Gene 515 (2013) 349–358

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Gene

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

Xiushuai Yang a,b, Dayong Xue a, Hongxiang Han a,⁎ a Key Laboratory of Zoological Systematics and Evolution, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China b Graduate University of the Chinese Academy of Sciences, Beijing 100049, China article info abstract

Article history: The complete mitochondrial genome (mitogenome) of the Chinese pistacia looper Biston panterinaria was Accepted 1 November 2012 sequenced and annotated (15,517 bp). It contains the typical 37 genes of mitogenomes and a high Available online 5 December 2012 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 Keywords: dehydrogenase 4 (ND4) use a single T. All transfer RNA (tRNA) genes have the typical clover-leaf structure with Mitochondrial genome the exception of tRNASer(AGN). We reconstructed a preliminary mitochondrial phylogeny of six ditrysian super- Biston panterinaria Molecular phylogeny families and performed comparative analyses of inference methods (Bayesian Inference (BI), Maximum Likeli- hood (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. © 2012 Elsevier B.V. All rights reserved.

1. Introduction 2009b; Hao et al., 2012; Jiang et al., 2009; Kim et al., 2009b, 2011; Mahendran et al., 2006), mitogenomes are also important in various Mitochondrial genomes (mitogenomes) of are typically scientific disciplines, such as comparative and evolutionary genomics double-stranded and circular molecules that span 14–20 kb. They con- (Ballard, 2000; Ballard and Rand, 2005; Papanicolaou et al., 2008), tain 13 protein coding genes (PCGs), 2 ribosomal RNA (rRNA) genes, molecular evolution (Brower, 1994; Zakharov et al., 2004), phylo- and 22 transfer RNA (tRNA) genes (Boore, 1999; Wolstenholme, geography (Avise, 2000), and population genetics (Avise et al., 1987; 1992), as well as a non-coding region known as the A+T rich or control Hurst and Jiggins, 2005). region, which participates in the initiation of transcription and replica- Lepidoptera is one of the world largest orders, second only tion (Taanman, 1999; Zhang and Hewitt, 1997; Zhang et al., 1995). In to Coleoptera, with approximately 157,424 described species (van addition to their application in the reconstruction of phylogenetic rela- Nieukerken et al., 2011). The controversial issues of the phylogenetic re- tionships among insects (Cameron and Whiting, 2008; Dowton et al., lationships in the Lepidoptera have been intensively discussed, at both deep-level and low-level (Fibiger et al., 2010; Mutanen et al., 2010; Sihvonen et al., 2011; Wahlberg et al., 2005; Zahiri et al., 2011, 2012). Abbreviations: Mitogenome, mitochondrial genome; PCGs, protein coding genes; Increasing numbers of molecular markers have been exploited for phy- – ATP6 and ATP8, ATP synthase subunits 6 and 8 genes; COX1-3, cytochrome c oxidase 1 logenetic analyses. These markers include mitochondrial genes large 3genes;CYTB,cytochromeb gene; ND1-6 and ND4L, NADH dehydrogenase subunits 1–6 and 4L genes; rRNA, ribosomal RNA; lrRNA and srRNA, large and small subunit ribo- subunit ribosomal RNA (lrRNA) (Pashley and Ke, 1992), small subunit somal RNA; tRNA, transfer RNA; PCR, polymerase chain reaction; DHU, dihydrouridine; ribosomal RNA (srRNA) (Niehuis et al., 2006), cytochrome c oxidase 1 TΨC, pseudouridine; BI, Bayesian Inference; ML, Maximum Likelihood; MP, Maximum (COX1) (Wahlberg et al., 2005; Wu et al., 2010), COX2 (Braby et al., Parsimony; BP, bootstrap percentage. 2005), cytochrome b (CYTB) (Li et al., 2005), NADH dehydrogenase 1 ⁎ Corresponding author at: Key Laboratory of Zoological Systematics and Evolution, (ND1) (Martin and Pashley, 1992; Weller et al., 1994), and ND5 (Yagi Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China. Tel.: +86 10 64807230; fax: +86 10 64807099. et al., 1999), as well as the nuclear genes 18S (Weller et al., 1992), 28S E-mail address: [email protected] (H. Han). (Abraham et al., 2001), CAD (Regier et al., 2008b; Zwick, 2008), DDC

0378-1119/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.gene.2012.11.031 350 X. Yang et al. / Gene 515 (2013) 349–358

