insects (Anabrus simplex, Fenn et al., 2007; Adoxophyes hnmai, Lee et al., 2006; Manduca sexta, Cameron and Whiting, 2008; O. lunifer, Salvato et al., 2008; Eriogyna pyretorum, Jiang et al., 2009; Phthonandria atrilineata, Yang et al., 2009; Hyphantria cunea, Liao et al., 2010; Artogeia melete, Hong et al., 2009; Antheraea yamamai, Kim SR et al., 2009; Eumenis autonoe, Kim et al., 2010). The tetranucleotides TTAG and hexanucleotide TATTAG have also been proposed as start codons for the cox1 gene (Parnassius bremeri, Kim et al., 2009; C. raphaelis, Kim et al., 2006; Antheraea pernyi, Liu et al., 2008; Ostrinia nubilalis, Ostrinia furnacalis, Coates et al., 2005; Bombyx mandarina, Yukuhiroetal., 2002; Papilio xuthus, Feng et al., 2010). However, TTAG lacks absolute conservation and may serve alternative functions, not always as an initiation codon. Alignment of the mitogenome sequence from all Lepidopterans had shown that an arginine (CGR) functions as the start codon for the cox1 gene (Margam et al., 2011). In our study the start codon of the cox1 gene is CGA according to the alignment of the lepidoterans (Fig. 1).

FIG. 1.

Alignment result of trnY and cox1 in 34 Lepidopterans. The dotted line and underline indicate the locations of cox1 and trnY, respectively. The overlapping base between trnY and cox1 is marked gray.

Transfer and ribosomal RNA genes

The 22 tRNA genes ranged from 63 to 75 nucleotides. Fourteen tRNAs are coded on the J-strand and 8 on the N-strand, as with other Lepidoptera. The trnK anticodon is TTT, which is unusual in this insect order. Complete cloverleaf secondary structures could be inferred for 21 of the 22 tRNAs. The secondary structure of trnS1(AGN) was incomplete, lacking the DHU arm (Fig. 2). A total of 43 unmatched base pairs were scattered throughout the 21 tRNA genes, including 15 pairs in the DHU stems, 11 pairs in the amino acid acceptor stems, 9 pairs in the TΨC stems, and 8 pairs in the anticodon stems. Twenty-one of these are G-U pairs, which form a stable hydrogen-bonded pair. The remaining were C-A, C-U, G-G, G-A, and U-U mismatches.

FIG. 2.

Putative secondary structures for the tRNA genes of Leucoptera malifoliella mitogenome.

As in the other insect mitogenome sequences, two rRNA genes were present in L. malifoliella. The rrnL gene (1351 bp) was found between trnL(CUN) and trnV, and the rrnS (770 bp) between trnV and the A+T-rich region. Both the secondary structure of rrnL and rrnS conform to the models proposed for other insects (Cameron and Whiting, 2008; Wei et al., 2009; Wei et al., 2010). Forty-nine helices are present in rrnL of L. malifoliella, as in G. molesta (Gong et al., 2011), M. sexta (Cameron and Whiting, 2008), Drosophila melanogaster (Schnare et al., 1996), and Apis mellifera (Gillespie et al., 2006). There is a large internal loop among H991, H1057, and H1087, which is similar to G. molesta, and differs from M. sexta. The microsatellite sequence of (TA)_n inserted in the loop region of H2347 in Adoxophyes honmai (Lee et al., 2006 ), Spilonota lechriaspis (Zhao et al., 2010), G. molesta, which belong to Tortricidae, is not present in L. malifoliella (Fig. 3). Twenty-nine helices present in rrnS of L. malifoliella belong to three domains, as in G. molesta, M. sexta, and A. mellifera. The structures of Helix H47, H673, H1303, H1047, H1068, H1074, and H1113 are different from M. sexta, but similar to G. molesta, with the exception of H47, which has a shorter loop length in L. malifoliella compared to G. molesta (Fig. 4).

FIG. 3.

Predicted rrnL secondary structure in Leucoptera malifoliella mitogenome.

FIG. 4.

Predicted rrnS secondary structure in Leucoptera malifoliella mitogenome. Tertiary interactions and base triples are shown connected by continuous lines. Base pairing is indicated as follows: Watson-Crick pairs by lines, wobble GU pairs by plus, and other noncanonical pairs by circles.

