The complete mitochondrial genome of the butterfly Apatura metis (Lepidoptera: Nymphalidae)

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1 Mol Biol Rep (2012) 39: DOI /s The complete mitochondrial genome of the butterfly Apatura metis (Lepidoptera: Nymphalidae) Min Zhang Xinping Nie Tianwen Cao Juping Wang Tao Li Xiaonan Zhang Yaping Guo Enbo Ma Yang Zhong Received: 24 July 2011 / Accepted: 24 January 2012 / Published online: 7 February 2012 Ó Springer Science+Business Media B.V Abstract 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 Electronic supplementary material The online version of this article (doi: /s ) contains supplementary material, which is available to authorized users. M. Zhang X. Nie T. Li E. Ma Research Institute of Applied Biology, Shanxi University, Taiyuan , People s Republic of China M. Zhang School of Life Sciences, Fudan University, Shanghai , People s Republic of China T. Cao (&) J. Wang Institute of Plant Protection, Shanxi Academy of Agriculture Science Research Institute of Applied Biology, Wucheng Road, Taiyuan , People s Republic of China ctwen@126.com X. Zhang Department of Immunology, Bethune Medical College, Jilin University, Changchun , China Y. Guo School of Life Sciences, Shanxi University, Taiyuan , People s Republic of China Y. Zhong (&) Institute of Biodiversity Science and Geobiology, Tibet University, Lhasa , People s Republic of China yangzhong@fudan.edu.cn 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. Keywords Apatura metis Genome architecture Lepidoptera Mitochondrial genome Nymphalidae Phylogeny Introduction To date, more than 180 insect mitochondrial genomes (mitogenomes) have been completely sequenced. These include 27 species from Lepidoptera, in which three are complete mitogenomes of Nymphalidae. The insect mtdna is generally a small double-stranded circular molecule of kb in length and has a remarkably conserved set of 37 genes: 13 proteincoding genes (PCGs), two ribosomal RNA genes (small- and large-subunit rrnas), and 22 trna genes [1]. In addition, it contains a major non-coding region known as the A? T-rich region. This region plays a role in the initiation of transcription and replication [1], and its length ranges from 70 to 13 kb [2 4].

2 6530 Mol Biol Rep (2012) 39: The structural organization of the insect mtdna A? T-rich region (also known as the control region) has also been investigated extensively [5 7]. Several advantages including their matrilineal inheritance, accelerated substitution rates, lack of extensive recombination, and being genome-level informative [8, 9] allow the mitochondrial genome to be widely used for population studies, phylogeographic analysis, molecular evolution at the genomic level, and phylogenetic relationships at various taxonomic levels across animal taxa, particularly in arthropods[10 14]. Nymphalidae, the largest family of butterfly, contains approximately 6,000 species and 12 subfamilies [15]. Though many researches have focused on the phylogenetic relationships in Nymphalidae by using mitochondrial and nuclear genes [16 19], the number of subfamilies and their phylogenetic relationships are still not very clear. In order to resolve the evolutionary relationships among Nymphalidae, mitogenome sequencing becomes a necessity rather than an alternative. As one of the most economically significant pests in the world, Apatura metis, also called Freyer s purple emperor, is very similar to the lesser purple emperor, A. ilia. It is smaller than A. ilia, always associated with water and certainly associated with the larval food plant slender leaved willow, Salix alba. A. metis is a species of the family Nymphalidae distributed in south-eastern Europe, Kazakhstan, Western Sibiria, Southwest China, Japan, Korea and Southern Russia [15]. In this study, as a representative of Nymphalidae, the complete mitogenome of A. metis was first documented. Then, the sequence and genome architecture of A. metis were also compared with Eumenis autonoe, Sasakia charonda, and Acraca issorie [20, 21], respectively. Finally, a phylogenetic analysis of 27 Lepidoptera species provides new perspectives for better understanding the relationship between Nymphalidae and Lepidoptera. Materials and methods Insect and genomic DNA extraction Adult A. metis was collected from Acheng, Heilongjiang Province, China in All samples were lived and initially preserved in small envelopes in the field, and then were transferred to -80 C for the long-term storage. We extracted the genomic DNA from muscle tissues of the thorax and legs through combining an OMEGA insect DNA kit and phenol chloroform extracting method together. The samples checked by 1% agarose gel electrophoresis were used for amplification. PCR amplification and sequencing Mitochondrial DNA fragments of the A. metis were amplified through using the long-and-accurate PCR kit (TaKaRa, China). 