(Regier et al., 2001, 2002, 2005), EF-1α (Mitchell et al., 1997, 2006), Table 1 enolase (Regier et al., 2008a), GAPDH (Wahlberg and Wheat, 2008; Statistics of the data matrices in the study. Wahlberg et al., 2009), IDH (Sihvonen et al., 2011; Zwick et al., 2011), Dataset name Number of nucleotides (bp) MDH (Zahiri et al., 2011, 2012), RpS5 (Mutanen et al., 2010), and PCG12MU 7790 wingless (Kawahara et al., 2009). Molecular phylogenetic studies of PCG12CW 7748 the Lepidoptera used to rely on a combination of two or more of the PCG12MA 7740 aforementioned molecular markers, and different markers or different PCG123MU 11,685 taxon samplings result in inconsistencies (Heikkilä et al., 2012; PCG123CW 11,622 PCG123MA 11,650 Mitchell et al., 1997, 2006; Mutanen et al., 2010; Regier et al., 2009; Wahlberg et al., 2005; Weller et al., 1994; Zahiri et al., 2011). Recently, the expansion of molecular markers and taxon samplings in the Lepi- doptera, especially in the superfamilies Bombycoidea, Noctuoidea, and in July 2011. Three legs from one side of the body were removed Papilionoidea, has greatly improved the resolution of some ambiguous and preserved in 100% ethanol during collection. After transport to phylogenetic relationships (Heikkilä et al., 2012; Mutanen et al., 2010; the laboratory, the legs were stored at −20 °C until DNA extraction, Regier et al., 2009; Zahiri et al., 2011; Zwick et al., 2011). However, and the specimens were deposited at the Museum of the Institute of many relationships remain poorly resolved. Zoology, Chinese Academy of Sciences. Total genomic DNA was Considerable efforts have been expended in the past decade extracted from the leg muscle tissue using a Qiagen DNeasy Blood & to reconstruct the evolutionary relationships using mitogenomes Tissue Kit (69506). (Cameron et al., 2007, 2009; Dowton et al., 2009a; Fenn et al., 2008; Kawaguchi et al., 2001; Minegishi et al., 2005; Miya et al., 2003; 2.2. Polymerase chain reaction (PCR) amplification and sequencing Song et al., 2010; Wiegmann et al., 2011). A range of phylogenetic approaches such as inference methods, partitioning strategies, align- The entire genome was amplified using standard PCR methods with ment methods, and gene exclusion have been tested to find the most a set of universal primers for the lepidopteran mitogenome (Folmer appropriate methods for analyzing mitogenomes, especially in insects et al., 1994; Lee et al., 2006; Simon et al., 1994, 2006; Zhao et al., (Cameron et al., 2009; Dowton et al., 2009a; Fenn et al., 2008; Song 2011; Zhu et al., 2010). Then, perfectly matched primers for internal et al., 2010; Wiegmann et al., 2011). In these studies, three of the four fragments were designed based on the sequences amplified by the most diverse holometabolous insect orders (Coleoptera, Diptera, Lepi- universal primers. Short PCRs were performed using Takara Taq™ doptera, and Hymenoptera) have been examined (Cameron et al., DNA polymerase (DR001B) under the following cycling parameters: 2007; Dowton et al., 2009a; Song et al., 2010). The number of complete 94 °C for 2 min; 35 cycles of 30 s at 94 °C, 30 s at 53 °C, and 1 min at or nearly complete lepidopteran mitogenomes in the GenBank has rap- 72 °C; and 72 °C for 10 min. Long PCRs were performed using Takara idly increased to 138, comprising 47 species from 6 superfamilies (as of LA Taq Hot Start Version DNA polymerase (DRR042A) under the follow- March, 2012). This large amount of public domain data offers greater ing cycling parameters: 94 °C for 2 min; 35 cycles of 30 s at 94 °C, 30 s possibilities for improving phylogenetic analyses. In the current paper, at 45–55 °C, and 12 min at 68 °C; and 68 °C for 10 min. The PCR prod- we present a comparative analysis of mitochondrial phylogeny within ucts were detected via 1.0% agarose gel electrophoresis, purified using a the Lepidoptera. 3S Spin PCR Product Purification Kit, and sequenced using ABI PRISM Biston panterinaria is one of the most economically important pests 3730xl capillary sequencers. The sequences of the universal primers widely distributed in China and Southeast Asia. The larvae feed on are listed in Table A.1. many field crops, fruit trees, and vegetables, causing serious damage. B. panterinaria belongs to the second largest lepidopteran family 2.3. Sequence analysis and annotation Geometridae, which contains approximately 23,002 described spe- cies, and has only one complete mitogenome available (Phthonandria The raw sequences were run using the NCBI BLAST program to de- atrilineata)(van Nieukerken et al., 2011). In this paper, we determined termine sequence homology, and were then aligned into contigs the complete mitogenome of B. panterinaria, and compared it with using ChromasPro (www.technelysium.com.au/ChromasPro.html). other lepidopteran mitogenomes to analyze characteristics, such as Thirteen PCGs were first identified using an open reading frame genome composition, nucleotide content, and codon usage. Phyloge- (ORF) finder (www.ncbi.nlm.nih.gov/gorf/orfig.cgi), specifying the netic analyses were conducted using 45 lepidopteran mitogenomes invertebrate mitochondrial genetic code. Then, the identified PCGs to resolve the intra-ordinal relationships. To enhance our understand- were calibrated by sequence similarity using published lepidopteran ing of the phylogenetic utility of the mitogenomes, we tested the ef- mitogenome sequences. Nineteen tRNA genes were identified using fects of three inference methods (Bayesian Inference (BI), Maximum tRNAscan-SE version 1.21 (lowelab.ucsc.edu/tRNAscan-SE/) (Lowe Likelihood (ML) and Maximum Parsimony (MP)), different dataset and Eddy, 1997), specifying mito/chloroplast DNA as the source and compositions (PCGs, including and excluding 3rd codon positions) choosing the invertebrate mito genetic code for tRNA isotype predic- and alignment methods (Muscle, Clustal W, and MAFFT). Finally, we tion. The other three tRNA genes that were not found by tRNAscan-SE created six datasets for the comparative analyses, which included were identified visually by comparing them with other lepidopteran three datasets consisting of 13 PCGs without 3rd codon positions mitogenomes, and the secondary structures were drawn manually. and aligned by Muscle (PCG12MU), Clustal W (PCG12CW), and The putative control region was defined by aligning it with the closely MAFFT (PCG12MA) and three datasets consisting of 13 PCGs with related species P. atrilineata (Yang et al., 2009). The nucleotide 3rd codon positions and aligned by Muscle (PCG123MU), Clustal W composition and codon usage were calculated using MEGA 5.05 (PCG123CW), and MAFFT (PCG123MA). The statistics of the data ma- (Tamura et al., 2011). trices are listed in Table 1. 2.4. Phylogenetic analyses 2. Materials and methods Phylogenetic analyses were conducted based on the 13 PCGs of the 2.1. Specimen sampling and DNA extraction complete mitogenome of B. panterinaria and other 44 lepidopteran taxa downloaded from the GenBank. Each of the 13 PCGs was aligned Adult specimens of B. panterinaria were collected from Tianmu by the three methods (Muscle, Clustal W, and MAFFT) under default Mountain (30° 32.441′ N, 119° 44.816′ E), Zhejiang Province, China, settings (Edgar, 2004; Katoh et al., 2002; Tamura et al., 2011; X. Yang et al. / Gene 515 (2013) 349–358 351