Codon usage

Relative synonymous codon usage values of the L. malifoliella mitogenome are summarized in Table 4. The codons CUG, ACG, and GCC were not represented in the coding sequences. The most frequent amino acids in L. malifoliella mitochondrial proteins are leucine (14.4%), isoleucine (12.8%), phenylalanine (10.9%), and serine (8.6%).

Table 4.

The Codon Number and Relative Synonymous Codon Usage in Leucoptera Malifoliella Mitochondrial Protein Coding Genes

Codon	Count	RSCU	Codon	Count	RSCU	Codon	Count	RSCU	Codon	Count	RSCU
UUU(F)	384	1.9	UCU(S)	118	2.97	UAU(Y)	184	1.8	UGU(C)	27	1.74
UUC(F)	21	0.1	UCC(S)	7	0.18	UAC(Y)	20	0.2	UGC(C)	4	0.26
UUA(L)	472	5.29	UCA(S)	85	2.14	UAA(*)	7	1.56	UGA(W)	85	1.79
UUG(L)	16	0.18	UCG(S)	1	0.03	UAG(*)	2	0.44	UGG(W)	10	0.21
CUU(L)	26	0.29	CCU(P)	53	1.83	CAU(H)	63	1.88	CGU(R)	16	1.31
CUC(L)	3	0.03	CCC(P)	13	0.45	CAC(H)	4	0.12	CGC(R)	4	0.33
CUA(L)	18	0.2	CCA(P)	49	1.69	CAA(Q)	55	1.86	CGA(R)	28	2.29
CUG(L)	0	0	CCG(P)	1	0.03	CAG(Q)	4	0.14	CGG(R)	1	0.08
AUU(I)	457	1.92	ACU(T)	76	2.16	AAU(N)	237	1.84	AGU(S)	19	0.48
AUC(I)	19	0.08	ACC(T)	4	0.11	AAC(N)	20	0.16	AGC(S)	1	0.03
AUA(M)	282	1.87	ACA(T)	61	1.73	AAA(K)	116	1.92	AGA(S)	76	1.91
AUG(M)	19	0.13	ACG(T)	0	0	AAG(K)	5	0.08	AGG(S)	11	0.28
GUU(V)	57	2.11	GCU(A)	72	2.62	GAU(D)	51	1.82	GGU(G)	59	1.25
GUC(V)	2	0.07	GCC(A)	0	0	GAC(D)	5	0.18	GGC(G)	3	0.06
GUA(V)	46	1.7	GCA(A)	35	1.27	GAA(E)	69	1.86	GGA(G)	110	2.33
GUG(V)	3	0.11	GCG(A)	3	0.11	GAG(E)	5	0.14	GGG(G)	17	0.36

A total of 3721 codons were analyzed, excluding the initiation and termination codons.

The amino acids encoded by codons are labeled according to the IUPAC-IUB single-letter amino acid codes.

RSCU, relative synonymous codon usage.

Noncoding and overlapping region

The L. malifoliella mitogenome harbors 16 noncoding regions, ranging from 1 to 43 bp. Intergenic spacer sequences have 5 regions with a length of more than 14 bp. The remaining intergenic spacers were less than 11 bp.

Spacer 1 (43 bp) is located between the trnQ and nad2 genes. This spacer can be taken as lepidopteran feature, not found in other insects. Kim et al. (2009) detected high sequence identity between the intergenic spacer sequence and the neighboring nad2 from several lepidopteran insects; this indicated that the spacer sequence may have originated from a partial duplication of the nad2 gene.

Spacer 2 (19 bp) is found between nad3 and trnA gene; the spacer is longest in lepidopterans sequenced, and in others it is only 1 to 2 bp. Additionally, this region is generally overlapped in other lepidopterans, such as B. mandarina, B. mori, M. sexta, O. furnacalis, O. nubilalis.

Spacer 3 (21 bp) is found between the nad5 and trnH genes; the spacer is also found in A. honmai (23 bp), E. pyretorum (18 bp), B. mandarina (18 bp), B. mori (21 bp), A. melete (18 bp), and C. raphaelis (16 bp). Spacer 4 (14 bp) is found between the cob and trnS2(UCN) genes. This spacer is also present in A. pernyi (15 bp), A. yamamai (24 bp), S. boisduvalii (41 bp), and M. sexta(21 bp), and it is shorter in other lepidopterans.