21 primers overlapping PCR fragments were shown in Table S1. Initially, some universal PCR primers and other primers that were relative to the species of Lepidoptera were synthesized after referred to Simon et al. and Hong et al. [22, 23]. Several primers including A? T-rich region were designed by the multiple sequence alignments of the complete mitochondrial genomes of the Lepidopterans, using Clustalx 1.83 [24] and Primer Premier 5.0 software [25], for the secondary PCRs. All PCRs were performed using TaKaRa LA Taq polymerase with the following cycling conditions: 1 min at 94 C, followed by 35 cycles of 20 s at 94 C; 1 min at C, 1 15 min at 68 C depending on the size of amplicons, and the subsequent final extension step at 72 C for 10 min. The quality of PCR products was evaluated via electrophoresis in 1.0% agarose gel. All fragments were purified by OMEGA PCR purification kit (OMEGA, USA). DNA sequencing was performed using an ABI 3730 semiautomated DNA sequencer (Applied BioSystems) at Invitrogen Inc. in Shanghai, China. All fragments were sequenced from both strands. Sequence analysis DNA sequences were assembled and proofread using the program Sequencher 4.14 (Gene Codes Corporation) [26]. Protein-coding and ribosomal RNA genes were identified based on their similarity to homologous genes in other species. Similarity searches of complete mtdna sequences in local databases and GenBank were performed using Clustalx 1.83 and NCBI BLAST network service [27], respectively. Identification of transfer RNA genes was conducted using trnascan-se 1.21 ( edu/trnascan-se/) [28]. Nucleotide composition, codon usage and length of the complete mitochondrial genome of A. metis were calculated by the Editseq program of DNASTAR (DNAStar Inc., Madison, WI, USA) and MEGA 4.0 program [29]. The putative control region was determined using Tandem Repeats Finder ( and DNAMAN software package (Version 5.2.2, Lynnon Biosoft, Canada) to locate regions with potential inverted repeats or palindromes. The GC- and AT-skews were used to determine the base compositional difference and strand asymmetry among the samples analyzed. Strand asymmetry was calculated using the formulas: AT-skew = [A - T]/ [A? T] and GC-skew = [G - C]/[G? C] [30]. Phylogenetic analysis Complete mitogenomes from GenBank of 27 Lepidoptera species (Table S2) were included in this analysis. The mitogenomes of Drosophila yakuba and Anopheles

3 Mol Biol Rep (2012) 39: gambiae were selected as outgroups. All the 13 proteinencoding genes were concatenated and aligned in Mega 4.0, removing the stop codons. Model selection was done with MrModeltest 2.3 and ModelTest 3.7 [31] for Bayesian inference and ML analysis, respectively. According to the Akaike information criterion (AIC), the GTR? I?G model was optimal for analysis with nucleotide alignments. MrBayes version 3.1.1[32] and a PHYML online web server [33] were employed to analyze this data set under the GTR? I?G model. In Bayesian inference, two simultaneous runs of 1,000,000 generations were conducted for the matrix. Each set was sampled as every 200 generations with a burnin of 25%. Trees inferred prior to stationarity were discarded as burn-in, and the remaining trees were used to construct a 50% majority-rule consensus tree. In ML analysis, the parameters were estimated during analysis and the node support values were assessed by bootstrap resampling calculated using 100 replicates. Results Genome organization and nucleotide composition The complete mtdna of A. metis is 15,236 bp in length. The typical 37 genes in insect mtdna, comprising 13 protein-encoding genes, two rrna genes (12S rrna and 16S rrna), 22 trna genes, and an A? T-riched region are found in the mitogenomes (Fig. 1; Table S3). DNA sequences were deposited in GenBank under accession number. 