Thompson et al., 1994). The nucleotide sequences of the 13 PCGs by MrModeltest 2.3, and node support was conducted with 100 boot- (excluding the stop codons) were translated into amino acid sequences strap replicates (Guindon et al., 2010). Nodes with the bootstrap using invertebrate mitochondrial genetic code before alignment with percentage (BP) of at least 70% were considered well-supported in MP Muscle and Clustal W (implemented in MEGA 5), and then back- and ML analyses (Hillis and Bull, 1993). translated into nucleotide sequences after alignment (Cameron et al., 2006; Fenn et al., 2008; Tamura et al., 2011). The alignments were 3. Results and discussion concatenated using the TREEFINDER program (Jobb et al., 2004). Phylo- genetic analyses were subsequently performed using BI, ML, and MP. 3.1. Genome organization and base composition BI analysis was conducted using MrBayes version 3.1.2 (Ronquist and Huelsenbeck, 2003). The datasets were divided into three parti- The complete mitogenome of B. panterinaria is a closed-circular tions: 1st, 2nd, and 3rd codon positions. Model selection was conducted molecule of 15,517 bp in length, with the typical gene content as via comparison of Akaike Information Criterion using MrModeltest other known lepidopterans: 13 PCGs, 22 tRNA genes, 2 rRNA genes 2.3 (Huelsenbeck and Ronquist, 2001; Nylander et al., 2004). The and 1 major non-coding region (control region) (Fig. 1). The sequence GTR+I+G model was chosen as the best-fit model for all partitions. file was imported into Geneious (Drummond et al., 2011) to map the Adoxophyes honmai (Tortricoidea) was set as the outgroup (Lee et al., complete mitogenome. The sequence was deposited in the GenBank 2006). Four independent runs were conducted for 1,000,000 genera- under the accession number JX406146. tions. Each set was sampled every 1000 generations with a burnin of The mitogenome gene order of B. panterinaria is the same as other 25%. Posterior probabilities over 0.9 were interpreted as strongly- previously sequenced ditrysians and also contains the typical rear- supported (Mutanen et al., 2010). MP analysis was conducted using rangement of tRNAMet (Cameron and Whiting, 2008; Kim et al., 2006, the program PAUP* 4.0 beta 10 Win with all sites weighted equally 2009a). However, the rearrangement is not found in the newly deter- (Swofford, 1993). Three Tortricoidea species were set as outgroups mined Hepialidae mitogenomes (Cao et al., 2012), which character- (Lee et al., 2006; Son and Kim, 2011; Zhao et al., 2011). A heuristic ize the ancestral gene order tRNAIle–tRNAGln–tRNAMet (Boore, 1999; search was initially performed to find the shortest tree using 1000 Flook et al., 1995). random taxon replicates. Gaps were treated as missing data. Nodal The J-strand of the B. panterinaria mitogenome is composed of 23 support was assessed with 1000 bootstrap replicates. ML analysis was genes, whereas the N-strand hosts the remaining 14 genes (Table 2). conducted using the PhyML program with the optimal model selected The nucleotide composition of the J-strand is 42.3% A, 37.2% T, 12.9%