Spacer 5 (19 bp) is between the trnS2(UCN) and nad1 genes, commonly detectable in lepidopterans with size 16–38 bp. This intergenic spacer is conserved for all insects. Most lepidopterans harbor the motif (ATACTAA), except for ATACTAT in Corcyra cephalonica (unpublished, HQ897685) and ATCATAT in Sesamia inferens (unpublished NC_015835). Similarly, in Hymenoptera there is a 6-bp conserved motif (THACWW) (Wei et al., 2010). In Coleoptera, there is a 5-bp conserved motif (TACTA) (Sheffield et al., 2008). The motif has been suggested to be a possible mitochondrial transcription termination peptide-binding site (Taanman, 1999).

Overlapping sequences had a total length of 25 bp from 1 to 8 bp, spread over 14 regions. The longest overlapping sequence AAGCCTTA (8 bp) is located between the trnW gene and the trnC gene. The seven-nucleotide overlap (ATGATAA) is located between atp8 and atp6, which is common in other insects. The remaining overlapping sequences are less than 3 bp.

A+T-rich region

The A+T-rich region of L. malifoliella mitogenome is located between rrnS and trnM, with 95.36% AT nucleotides and a length of 733 bp. There is a motif ATAGA downstream of rrnS, but not followed by the typical poly (T) stretch, but replaced by a stem-loop structure (Fig. 5). There are three long repeat (154 bp) sequences followed by one short repeat (56 bp), each preceded by (TA)_n microsatellite regions (Fig. 5). Finally, a 10-bp poly-A is present upstream of trnM, a feature common across lepidopterans.

FIG. 5.

The structure of the A+T-rich region of Leucoptera malifoliella mitogenome.

The stem-loop structure in the A+T-rich region was also observed in other insect orders, including Orthoptera, Diptera, Plecoptera, Hymenoptera, and Phthiraptera (Brehm et al., 2001; Schultheis et al., 2002; Cameron et al., 2007; Cha et al., 2007; Ye et al., 2008). The stem-loop structure in the A+T-rich region of Drosophila was suggested as the site of the initiation of light strand synthesis (Clary and Wolstenholme, 1987), but the position of the stem-loop structure in L. malifoliella is found to be same as the poly (T) stretch of other lepidopterans (Fig. 6), a feature only found in Leucoptera. Two species (A. yamamai and S. boisduvalii) in Lepidoptera have a stem-loop structure, but also possess a poly (T) stretch, and the flanking sequence of the stem-loop structure are conserved, with consensus TATA sequences at the 5′ and G(A)_nT at the 3′. The feather is also among other insects (Zhang et al., 1995; Schultheis et al., 2002). In contrast to these insects, there are no conserved sequences flanking both sides of the L. malifoliella stem-loop structure, and the location of stem-loop structure is closer to rrnS. Ye et al. (2008) suggested that the stem-loop structure might have the same function as the poly (T) stretch, if the latter feature is absent. Therefore, the stem-loop structure in L. malifoliella may play an important role in recognition of the light strand replication origin, but determining the function needs additional research.

FIG. 6.

Alignment of motif and Poly(T) in the A+T-rich region of 34 lepidopterans. The Poly(T) stretch is marked gray. Marked box is motif ATAGA. Underline is the stem-loop structure of Leucoptera malifoliella.

Phylogenetic Relationships

To place the L. malifoliella mitogenome relative to other lepidopterans mitogenomes and investigate the phylogenetic relationships among the superfamilies in Lepidoptera, two data sets containing the concatenated amino acid sequences and nucleotide sequences of 13 PCGs were generated. These 34 sequences represent seven superfamilies: Bombycoidea, Geometroidea, Noctuoidea, Papilionoidea, Pyraloidea, Tortricoidea, and Yponomeutoidea. According to the most recent consensus view of lepidopteran relationships in Kristensen and Skalski (1999), Papilionoidea, Bombycoidea, Noctuoidea, and Geometroidea are designated as the Macrolepidoptera; Pyraloidea together with Macrolepidoptera are designated as Obtectomera; Tortricoidea together with Obtectomera are designated as Apoditrysia; Yponomeutoidea is the sister to the remaining lepidopteran superfamilies covered in the present study. The BI and ML analyses generate similar topologies, and most major groups were consistently monophyletic apart from Pyraloidea. Three trees all support that C. cephalonica is grouped with Pyraloidea; this is same as traditional classifications (Solis, 1997). However, in the BI tree inferred from amino acid sequences, C. cephalonica is sister to the clade (Pyraloidea+(Noctuoidea+(Geometroidea+Bombycoidea))).