14 genes including 4 PCGs (ND5, ND4, ND4L, ND1), eight trna genes (trna Gln, trna Cys, trna Tyr, trna Phe, trna His, trna Pro, trna Leu(CUN), trna Val ) and two rrna genes are encoded on minority-strand (Table S3). The rested 23 genes are found in the majority-strand. One 394 bp long sequences which are identified as A? T- riched region are present between rrns and trna Met. In the A. metis mitogenome, a total of 36 bp gene overlap at 11 locations ranging in size from 1 to 8 bp. The intergenic spacer sequence in A. metis mitogenome is totally 162 bp in 12 regions, varying from 1 to 60 bp. The largest spacer consists of 60 bp, and it is located between trna Gln and ND2. The nucleotide composition of the A. metis is as follows: the 13 PCGs sequences have a A? T content of 78.9%, the 22 trna genes have 81.5% AT, the rrnl and rrns genes have 84.5 and 84.8% AT, respectively, and the control region has a A? T content of 92.9% (Table S4). The overall base composition of the whole mitochondrion sequence was A: 39.8%, T: 40.7%, C: 11.8%, and G: 7.7% in A. metis. Genes encoded on majority-strand and minority-strand had negative AT-skews (-0.4 and ) in A. metis (Table S4). Genes encoded on Fig. 1 Map of the mitogenome of A. metis. The trnas are denoted by the color blocks and are labeled according to the IUPACIUB single-letter amino acid codes. Gene name without underline indicates the direction of transcription from left to right, and with underline indicates right to left. Overlapping lines within the circle denote PCR fragments amplified used for cloning and sequencing minority-strand showed positive GC-skews while genes encoded on majority-strand had a negative GC-skews (Table S4). These results indicated the preference for these two nucleotides, a widespread characteristic of insect mtdnas. Protein coding genes Start codons in all protein-coding genes of A. metis follow the ATN rule, except cox1 gene, which has a CGA start codon. The start codon of CGA for the cox1 gene also presents in Artogeia melete, Bombyx mori, Eumenis autonoe, and so on. Seven protein-coding genes start with ATG (cox2, atp6, cox3, nad4, nad4 l, cytb, and nad1), four with ATT (nad2, atp8, nad3, and nad5), and one with ATA (nad6). Nine genes (cox1, atp8, atp6, cox3, nad4, nad6, cytb, nad1, andnad2) use the complete stop codon TAA (Table S3), and one gene (nad3) uses the complete stop codon TAG, whereas three genes have incomplete stop codons, T_(cox2, nad5, nad4). Transfer RNAs and ribosomal RNA genes The mtdna of A. metis includes standard 22 trna genes with anticodons representing 20 different amino acids. The respective predicted secondary structure of 22 trna genes are shown in Fig. S1. The 22 trna gene sequences in A. metis mitogenome ranged from 62 to 74 bp. All the secondary structure of trna genes, except trna Ser(AGN) that has a reduced dihydrouridine arm, fold into a typical

4 6532 Mol Biol Rep (2012) 39: clover-leaf structure. Wobble base pairs are fundamental in RNA secondary structure and are critical for the proper translation of the genetic code. A total of 25 unmatched base pairs present in 14 trna genes of the A. metis. 19of them are G U pairs, which form a weak bond. The remaining are U U mismatches at the AA stem of trna Ala and trna leu(uur), and the AC stem of trna ser(ucn) and trna Thr, respectively (Fig. S1). The lengths of the SrRNA and lrrna genes presented in A. metis are 779 and 1,333 bp, respectively. The A? T content were 84.5% for lrrna and 84.8% for srrna. Like all other sequenced insect mitogenomes, lrrna and srrna are located at trna Leu(CUN), trna Val and trna Val control region, respectively (Fig. S1; Table S3). Control region The A? T-rich region of A. metis mitogenome, flanked by SrRNA and trna Met, has 394 nucleotides (Fig. 2). The A? T-rich region harbors the highest A? T content (92.9%) of any region of the A. metis mitogenome. The size of the A? T-rich region is well within the range detected in the observed Nymphalidae insects ( bp). In the A. metis, a duplicated 27 bp repeat element is present from 15,049 to 15,112 and repeats two times. The majority of the A. metis A? T-rich region contains a poly thymidine stretch that is highly conserved in Lepidoptera, a poly-a stretch, a microsatellite-like TA repeat and non-repetitive sequence. In A. metis, the origin of minority or light strand replication (O N ) is located downstream from srrna gene, which contains the motif ATAGA followed by a 19 bp poly-t stretch. A microsatellite (AT) 10 element presents in the 3 0 end of the A. metis A? T-rich region, which is also found in other lepidopteran mitogenomes. Finally, the poly-a stretch that is located at the 5 0 -end of the A? T-rich region (upstream of trna Met ), is comprised of a total of nine A nucleotides. Phylogenetic analysis For the 27 species, there are 11,202 sites in the matrix (containing all three codon positions for PCGs) to reconstruct phylogenetic relationships of superfamily level in Lepidoptera, and phylogenetic relationships of family level in Papilionoidea. The ML tree obtained by Phylip is identical to the Bayesian tree obtained by MrBayes (Figs. 3, 4). Using Drosophila yakuba and Anopheles gambiae as outgroups, the phylogenetic tree revealed five main clades. The species of the