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Complete Mitochondrial Genome of Papilio protenor (Lepidoptera, Papilionidae) and Implications for Papilionidae Taxonomy

Abstract The complete mitochondrial genome (mitogenome) of Papilio protenor (Lepidoptera: Papilionidae) was sequenced and annotated in this study. The mitogenome comprised a typical circular, double-stranded DNA molecule of 15,268 bp in length including 13 protein-coding genes (PCGs), two ribosomal RNA genes, 22 transfer RNA (tRNA) genes, and an A+T-rich region. The gene order and mitogenome orientation were similar to those of all known Papilionidae species. The nucleotide composition of the P. protenor mitogenome exhibits considerable A+T bias (80.5%) and the AT (−0.019) and GC (−0.231) skewness is slightly negative. All of the PCGs except for cytochrome c oxidase (COI) start with a canonical ATN start codon, whereas the COI gene is tentatively designated by the CGA codon. Of the 13 PCGs, 11 contain the complete stop codon TAA or TAG, whereas COI and COII were terminated with a single T nucleotide. All tRNAs exhibit the typical cloverleaf structure, except for tRNASer(AGN), which does not contain the dihydrouridine arm. The 458 bp A+T-rich region is comprised of nonrepetitive sequences including the motif ATAGA followed by a poly-T stretch and a microsatellite-like (AT)6 element preceded by the ATTTA motif. Phylogenetic analysis of the 13 PCGs data using Bayesian inference, maximum likelihood methods, and maximum parsimony support the view that the subfamily Parnassiinae is regarded as an independent subfamily within Papilionidae and that Zerynthiini should be treated as one of the two clades of the subfamily Parnassiinae along with Parnasiini. In addition, the analysis strongly supports the monophyly of the subfamily Parnassiinae.

Lepidoptera (moths and butterflies) is the second largest order in Insecta, accounting for more than 16,000 species (Dai et al. 2015). Among these, the Papilionidae family comprises over 570 species worldwide (Shen et al. 2015). However, despite the huge taxonomic diversity, current information regarding the lepidopteran mitogenome is very limited. To date, complete or nearly complete mitogenomes have been sequenced for approximately 100 species of lepidoptera whereas only 24 complete mitogenomes are available in GenBank (http://www.ncbi.nlm.nih.gov/genbank/) for Papilionidae species.
The insect mitochondrial genome (mitogenome) consists of a closed double-strand circular DNA molecule, ranging from 14 to 19 kb in length, which encodes an extremely highly conserved set of 37 genes, including 13 protein-coding genes (PCGs) [ATPase subunits (ATP) 6 and 8, cytochrome c oxidase subunits 1-3 (COI-III), cytochrome (Cyt) B, and NADH dehydrogenase subunits (ND) 1-6 and 4L], two ribosomal RNA genes (12S rRNA and 16S rRNA, encoding the small and large rRNA subunits, respectively), and 22 tRNA genes (Wolstenholme 1992, Boore 1999). In addition, it contains an A+T-rich region, which takes part in the regulation and initiation of mitogenome replication and transcription (Shadel and Clayton 1993). Compared with nuclear genes, the mitogenome is a compact structure, exhibits maternal inheritance, lacks genetic recombination, and undergoes a relatively fast evolutionary rate of variation. Because of these unique features, mitogenome genes and genomes have been widely used as a molecular marker to study molecular evolution, population genetics, phylogeography, and phylogenetics (Harrison 1989, Boore 1999, Ingman et al. 2000, Hurst and Jiggins 2005, Cameron et al. 2006, Timmermans et al. 2014. Swallowtail butterflies (Papilionidae) are among the most popular insect taxa and have greatly contributed to studies of ecology, behavior, and evolution in insects. Although numerous studies have been published regarding Swallowtail butterflies, the taxonomic status of Parnassiinae and Zerynthiinae has been particularly controversial at the subfamily level. Many authors suggest that the clades Parnasiini and Zerynthiini should be treated as two independent subfamilies (Talbot andSewell 1939, Wang 2001). On the other hand, some authors support that Parnasiini should be regarded as a tribe of the subfamily Parnassiinae, which would thus consist of two clades: Parnasiini (including Archon, Hypermnestra, and Parnassius), and Zerynthiini (including Allancastria, Sericinus, Zerynthia, Luehdorfia, and Bhutanitis), based on morphological and molecular data (Ehrlich 1958, Igarashi 1984, Huang et al. 2015. However, these views have been challenged by several authors, who believe that the subfamily Parnassiinae should not belong to Papilionidae at all but rather should be upgraded as a separate family, including the subfamilies Parnassiinae and Zerynthiinae (Li and Zhu 1992). Furthermore, the monophyly of the subfamily of Parnassiinae is increasingly controversial (Häuser 1993, Yagi et al. 1999, Caterino et al. 2001. Taxonomic disputes mainly focus on: 1) taxonomic status of the subfamily Parnassiinae; 2) taxonomic status of the subfamily Zerynthiinae; and 3) phylogenetic relationships with the subfamily Parnassiinae.
To address these issues, in the present study, we determined the complete mitogenome sequence of Papilio protenor (Lepidoptera: Papilionidae) and compared its gene structure with that of other known Papilionidae species. Furthermore, we performed a phylogenetic analysis based on the available complete mitogenome sequences to provide insight into the phylogenetic relationship of Papilionidae.