In our phylogenetic results, the placement of Yponomeutoidea (as represented by L. malifoliella) is the same as the traditional classification, basal to all Lepidoptera, with full nodal support in BI (100%/100%) and ML analyses (100%/100%). Bombycoidea and Geometroidea are sister groups with high nodal support on BI (100%/100%) and ML analyses (90%/92%) (Fig. 7A, B), which is consistent with Yang et al. (2009), but differs to the typical morphological results, which give a sister group relationship between the Papilionoidea and Geometroidea. Papilionoidea is the sister of the remaining macrolepidopteran families, in accordance with other studies (Jiang et al., 2009; Yang et al., 2009; Liao et al., 2010). Pyraloidea has a closer relationship to most Macrolepidoptera than Papilionoidea (butterflies), a result confirmed by a recent study (Regier et al., 2009), but different from the traditional classification.

FIG. 7.

Phylogeny of lepidopteran insects. (A) Phylogenetic trees inferred from amino acid sequences and nucleotide sequences of 13 protein-coding genes (PCGs) of the mitogenome using ML analysis; the numbers above branches are bootstrap percentages; the first and second values are from amino acid sequences and nucleotide sequences, respectively. Bactrocera oleae and Anopheles gambiae were used as outgroups. (B) Phylogenetic trees inferred from amino acid sequences and nucleotide sequences of 13 PCGs of the mitogenome using Bayesian Inference (BI) analysis; the numbers above branches give posterior probabilities. In the BI tree inferred from amino acid sequences C. cephalonica is sister to the clade (Pyraloidea+(Noctuoidea+(Geometroidea+Bombycoidea))); the posterior probabilities are 100% and 90%, and the posterior probabilities of C. cephalonica in the BI tree from nucleotide sequences is 100% (not labeled). The other information on the tree is the same as in (A).

Footnotes

Acknowledgments

Prof. Qi-lian Qin and his lab members (Institute of Zoology, Chinese Academy of Sciences) kindly provided advice and facilities in sequence cloning. We also thank Shu-jun Wei (Institute of Plant and Environmental Protection, Beijing Academy of Agriculture and Forestry Sciences) and Xiao-he Wang (Institute of Zoology, Chinese Academy of Sciences) for their kind help in data analysis.

This work was supported mainly by grants from the Knowledge Innovation Program of Chinese Academy of Sciences (Grant No. KSXC2-EW-B-02), Public Welfare Project from the Ministry of Agriculture, China (Grant No. 201103024), the National Science Foundation, China (NSFC Grant No. 30870268, 31172048, J0930004) to Chao-dong Zhu and NSFC Grant (No. 31172129) to Chun-Sheng Wu.

Disclosure Statement

No competing financial interests exist.

References

Abascal

, Zardoya

, Posada

2005. ProTest: selection of best-fit models of protein evolution. Bioinformatics, 21:2104–2105.

Adachi

, Hasegawa

1996. Model of amino acid substitution in proteins encoded by mitochondrial DNA. J Mol Evol, 42:459–468.

Akaike

1974. A new look at the statistical model identification. IEEE Trans Autom Contr, 19:716–723.

Arunkumar

K.P.

, Metta

, Nagaraju

2006. Molecular phylogeny of silkmoths reveals the origin of domesticated silkmoth, Bombyx mori from Chinese Bombyx mandarina and paternal inheritance of Antheraea proylei mitochondrial DNA. Mol Phylogenet Evol, 40:419–427.

Beard

C.B.

, Mills

, Collins

F.H.