Fig. 1. Map of the B. panterinaria mitogenome. Gene orientations are indicated by the arrow directions. PCGs are denoted in blue, transfer RNA genes in pink, rRNA genes in green and the control region in red. The abbreviations for the genes are as follows: ATP6 and ATP8 refer to ATP synthase subunits 6 and 8 genes, COX1-3 refer to the cytochrome oxidase subunit 1–3 genes, CYTB refers to cytochrome b gene, and ND1-6 and ND4L refer to NADH dehydrogenase subunit 1–6 and 4L genes. tRNA genes are denoted as three-letter symbols according to the IUPAC-IUB amino acid codes. 352 X. Yang et al. / Gene 515 (2013) 349–358

Table 2 Organization of the B. panterinaria mitochondrial genome.

Gene Direction Location Size(bp) Intergenic Anticodon Start Stop codon nucleotides codon

tRNAMet F1–68 68 CAT tRNAIle F69–133 65 0 GAT tRNAGln R 138–206 69 4 TTG ND2 F 261–1274 1014 54 ATT TAA tRNATrp F 1279–1347 69 4 TCA tRNACys R 1340–1409 70 −8 GCA tRNATyr R 1410–1476 67 0 GTA COX1 F 1485–3020 1536 8 CGA TAA tRNALeu(UUR) F 3016–3086 71 −5 TAA COX2 F 3087–3768 682 0 ATG T tRNALys F 3769–3839 71 0 CTT tRNAAsp F 3840–3905 66 0 GTC ATP8 F 3906–4070 165 0 ATT TAA ATP6 F 4064–4741 678 −7 ATG TAA COX3 F 4756–5544 789 14 ATG TAA tRNAGly F 5551–5615 65 6 TCC ND3 F 5616–5969 354 0 ATT TAG tRNAAla F 5992–6061 70 22 TGC tRNAArg F 6068–6130 63 6 TCG tRNAAsn F 6138–6203 66 7 GTT tRNASer(AGN) F 6206–6271 66 2 GCT tRNAGlu F 6299–6366 68 27 TTC tRNAPhe R 6365–6430 66 −2 GAA ND5 R 6414–8168 1755 −17 ATT TAA tRNAHis R 8169–8237 69 0 GTG ND4 R 8238–9576 1339 0 ATG T ND4L R 9592–9882 291 15 ATG TAA tRNAThr F 9885–9949 65 2 TGT tRNAPro R 9950–10,014 65 0 TGG ND6 F 10,017–10,553 537 2 ATT TAA CYTB F 10,592–11,743 1152 38 ATG TAA tRNASer(UCN) F 11,750–11,815 66 6 TGA ND1 R 11,838–12,782 945 22 ATA TAA tRNALeu(CUN) R 12,777–12,837 61 −6 TAG lrRNA R 12,838–14,311 1474 0 tRNAVal R 14,312–14,381 70 0 TAC srRNA R 14,382–15,168 787 0 Control region 15,169–15,517 349 0

C, and 7.6% G. The A+T content of B. panterinaria is 79.5%, which is study (Salvato et al., 2008). Codon usage of PCGs shows a significant within the range of known lepidopteran mitogenomes from 77.8% bias of high A+T content, which plays a major role in the A+T bias (Ochrogaster lunifer) to 82.7% (Coreana raphaelis). The B. panterinaria of the entire mitogenome. mitogenome harbors 17 short non-coding regions with a total of 243 bp. Six gene junctions with 45 bp overlap are observed. The largest 3.3. Transfer and ribosomal RNA genes one is a 17 bp overlap located between tRNAPhe and ND5 genes. A sim- ilarly sized overlap in the same location has been reported in some The typical set of 22 tRNA genes that ranged from 61 to 71 bp were other lepidopteran mitogenomes (Hong et al., 2008; Jiang et al., 2009; identified in the mitogenomes of B. panterinaria. Fourteen tRNA genes Son and Kim, 2011; Zhao et al., 2011). The other five overlaps span are located on the J-strand and eight on the N-strand. Anticodons of less than 10 bp each. all tRNA genes are identical across the Lepidoptera. Twenty-one tRNA genes were predicted with a characteristic cloverleaf secondary struc- 3.2. Protein-coding genes and codon usage ture except tRNASer(AGN), which lacked the dihydrouridine (DHU) arm (Fig. A.1). A total of 28 pairs of mismatches were found in 19 tRNA The mitogenome of B. panterinaria contains the canonical PCGs genes, with 9 in the amino acid acceptor stems, 8 in the DHU stems, 7 of metazoans with the typical order in insects (Boore, 1999; in the pseudouridine (TΨC) stems and 4 in the anticodon stems. The Wolstenholme, 1992). All PCGs are predicted to utilize the standard mismatched bases show significant bias, with 18 U–G, 5 U–U, 3 A–A, ATN start codons except for COX1, which initiates with CGA (Table 2). and 2 A–Cmismatches. This is the most common putative start codon for the COX1 gene of Two rRNA genes were identified on the N-strand in the the lepidopteran mitogenomes (Liao et al., 2010; Yang et al., 2009; B. panterinaria mitogenome with the lrRNA gene located between Yukuhiro et al., 2002; Zhu et al., 2010). Eleven PCGs are predicted to uti- tRNALeu(CUN) and tRNAVal, and the srRNA gene between tRNAVal lize complete stop codons TAA or TAG, and the other two genes (COX2 and the control region. The lrRNA gene is 1474 bp long, which is the and ND4) only have a T-nucleotide as stop codons (Table 2). longest one of the known mitogenomes in the Lepidoptera with 85.5% Codon usage of the B. panterinaria mitogenome is presented in A+T content. The shortest lrRNA gene of the known species is Table 3. Two codons (ACG and CGC) were not presented. The mostly 1234 bp (Troides aeacus). The srRNA gene is 787 bp long with 84.8% used codons (ATT-Ile, TTT-Phe, TTA-Leu, AAT-Asn, TAT-Tyr, and A+T content. The length of the srRNA gene among the lepidopteran ATA-Met) are composed of T or A+T, whereas the least used codons mitogenomes ranges from 434 bp (Ostrinia nubilalis) to 830 bp (Phalera (CTC-Leu, CCG-Pro, AGC-Ser, AGG-Ser and so on) have a high content flavescens)(Coates et al., 2004). Only one (TA)n microsatellite was of G+C. The relative synonymous codon usage (RSCU) exhibits exten- found in the lrRNA gene with 10 replications and none in the srRNA sive similarity with other lepidopteran mitogenomes in a previous gene. X. Yang et al. / Gene 515 (2013) 349–358 353