superfamilies Noctuoidea, Geometroidea, Bombycoidea, Pyraloidea, Tortricoidea, and Papilionoidea cluster monophyletic groups, respectively, with strongly supported (bootstrap values [92% and Bayesian posterior probability values are 1.00). Notuoidea (clade 1) contains species of the families Arctiidae, Lymantriidae, Notodontidae, and Noctuidae. The phylogenetic relationships of the four families are shown as ((Notodontidae? Lymantriidae)? Arctiidae)? Noctuidae. Clade 2 contains species of the superfamilies Geometroidea and Bombycoidea. These two superfamlies are sister groups. The Bombycoidea clade is divided into three subclades and two of which are strongly supported (bootstrap values are 100% and Bayesian posterior probability values are 1.00). The three subclades are the Manduca sexta of Sphingidae, the Bombyx mori and Japanese Bombyx mandarina of the Bombycidae, and the Saturnia boisduvalii of Sphingidae and the three species of Saturniidae. In the Pyraloidea (clade 3), The Diatraea saccharalis of Crambidae is closer to the Ostrinia furnacalis and O. nubilalis which are also species of Crambidae. Papilionoidea (clade 4) includes eight species from four families. The phylogenetic relationships of the four families are ((Pieridae? Lycaenidae)? Nymphalidae)? Papilionidae. And finally, the species Adoxophyes honmai, Grapholita molesta, and Spilonota lechriaspis of the family Tortricidae clustered together to form the Tortricoidea clade (clade 5). Fig. 2 The structure of the A? T-rich region of the A. metis mitochondrial genome

5 Mol Biol Rep (2012) 39: Fig. 3 Phylogenetic tree of 27 species of Lepidoptera. ML analysis inferred from all codon positions of 13 PCGs. Bootstrap support values are indicated at each node Fig. 4 Phylogenetic tree of 27species of Lepidoptera. Bayesian analysis inferred from all codon positions of 13 PCGs. Bayesian posterior probabilities are indicated at each node Discussion The size of the complete mitogenome of A. metis is well within the range observed in the Nymphalidae species, with size varying from 15,245 in Acraea issoria and Sasakia charonda to 15,489 in Eumenis autonoe (Table S5) [20, 21]. The variable number of repeats in the control regions mainly results in the size differences among sequenced Nymphalidae mitogenomes. The mitogenomes of the four Nymphalidae butterflies have the same genome content (37 genes and one control region), gene order and orientation. The mitochondrial gene arrangement of trni, trnq, and

6 6534 Mol Biol Rep (2012) 39: trnm in Nymphalidae, which differs from those of D. yakuba, is: trna Met, trna Ile, and trna Gln. This result supports the viewpoint of that the mitochondrial gene arrangement in lepidopteran insects evolved independently after splitting from its stem lineage [20, 21, 34, 35]. The A? T content (80.5%) of the A. metis mitogenome is much closer to the observed for other lepidopteran insects. The A? T-rich regions of the four sequenced Nymphalidae species have the highest A? T content in the different mitochondrial regions. The A? T content of trna and rrna genes of other three Nymphalidae species are higher than those of protein-coding genes (Table S5). The analysis of the base composition at each codon position of the A. metis mitogenome demonstrates that the A? T content of the third codon positions (91.5%) is higher than the first codon positions (74.6%) and the second codon positions (70.7%). These results suggest that the A? T bias may be caused by GC towards AT mutation and nature selective. Six of the protein-coding genes of A. metis (ND2, COI, COII, ND3, ND5, and ND1) are flanked by trna genes on both the and ends. Four of the PCGs of the A. metis (ATP8, ATP6, ND4L, and ND6) are next to another PCG at their 3 0 -end. These four PCGs and their adjacent PCGs are arranged as ATP8-ATP6, ATP6-COIII, ND4L-ND4, and ND6-CytB. These results are similar to those observed from other Nymphalidae species. In addition, in the mitogenomes of the species A. metis, E. autonoe, and A. issoria, seven of the PCGs (COII, ATP6, COIII, ND4, ND4L, CytB, and ND1) have the same Met start codons (ATG) (Table S6), and one of the PCGs (ND2) starts with the ATT codons. In A. metis and E. autonoe, the start codons for ATP8, ND3, and ND5 are the Ile start codons (ATT or ATC). The protein-coding genes ATP8 and ND5 of the A. issoria have the Met start codons (ATG or ATA). However, the ND3 gene uses ATT as the start codon. ND6 of A. metis and A. issoria look forward the Met codon (ATA) as their start codons, while E. autonoe sees Ile codon (ATT) as its start codon. However, the initiation codons of COI gene are different from those of other