1993. The mitochondrial genome of the mosquito Anopheles gambiae: DNA sequence, genome organization and comparisons with mitochondrial sequences of other insects. Insect Mol Biol, 2:103–124.

Boore

J.L.

1999. Animal mitochondrial genomes. Nucleic Acids Res, 27:1767–1780.

Brehm

, Harris

D.J.

, Hernández

, Cabrera

V.M.

, Larruga

J.M.

, Pinto

F.M.

, González

A.M.

2001. Structure and evolution of the mitochondrial DNA complete control region in the Drosophila subobscura subgroup. Insect Mol Biol, 10:573–578.

Cameron

S.L.

, Johnson

K.P.

, Whiting

M.F.

2007. The mitochondrial genome of the screamer Louse Bothriometopus (Phthiraptera: Ischnocera): effects of extensive gene rearrangements on the evolution of the genome. J Mol Evol, 65:589–604.

Cameron

S.L.

, Whiting

M.F.

2008. The complete mitochondrial genome of the tobacco hornworm, Manduca sexta, (Insecta: Lepidoptera: Sphingidae), and an examination of mitochondrial gene variability within butterflies and moths. Gene, 408:112–123.

10.

Cannone

J.J.

, Subramanian

, Schnare

M.N.

, Collett

J.R.

, D'Souza

L.M.

, Du

, Feng

, Lin

, Madabusi

L.V.

, Muller

K.M.

2002. The comparative RNA web (CRW) site: an online database of comparative sequence and structure information for ribosomal, intron, and other RNAs. BMC Bioinformatics, 3:1471–2105.

11.

Cha

S.Y.

, Yoon

H.J.

, Lee

E.M.

, Yoon

M.H.

, Hwang

J.S.

, Jin

B.R.

, Han

Y.S.

, Kim

2007. The complete nucleotide sequence and gene organization of the mitochondrial genome of the bumblebee, Bombus ignitus (Hymenoptera: Apidae) Gene, 392:206–220.

12.

Clary

D.O.

, Wolstenholme

D.R.

1985. The mitochondrial DNA molecular of Drosophila yakuba: nucleotide sequence, gene organization, and genetic code. J Mol Evol, 22:252–271.

13.

Clary

D.O.

, Wolstenholme

D.R.

1987. Drosophila mitochondrial DNA: conserved sequences in the A+T-rich region and supporting evidence for a secondary structure model of the small ribosomal RNA. J Mol Evol, 25:116–125.

14.

Coates

B.S.

, Sumerford

D.V.

, Hellmich

R.L.

, Lewis

L.C.

2005. Partial mitochondrial genome sequence of Ostrinia nubilalis and Ostrinia furnicalis. Int J Biol Sci, 1:13–18.

15.

Edgar

R.C.

2004. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res, 32:1792–1797.

16.

Feng

, Liu

D.F.

, Wang

N.X.

, Zhu

C.D.

, Jiang

G.F.

2010. The mitochondrial genome of the butterfly Papilio xuthus (Lepidoptera: Papilionidae) and related phylogenetic analyses. Mol Biol Rep, 37:3877–3888.

17.

Fenn

J.D.

, Cameron

S.L.

, Whiting

M.F.

2007. The complete mitochondrial genome of the Mormon cricket (Anabrus simplex: Tettigoniidae: Orthoptera) and an analysis of control region variability. Insect Mol Biol, 16:239–252.

18.

Francke

, Franke

, Toth

, Szöcs

, Guerin

, Arn

1987. Identification of 5,9-dimethylheptadecane as a sex pheromone of the moth Leucoptera scitella. Naturwissenschaften, 74:143–144.

19.

Gillespie

J.J.

, Johnston

J.S.

, Cannone

J.J.

, Gutell

R.R.

2006. Characteristics of the nuclear (18S, 5.8S, 28S and 5S) and mitochondrial (12S and 16S) rRNA genes of Apis mellifera (Insecta: Hymenoptera): structure, organization, and retrotransposable elements. Insect Mol Biol, 15:657–686.

20.

Gong

Y.J.

, Shi

B.C.

, Kang

Z.J.

, Zhang

, Wei

S.J.

2011. The complete mitochondrial genome of the oriental fruit moth Grapholita molesta (Busck) (Lepidoptera: Tortricidae) Mol Biol Rep, 39:2893–2900.