Table 3 Codon usage of protein-coding genes in the B. panterinaria mitochondrial genome.

Codon No. of RSCU Codon No. of RSCUa codons codons

TTT(Phe) 302 1.73 TAT(Tyr) 171 1.70 TTC(Phe) 48 0.27 TAC(Tyr) 30 0.30 TTA(Leu) 439 4.82 TAA(b) 12 1.82 TTG(Leu) 38 0.42 TAG(b) 1 0.18 CTT(Leu) 22 0.24 CAT(His) 44 1.31 CTC(Leu) 1 0.01 CAC(His) 23 0.69 CTA(Leu) 45 0.49 CAA(Gln) 58 1.87 CTG(Leu) 2 0.02 CAG(Gln) 4 0.13 ATT(Ile) 393 1.78 AAT(Asn) 227 1.69 ATC(Ile) 48 0.22 AAC(Asn) 41 0.31 ATA(Met) 267 1.73 AAA(Lys) 95 1.81 ATG(Met) 42 0.27 AAG(Lys) 10 0.19 GTT(Val) 78 2.08 GAT(Asp) 44 1.49 Fig. 2. Organization of the B. panterinaria mitochondrial control region. GTC(Val) 2 0.05 GAC(Asp) 15 0.51 GTA(Val) 62 1.65 GAA(Glu) 61 1.63 GTG(Val) 8 0.21 GAG(Glu) 14 0.37 TCT(Ser) 86 2.15 TGT(Cys) 38 1.85 TCC(Ser) 12 0.30 TGC(Cys) 3 0.15 inference methods, dataset compositions, and alignment methods. TCA(Ser) 99 2.47 TGA(Trp) 85 1.81 Among these three factors, inference methods and dataset compo- TCG(Ser) 4 0.10 TGG(Trp) 9 0.19 sitions significantly affected our analyses, whereas the alignment CCT(Pro) 68 2.25 CGT(Arg) 18 1.36 methods had limited effect. CCC(Pro) 18 0.60 CGC(Arg) 0 0 CCA(Pro) 34 1.12 CGA(Arg) 33 2.49 By comparing the nodal support values across all datasets, BI analy- CCG(Pro) 1 0.03 CGG(Arg) 2 0.15 sis was found to be superior to ML analysis, which was superior to MP ACT(Thr) 71 1.86 AGU(Ser) 41 1.02 analysis, and these conclusions were already proved in the Diptera ACC(Thr) 14 0.37 AGC(Ser) 1 0.03 (Cameron et al., 2007). Topological differences among the three infer- ACA(Thr) 68 1.78 AGA(Ser) 76 1.90 ence methods were also apparent. Phylogenetic analyses of all datasets ACG(Thr) 0 0 AGG(Ser) 1 0.03 GCT(Ala) 57 1.85 GGT(Gly) 58 1.20 performed by BI analysis consistently revealed the most traditional GCC(Ala) 13 0.42 GGC(Gly) 10 0.21 intra-ordinal relationships (Fig. 3). In ML analysis, the datasets exclud- GCA(Ala) 48 1.56 GGA(Gly) 81 1.67 ing 3rd codon positions revealed the same tree topology as the major- GCG(Ala) 5 0.16 GGG(Gly) 45 0.93 ity in BI analysis, whereas those that include the 3rd codon positions a Relative synonymous codon usage. revealed a different topology with respect to the Libytheinae branch b Stop codon. within Nymphalidae clade (Fig. A.2). Different datasets resulted in topologies with prominent differences in MP analysis, and none of them produced a robust tree topology (Fig. A.3). 3.4. Non-coding regions Dataset compositions also appeared to affect significantly both the phylogenetic relationships and nodal support values. The topologies The mitogenome of B. panterinaria contains 17 non-coding regions varied notably among the datasets in MP analysis. For example, the (excluding the control region) ranging from 2 bp to 54 bp. The well-accepted monophyletic Pyraloidea was not recovered by the longest spacer of B. panterinaria locates between tRNAGln and ND2 datasets that included 3rd codon positions (Fig. A.3). Furthermore, genes and is suggested to be constitutively synapomorphic and the datasets excluding 3rd codon positions provided tree topologies restricted to ditrysian mitogenomes (Cameron and Whiting, 2008; with higher support values than those that included 3rd codon posi- Hao et al., 2012), because no spacer is found in the newly determined tions in BI analysis (Fig. 3). As in previous studies (Baker et al., 2001; Hepialidae species (Cao et al., 2012). Damgaard and Cognato, 2003; Simmons et al., 2004), 3rd codon posi- The control region of B. panterinaria is 349 bp with 93.1% A+T con- tions were generally assumed to be more homoplastic, and contained tent. Among all known lepidopteran mitogenomes, the control regions more noise and less signal than the other two, as shown by the lepidop- have the largest variation ranging from 311 bp (Sesamia inferens)to teran mitogenomes at deep-level phylogeny in our analyses. However, 1270 bp (Papilio maraho). The conserved motif “ATAGA+poly T”, despite the negative effect of 3rd codon positions, some low-level which was suggested as the origin of the N-strand DNA replication nodes were improved, such as the Saturniinae, Pyraloidea, and (Jiang et al., 2009; Taanman, 1999), was found near the beginning of Tortricoidea clades (Figs. A.2 and A.3). In ML analysis, all PCG datasets the control region with 17 poly T. No tandem repeats were found in failed to reveal a robust relationship of Libytheinae with other subfam- the control region of B. panterinaria, whereas two short repeat units ilies (Fig. 2). scattered in the control region. One is 16 bp long with a 211 bp Alignment methods had little influence on the phylogenetic rela- interspacer between two copies, and the other is inverted and 18 bp tionships or support values; however, their superiority varied among long (Fig. 2). The conserved structural elements in the mitochondrial different inference methods. In BI analysis, MAFFT was superior to control region found in other insect orders, such as Diptera or Muscle and Clustal W for the PCG datasets that excluded 3rd codon Hemiptera (Li et al., 2011a; Zhang and Hewitt, 1997), are difficult to positions, whereas Muscle was a little superior to the other two identify in the Lepidoptera except poly T, which could be because the for those that included 3rd codon positions (Fig. 3). MAFFT and mitochondrial control regions of most lepidopterans are actually Muscle were slightly superior to Clustal W in inferring the nodes of short. More comparative studies on the structural elements of the mito- Geometroidea+Bombycoidea and Sphingidae+Bombycidae in ML chondrial control regions are needed in the future. analysis (Fig. A.2). A comparison about the relative alignment superior- ity using MP analysis was difficult, because many nodes were weakly 3.5. Methodological effects of various approaches supported (Fig. A.3). No matter how much the alignment methods were improved, non-homologous positions still existed in all datasets, After comparing all the tree topologies and nodal support values, which affected the branch lengths and support values but not the tree we assessed the effects of the three factors studied in our analyses: topologies. 354 X. Yang et al. / Gene 515 (2013) 349–358

Fig. 3. Phylogenetic tree inferred from the PCG12MA dataset using BI analysis. A. honmai (Tortricoidea) was specified as the outgroup. The posterior probabilities of all datasets are drawn on the tree topology. Nodes marked with black spots are all supported with the highest value of 1.00 for each dataset A–C and a–c. Support values for the other nodes are drawn on the tree in the following order: A, PCG12MU; B, PCG12CW; C, PCG12MA; a, PCG123MU; b, PCG123CW; and c, PCG123MA.