genes in insects. The COI in lepidopteran insects often starts with CGA. Some researchers argued that the initiation codon CGA of COI gene is used to encode the amino acid Arg. In this study, the COI of A. metis and E. autonoe start with CGA, and that of A. issoria starts with TTG, respectively. The stop codons TAA and TAG were inferred for all coding sequences except cox2, nad5, and nad4 in A. metis and E. autonoe as well as cox1 in A. issoria. The typical 22 trna genes in four Nymphalidae species have the same gene arrangement. The length of trnas in four Nymphalidae species ranges from 60 to 74 bp. The anticodons of the A. metis trnas are identical to those in E. autonoe, A. issoria, and S. charonda as well as other insects [20, 21]. The trnas of the four Nymphalidae species show conservation especially in their acceptor and anticodon stems and being more variable in the D and T loops. The trna Ser(AGN) of four Nymphalidae species have the absence of the dihydrouridine (DHU) arm as other insects [36]. Most of the variational stems from the DHU and T arms have the common stem size (3 5 bp, except for trna Ser(AGN) ). The unmatched base pairs (U U and U G) are scattered in the DHU arms and T w C arms of different trnas. At present, some scholars argued that these unmatched phenomenons in the mitogenome may be induced by the deficiency of reorganization mechanism in the mitochondrial DNA, which was difficult to remove these mutations [37]. The lengths of the two rrnas in A. metis are similar to those of other lepidopterans. And, the sizes of rrnl and rrns in A. metis (1,333 and 779 bp, respectively) are similar to those of E. autonoe (1,335 and 775 bp), S. charonda (1,311 and 775 bp), and A. issoria (1,331 and 788 bp) [20, 21]. However, the sequence variation in some regions seems too high to carry out meaningful structural comparisons. The 394 bp long A? T-rich region of the A. metis mitogenome is the second shortest among the four completely sequenced Nymphalidae species, after the 380 bp long S. charonda. In this study, we found that the complete mitogenome is long when the A? T-rich region is long, suggesting that length of A? T-rich region plays an important role in affecting length of mitogenome. The A? T contents of this region in the A. metis, S. charonda, and E. autonoe are more than 90%, whereas that of A. issoria is 88.2%. In four Nymphalidae species, the O N includes a motif ATAGA or ATAGAA followed by a poly- T stretch (17 19 bp). A microsatellite (AT) 10 element, which is also found in other Nymphalidae species mitogenomes, harbors in the 3 0 end of the A. metis A? T-rich region. The microsatellite-like TG (TA) 9 element is observed in A. issoria and S. charonda. The poly-a stretches in the A. metis, A. issoria, and S. charonda vary in length ranging from 6 to 11 bp whereas the E. autonoe poly-a stretch is 11 bp in length, which is interrupted by one G nucleotide. In E. autonoe, the A? T-rich region has a tandem repeat consisting of 10 duplicated and identical 27 bp copies. Only two 27 bp repeats are observed in A. metis, two 17 bp repeats harbor in S. charonda, and two 22 bp repeats are found in A. issoria, respectively. In this study, we reconstructed the phylogenetic tree of 27 lepidopteran species based on the 13 PCGs of complete mitogenomes. The phylogenetic relationship of the six superfamilies is shown as: (((Noctuoidea? (Geometroidea? Bombycoidea))? Pyraloidea)? Papilionoidea)? Tortricoidea. This result is identical to the point of Kim et al. [21] who reconstructed the phylogenetic tree of 17 lepidopteran insects. In clade 1, Lymantriidae is sister to Notodontidae with very strong support (bootstrap value is

7 Mol Biol Rep (2012) 39: % and Bayesian posterior probability values are 1.00). Arctiidae clusters to the two families Lymantriidae and Notodontidae, and they have a further relationship with Noctuidae. Based on morphological classification, most of researches accepted that the Pieridae were considered as a sister to the Nymphalidae and Lycaenidae group. Kim et al. [21] obtained a result deviated from the traditional view, in which the Pieridae was sister to Lycaenidae with good support (100% by BI and 99 or 78% by ML). In Papilionoidea group, our conclusion is ((Lycaenidae? Pieridae)? Nymphalidae)? Papilionidae, which is the same as Kim et al. [21]. We investigated nine species placed in the four Papilionoidea families, while Kim involved four species belonging to the same four families. However, the node support at the (Lycaenidae? Pieridae) clade reduced from 1.00 to 0.74 by BI as well as from 78 to 58% by ML. 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