21.

Hall

T.A.

1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser, 41:95–98.

22.

Hong

G.Y.

, Jiang

S.T.

, Yu

, Yang

, Li

, Xue

F.S.

, Wei

Z.J.

2009. The complete nucleotide sequence of the mitochondrial genome of the cabbage butterfly, Artogeia melete (Lepidoptera: Pieridae) Acta Biochim Biophys Sin, 41:446–455.

23.

Hong

M.Y.

, Lee

E.M.

, Jo

Y.H.

, Park

H.C.

, Kim

S.R.

, Hwang

J.S.

, Jin

B.R.

, Kang

P.D.

, Kim

K.G.

, Han

Y.S.

, Kim

2008. Complete nucleotide sequence and organization of the mitogenome of the silk moth Caligula boisduvalii (Lepidoptera: Saturniidae) and comparison with other lepidopteran insects. Gene, 30 413:49–57.

24.

, Zhang

D.X.

, Hao

J.S.

, Huang

D.Y.

, Cameron

, Zhu

C.D.

2010. The complete mitochondrial genome of the yellow coaster, Acraea issoria (Lepidoptera: Nymphalidae: Heliconiinae: Acraeini): sequence, gene organization and a unique tRNA translocation event. Mol Biol Rep, 37:431–3438.

25.

Huelsenbeck

J.P.

, Ronquist

2001. MrBayes: bayesian inference of phylogeny. Bioinformatics, 17:754–755.

26.

Jiang

, Hong

, Yu

, Li

, Yang

, Liu

, Wei

2009. Characterization of the complete mitochondrial genome of the giant silkworm moth, Eriogyna pyretorum (Lepidoptera: Saturniidae) Int J Biol Sci, 5:351–365.

27.

Junqueira

A.C.M.

, Lessinger

A.C.

, Torres

T.T.

, da Silva

F.R.

, Vettore

A.L.

, Arruda

, Espin

A.M.L.A.

2004. The mitochondrial genome of the blowfly Chrysomya chloropyga (Diptera: Calliphoridae) Gene, 339:7–15.

28.

Kim

, Lee

E.M.

, Seol

K.Y.

, Yun

E.Y.

, Lee

Y.B.

, Hwang

J.S.

, Jin

B.R.

2006. The mitochondrial genome of the Korean hairstreak, Coreana raphaelis (Lepidoptera: Lycaenidae) Insect Mol Biol, 15:217–225.

29.

Kim

M.I.

, Baek

J.Y.

, Kim

M.J.

, Jeong

H.C.

, Kim

K.G.

, Bae

C.H.

, Han

Y.S.

, Jin

B.R.

, Kim

2009. Complete nucleotide sequence and organization of the mitogenome of the red-spotted apollo butterfly, Parnassius bremeri (Lepidoptera: Papilionidae) and comparison with other Lepidopteran insects. Mol Cells, 28:347–363.

30.

Kim

M.J.

, Wan

X.L.

, Kim

K.G.

, Hwang

J.S.

, Kim

2010. Complete nucleotide sequence and organization of the mitogenome of endangered Eumenis autonoe (Lepidoptera: Nymphalidae) African J Biotech, 9:735–754.

31.

Kim

S.R.

, Kim

M.I.

, Hong

M.Y.

, Kim

K.Y.

, Kang

P.D.

, Hwang

J.S.

, Han

Y.S.

, Jin

B.R.

, Kim

2009. The complete mitogenome sequence of the Japanese oak silkmoth, Antheraea yamamai (Lepidoptera: Saturniidae) Mol Biol Rep, 36:1871–1880.

32.

Koutinkova

, Andreev

, Subchev

, Tóth

, Szôcs

1999. Monitoring of the leafminer Leucoptera scitella Zell (Lepidoptera: Lyonetidae) by pheromene traps in Bulgaria. Acta Phytopathologica et Entomologica Hungarica, 34:327–331.

33.

Kristensen

N.P.

, Scoble

M.J.

, Karsholt

2007. Lepidoptera phylogeny and systematics: the state of inventorying moth and butterfly diversity. Zootaxa, 1668:699–747.

34.

Kristensen

N.P.

, Skalski

A.W.