From the above-mentioned reasons, we present a tree produced by relationship with Bombycoidea than Noctuoidea. According to the pre- BI analysis using the PCG12MA dataset, which shows the highest nodal vious studies by Mutanen et al. (2010), the monophyly of Geometroidea support and the most identical phylogeny in our analyses (Fig. 3). is uncertain, and we suggest that more sampling of the representative taxa by complete mitogenomic analysis could help in solving this prob- 3.6. Phylogenetic analyses lem. In addition, the relationships within the Noctuoidea clade were verified, and the relationships of the three families in our analyses All 45 lepidopteran taxa in the analyses are from the Ditrysia, which were in agreement with recent studies (Mutanen et al., 2010; Regier currently contains 29 superfamilies, whereas the ones in which et al., 2009; Zahiri et al., 2011). With Oenosandridae at the base, mitogenomes are available are as follows: Tortricoidea, Pyraloidea, Notodontidae was the sister group of the rest of Noctuoidea including Papilionoidea (including Hesperiidae), Bombycoidea, Geometroidea Nolidae, Euteliidae, Noctuidae, and Erebidae, as recently advocated and Noctuoidea (van Nieukerken et al., 2011). According to the (Mutanen et al., 2010; Zahiri et al., 2011). The relationships of trifine new Lepidoptera system revised by van Nieukerken et al. (2011), and quadrifine noctuoids have been contrasted in recent studies Bombycoidea, Geometroidea, and Noctuoidea are relegated to the (Fibiger et al., 2010; Lafontaine and Fibiger, 2006; Lafontaine and Macroheterocera clade, Tortricoidea to the lower part of the Apoditrysia Schmidt, 2011; Mitchell et al., 2006; Regier et al., 2009; Zahiri et al., clade, and Papilionoidea and Pyraloidea to the upper apoditrysian 2012). Some of the analyses support the monophyly of quadrifine Obtectomera clade. The 44 lepidopteran taxa with complete noctuoids (Mitchell et al., 1997, 2000, 2006; Weller et al., 1994), where- mitogenomes downloaded from the GenBank are listed in Table 4. as others have suggested that quadrifine noctuoids are part of the Regardless of the limited taxon sampling, the monophyly of the six Noctuidae family (Lafontaine and Fibiger, 2006). Recent studies based superfamilies were well established in our analyses according to the on the combination of eight molecular markers proposed a new system new Lepidoptera family classification system by van Nieukerken et al. of six families in Noctuoidea (Zahiri et al., 2011, 2012), although (2011). The monophyly of the Papilionoidea, Pyraloidea, Noctuoidea, Erebidae lacked either synapomorphies or clear molecular shared char- and Tortricoidea seemed to be quite convincing for the relatively wide acteristics so far. With advances in our understanding of the phylogeny taxonomic coverage, whereas further taxon sampling was still needed within Noctuoidea, such controversy is bound to continue, and more at the family and superfamily levels. detailed studies from different characteristic systems are certainly The relationships at the superfamily level in our analyses were sim- needed. ilar to the recent studies by Regier et al. (2009) and Mutanen et al. Bombycoidea contains 10 families and 497 genera in Bombycoidea, (2010), except that Geometroidea was found to have a closer lineage and their phylogenetic relationships are hard to infer as all families X. Yang et al. / Gene 515 (2013) 349–358 355

Table 4 List of taxa in the phylogenetic analyses with their GenBank accession numbers.