1999. Phylogeny and paleontology. Lepidoptera: Moths and Butterflies, Evolution, Systematics, and Biogeography, Handbook of ZoologyVol IV, Part 35 Kristensen

N.P.

De Gruyter: Berlin and New York, 7–25.

35.

Lanave

, Preparata

, Saccone

, Serio

1984. A new method for calculating evolutionary substitution rates. J Mol Evol, 20:86–93.

36.

Lee

E.S.

, Shin

K.S.

, Kim

M.S.

, Park

, Cho

, Kim

C.B.

2006. The mitochondrial genome of the smaller tea tortrix Adoxophyes honmai (Lepidoptera: Tortricidae) Gene, 373:52–57.

37.

, Zhang

, Fan

, Yue

, Huang

, King

, Ran

2010. Structural characteristics and phylogenetic analysis of the mitochondrial genome of the sugarcane borer, Diatraea saccharalis (Lepidoptera: Crambidae) DNA Cell Biol, 00:1–6.

38.

Liao

, Wang

, Wu

, Li

Y.P.

, Zhao

, Huang

G.M.

, Niu

C.J.

, Liu

Y.Q.

, Li

M.G.

2010. The complete mitochondrial genome of the fall webworm, Hyphantria cunea (Lepidoptera: Arctiidae) Int J Biol Sci, 6:172–186.

39.

Liu

Y.Q.

, Li

Y.P.

, Pan

M.H.

, Dai

F.Y.

, Zhu

X.W.

, Lu

, Xiang

Z.H.

2008. The complete mitochondrial genome of the Chinese oak silkmoth, Antheraea pernyi (Lepidoptera: Saturniidae) Acta Biochim Biophys Sin, 40:693–703.

40.

Lowe

T.M.

, Eddy

S.R.

1997. tRNAscan-SE: a program for improved detection of transfer RNA genes in genomic sequence. Nucleic Acids Res, 25:955–964.

41.

Margam

V.M.

, Coates

B.S.

, Hellmich

R.L.

, Agunbiade

, Seufferheld

M.J.

, Sun

, Ba

M.N.

, Sanon

, Binso-Dabire

C.L.

, Baoua

, Ishiyaku

M.F.

, Covas

F.G.

, Srinivasan

, Armstrong

, Murdock

L.L.

, Pittendrigh

B.R.

2011. Mitochondrial genome sequence and expression profiling for the legume pod borer Maruca vitrata (Lepidoptera: Crambidae) PLoS One, 6:e16444.

42.

Mey

1994. Taxonomische Bearbeitung der westpalaearktischen Arten der Gattung Leucoptera Hübner, [1825], s.l. (Lep.: Lyonetiidae) Dtsch Entomologische Z N F, 41:173–234.

43.

Nardi

, Caeapelli

, Dallai

, Frati

2003. The mitochondrial genome of the olive fly Bactrocera oleae: two haplotypes from distant geographic locations. Insect Mol Biol, 12:605–611.

44.

Pan

M.H.

, Yu

Q.Y.

, Xia

Y.L.

, Dai

F.Y.

, Liu

Y.Q.

, Lu

, Zhang

, Xiang

Z.H.

2008. Characterization of mitochondrial genome of Chinese wild mulberry silkworm, Bomyx mandarina (Lepidoptera: Bombycidae) Sci China Ser C-Life Sci, 51:693–701.

45.

Posada

, Crandal

K.A.

1998. Modeltest: testing the model of DNA substitution. Bioinformatics, 14:817–818.

46.

Regier

J.C.

, Zwick

, Cummings

M.P.

, Kawahara

A.Y.

, Cho

, Weller

, Roe

, Baixeras

, Brown

J.W.

, Parr

, Davis

D.R.

, Epstein

, Hallwachs

, Hausmann

, Janzen

D.H.

, Kitching

I.J.

, Solis

M.A.

, Yen

S.H.

, Bazinet

A.L.

, Mitter

2009. Toward reconstructing the evolution of advanced moths and butterflies (Lepidoptera: Ditrysia): an initial molecular study. BMC Evol Biol, 9:280.

47.

Salvato

, Simonato

, Battisti

, Negrisolo

2008. The complete mitochondrial genome of the bag-shelter moth Ochrogaster lunifer (Lepidoptera: Notodontidae) BMC Genomics, 9:331–345.

48.

Schnare

M.N.

, Damberger

S.H.

, Gray

M.W.

, Gutell

R.R.

1996. Comprehensive comparison of structural characteristics in eukaryotic cytoplasmic large subunit (23S-like) ribosomal RNA. J Mol Biol, 256:701–719.

49.

Schultheis

A.S.

, Weigt

L.A.

, Hendricks

A.C.

2002. Arrangement and structural conservation of the mitochondrial control region of two species of Plecoptera: utility of tandem repeat-containing regions in studies of population genetics and evolutionary history. Insect Mol Biol, 11:605–610.

50.

Sheffield

N.C.

, Song

, Cameron

, Whiting

M.F.

2008. A comparative analysis of mitochondrial genomes in Coleoptera (Arthropoda: Insecta) and genome descriptions of six new beetles. Mol Biol Evol, 25:2499–2509.

51.

Simon

, Frati

, Bekenbach

, Crespi

, Liu

, Flook

. 1994. Evolution, weighting, and phylogenetic utility of mitochondrial gene sequences and a compilation of conserved polymerase chain-reaction primers. Ann Entomol Soc Am, 87:651–701.

52.

Solis

M.A.

1997. Michaelshaffera gen. n. - a pyraloid taxon lacking an abdominal tympanal organ (Lepidoptera: Pyralidae) Entomol Scand, 28:391–402.

53.

Stamatakis

2006. RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models. Bioinformatics, 22:2688–2690.

54.

Steinberg

, Cedergren

1994. Structural compensation in a typical mitochondrial tRNAs. Nat Struct Biol, 1:507–510.

55.

Taanman

J.W.

1999. The mitochondrial genome: structure, transcription, translation and replication. Biochim Biophys Acta, 1410:103–123.

56.

Tamura

, Peterson

, Stecher

, Nei

, Kumar

2011. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol, 28:2731–2739.

57.

Wei

S.J.

, Shi

, He

J.H.

, Sharkey

M.J.

, Chen

X.X.

2009. The complete mitochondrial genome of Diadegma semiclausum (Hymenoptera: Ichneumonidae) indicates extensive independent evolutionary events. Genome, 52:308–319.

58.

Wei

S.J.

, Tang

, Zheng

L.H.

, Shi

, Chen

X.X.

2010. The complete mitochondrial genome of Evania appendigaster (Hymenoptera: Evaniidae) has low A+T content and a long intergenic spacer between atp8 and atp6. Mol Biol Rep, 37:1931–1942.

59.

Wolstenholme

D.R.

1992. Animal mitochondrial DNA: structure and evolution. Int Rev Cytol, 141:173–216.

60.

Yang

, Wei

Z.J.

, Hong

G.Y.

, Jiang

S.T.

, Wen

L.P.

2009. The complete nucleotide sequence of the mitochondrial genome of Phthonandria atrilineata (Lepidoptera: Geometridae) Mol Biol Rep, 36:1441–1449.

61.

, Dang

J.P.

, Xie

L.D.

, Huang

2008. Complete mitochondrial genome of Teleogryllus emma (Orthoptera:Gryllidae) with a new gene order in Orthoptera. Zoolog Res, 29:236–244.

62.

Yukuhiro

, Sezutsu

, Itoh

, Shimizu

, Banno

2002. Significant levels of sequence divergence and gene rearrangements have occurred between the mitochondrial genomes of the wild mulberry silkmoth, Bombyx mandarina, and its close relative, the domesticated silkmoth, Bombyx mori. Mol Biol Evol, 19:1385–1389.

63.

Zhang

D.X.

, Szymura

J.M.

, Hewitt

G.M.

1995. Evolution and structural conservation of the control region of insect mitochondrial DNA. J Mol Evol, 40:382–391.

64.

Zhao

J.L.

, Zhang

Y.Y.

, Luo

A.R.

, Jiang

G.F.

, Cameron

S.L.

, Zhu

C.D.

2010. The complete mitochondrial genome of Spilonota lechriaspis Meyrick (Lepidoptera: Tortricidae) Mol Biol Rep, 38:3757–3764.

65.

Zuker

2003. Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res, 31:3406–3415.