Superfamily Family Subfamily Species GBAN a Reference

Bombycoidea Bombycidae Bombycinae Bombyx mori NC_002355 DS b Bombycoidea Bombycidae Bombycinae Bombyx mandarina NC_003395 Yukuhiro et al. (2002) Bombycoidea Saturniidae Saturniinae Saturnia boisduvalii NC_010613 Hong et al. (2008) Bombycoidea Saturniidae Saturniinae Antheraea yamamai NC_012739 Kim et al. (2009c) Bombycoidea Saturniidae Saturniinae Antheraea pernyi NC_004622 Liu et al. (2008) Bombycoidea Saturniidae Saturniinae Eriogyna pyretorum NC_012727 Jiang et al. (2009) Bombycoidea Saturniidae Saturniinae Samia cynthia ricini JN215366 Kim et al. (2012) Bombycoidea Sphingidae Sphinginae Manduca sexta NC_010266 Cameron and Whiting (2008) Geometroidea Geometridae Ennominae Phthonandria atrilineata NC_010522 Yang et al. (2009) Noctuoidea Erebidae Lymantriinae Lymantria dispar NC_012893 Zhu et al. (2010) Noctuoidea Erebidae Arctiinae Hyphantria cunea NC_014058 Liao et al. (2010) Noctuoidea Noctuidae Heliothinae Helicoverpa armigera NC_014668 Yin et al. (2010) Noctuoidea Noctuidae Amphipyrinae Sesamia inferens NC_015835 Un c Noctuoidea Notodontidae Phalerinae Phalera flavescens NC_016067 Un c Noctuoidea Notodontidae Thaumetopoeinae Ochrogaster lunifer NC_011128 Salvato et al. (2008) Papilionoidea Hesperiidae Pyrginae Ctenoptilum vasava NC_016704 DS b Papilionoidea Aphnaeinae Spindasis takanonis NC_016018 Kim et al. (2011b) Papilionoidea Lycaenidae Coreana raphaelis NC_007976 Kim et al. (2006) Papilionoidea Lycaenidae Theclinae Protantigius superans NC_016016 Kim et al. (2011b) Papilionoidea Nymphalidae Apaturinae Sasakia charonda NC_014224 Un c Papilionoidea Nymphalidae Apaturinae Apatura metis NC_015537 Un c Papilionoidea Nymphalidae Apaturinae Apatura ilia NC_016062 Un c Papilionoidea Nymphalidae Calinaginae Calinaga davidis NC_015480 Mao et al. (2011) Papilionoidea Nymphalidae Danainae Euploea mulciber NC_016720 Un c Papilionoidea Nymphalidae Heliconiinae Acraea issoria NC_013604 Hu et al. (2010) Papilionoidea Nymphalidae Heliconiinae Argynnis hyperbius NC_015988 Wang et al. (2011) Papilionoidea Nymphalidae Heliconiinae Fabriciana nerippe NC_016419 Kim et al. (2011a) Papilionoidea Nymphalidae Libytheinae Libythea celtis NC_016724 Un c Papilionoidea Nymphalidae Nymphalinae Kallima inachus NC_016196 Un c Papilionoidea Nymphalidae Satyrinae Hipparchia autonoe NC_014587 Kim et al. (2010) Papilionoidea Papilionidae Parnassiinae Parnassius bremeri NC_014053 Kim et al. (2009b) Papilionoidea Papilionidae Papilioninae Teinopalpus aureus NC_014398 Un c Papilionoidea Papilionidae Papilioninae Papilio maraho NC_014055 Un c Papilionoidea Pieridae Pierinae Pieris melete NC_010568 Hong et al. (2009) Papilionoidea Pieridae Pierinae Pieris rapae NC_015895 Mao et al. (2010) Pyraloidea Crambidae Crambinae Chilo suppressalis NC_015612 Un c Pyraloidea Crambidae Crambinae Diatraea saccharalis NC_013274 Li et al. (2011b) Pyraloidea Crambidae Pyraustinae Ostrinia nubilalis NC_003367 Coates et al. (2004) Pyraloidea Crambidae Pyraustinae Ostrinia furnacalis NC_003368 Coates et al. (2005) Pyraloidea Crambidae Spilomelinae Cnaphalocrocis medinalis NC_015985 Un c Pyraloidea Pyralidae Galleriinae Corcyra cephalonica NC_016866 Un c Tortricoidea Tortricidae Tortricinae Adoxophyes honmai NC_008141 Lee et al. (2006) Tortricoidea Tortricidae Olethreutinae Grapholita molesta NC_014806 Son and Kim (2011) Tortricoidea Tortricidae Olethreutinae Spilonota lechriaspis NC_014294 Zhao et al. (2011)

a GenBank accession number. b Direct submission. c Unpublished. shared a few synapomorphies (Minet, 1994). Therefore, molecular rhopaloceran “club-horns”, except when Hedylidae was included) was markers are likely to be more reliable in solving the problems. The supported by our analyses. However, a sister group relationship be- phylogenetic relationships in the “SBS” group of Bombycoidea tween Papilionoidea and Hesperioidea established from a combination [Saturniidae+(Sphingidae+Bombycidae)] were well supported in of morphological characters and molecular data by Wahlberg et al. was our analyses, which were consistent with some previous studies not supported here, which was also in contrast with other previous (Jiang et al., 2009; Mutanen et al., 2010; Regier et al., 2009) but in con- studies and with subsequent ones (DeJong et al., 1996; Heikkilä et al., flict with some others (Zwick, 2008; Zwick et al., 2011). However, phy- 2012; Mutanen et al., 2010; Regier et al., 2009; Wahlberg et al., 2005). logenetic hypotheses resulting from molecular studies contradict The most prominent problem in our analyses was the narrow taxon significantly with morphological data, and the relationships with sampling with only 1 species from the former Hesperioidea, absence weak support still require more research. The most recent study by of former Hedyloidea, and only 7 of 43 lepidopteran superfamilies Zwick et al. (2011) already made a great progress on the phylogeny (van Nieukerken et al., 2011). However, the phylogenetic relationships within Bombycoidea, which had the widest taxa and nuclear gene sam- of the five Papilionoidea families (Papilionidae, Hesperiidae, Pieridae, pling in the Lepidoptera so far. They reconstructed a stable tree topology Lycaenidae, and Nymphalidae), excluding the former Hedyloidea were within Bombycoidea as well as the relationship with its sister group the same as the recent studies published by Heikkilä et al. (2012),as Lasiocampidae. Most relationships among deep-level groups were well as the basal position of Papilionidae within butterflies. The phylo- strongly supported except for Phiditiinae+Carthaeidae+Anthelidae genetic relationships within Nymphalidae based on BI in our analyses and Saturniidae+Bombycinae. Furthermore, with the increase in addi- were congruent with the tree conducted on the combined molecular tional nuclear genes or taxa for further improvement, the analyses of the and morphological dataset based on BI analysis in Wahlberg et al. mitogenomes may present a new solution. (2005), and mostly consistent with that of Heikkilä et al. (2012) except The most controversial relationships occurred in the butterfly for the Libytheinae branch. clade. The monophyly of “butterflies” (those families previously called The monophyly of Pyraloidea and the relationships with other su- Papilionoidea)+“skippers” (Hesperiidae) (comprising the traditional perfamilies were well-supported, as well as the two-family system 356 X. Yang et al. / Gene 515 (2013) 349–358

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