Sample Collection and DNA Extraction
Adult individuals of P. protenor were collected in Yaoluoping nature reserve, Yuexi County, Anhui province, China, in July 2015. After collection, the samples were immediately preserved in 100% ethyl alcohol and stored at −20°C prior to DNA extraction. Whole genomic DNA was extracted from the thorax muscle tissues of a single sample using a standard phenol-chloroform protocol (Sambrook and Russell 2001). DNA yield and quality were examined by 1.5% agarose gel/ethidium bromide.

Polymerase Chain Reaction Amplification and Sequencing
To amplify the whole mitogenome of P. protenor, we designed three long polymerase chain reaction (PCR) primers using Primer Premier 5.0 software (Singh et al. 1998) and were based on three fragments of COI, ND5, and 16S rRNA of P. protenor that were released by the National Center for Biotechnology Information. All PCRs were performed in a 50-μl reaction volume containing 33.5-μl sterilized distilled water, 5-μl 10 × Taq buffer (Mg 2+ plus, TaKaRa, Shiga, Japan), 8-μl dNTPs, 1-μl each primer, 0.5-μl LA Taq polymerase (5 U/μL, TaKaRa), and 1-μl DNA template. The conditions for PCR amplification were as follows: initial denaturation at 95°C for 5 min, followed by 30 cycles at 98°C for 10 s, 68°C for 5 min (depending on primer combinations and putative length of the fragments), and a final extension step of 72°C for 10 min. The quality of the PCR products was detected by 1.5% agarose-gel electrophoresis, and products were purified using a DNA gel extraction kit (TaKaRa). The purified PCR products were sequenced after cloning into the pMD-19T Vector (TaKaRa, Dalian, China). Two vector-specific primers and internal primers were used to complete the sequences by primer walking.

Sequence Analysis and Gene Annotation
The mtDNA sequences of P. protenor were assembled using the Seqman II program from the Lasergene package DNAStar (Madison, WI). The 13 PCGs, 2 rRNAs, and the A+T-rich region were annotated manually and determined by comparison with the close relative insects for which published complete mitogenome sequences were available. The nucleotide sequences of the 13 PCGs were translated into amino acid sequences based on the invertebrate mitochondrial genetic code. Nucleotide composition and relative synonymous codon usage (RSCU) were calculated using MEGA version 5.0 (Tamura et al. 2011). tRNA genes were identified using the tRNAs-canSE Search Server (http://lowelab.ucsc.edu/tRNAscan-SE/) (Lowe and Eddy 1997

Phylogenetic Analysis
To study the phylogenetic relationships among Papilionidae, phylogenetic trees were reconstructed based on the 13 PCGs of the complete mitogenomes of the 36 known butterfly sequences. The mitogenomes of Argynnes hyperbius (NC_015988.1) and Timelaea maculata (NC_021090.1) were selected as outgroups. The concatenated nucleotide alignments of the 13 PCGs yielded a nucleotide matrix of 11,017 bp in length that was used for phylogenetic analysis with Bayesian inference (BI), maximum likelihood (ML), and maximum parsimony (MP) methods.
The ML analysis was performed by RAxML with 1,000 bootstrap replicates and using the rapid bootstrap feature (random seed value 12,345) (Stamatakis et al. 2008). BI analysis was implemented with MrBayes version 3.1.2 (Ronquist and Huelsenbeck 2003). MrModeltest 2.3 (Posada and Crandall 1998) was used in Fig. 1. Map of the circular P. protenor mitochondrial genome. Gene names on the thick line side indicated that these genes were located on H strand, whereas the others were located on L strand. Color codes for different genes were detail listed on the map. combination with PAUP* 4b10 to select an appropriate nucleotide substitution model for BI. The GTR + I + G model was chosen as the best-fit model following the Akaike information criterion. In Bayesian analysis, The Monte Carlo Markov chain length was run for 10,000,000 generations. When the average standard deviation of split frequencies was below 0.01, we considered that stationarity was reached and stopped the run. Each set was sampled every 1,000 generations with a burn-in of 25%. Posterior probabilities over 0.9 were considered as strongly supported (Mutanen et al. 2010). MP analysis was performed with PAUP* 4b10 first under the heuristic search strategy with all sites weighted equally, gaps treated as missing data, and 1,000 random addition sequences and tree bisection-reconnection branch swapping. To assess the support for branching events, nonparametric bootstrapping was performed with 1,000 pseudoreplicates under a heuristic search strategy and 100 random-addition sequences in each pseudoreplicate.

Genome Structure and Organization
The P. protenor mitogenome is a typical circular, double-stranded DNA molecule of 15,268 bp in length (GenBank accession No.KY272622.1), consistent with other known Papilionidae mitogenomes, which range from 15,185 bp in P. machaon to 16,094 bp in Agehana maraho. It contains 37 genes (13 PCGs, 22 tRNAs, and 2 rRNAs) and an A+T-rich region (Fig. 1). Among these, 14 genes are encoded on the L strand: four PCGs (ND1, ND4, ND4L, and ND5), two rRNA genes (16S rRNA and 12S rRNA), and eight tRNA genes (tRNA Glu , tRNA Cys , tRNA Tyr , tRNA Phe , tRNA His , tRNA Pro , tRNA Leu(CUN) , and tRNA Val ). The remaining 23 genes and the A+Trich region are encoded on the H strand (Table 1). The gene order and orientation of P. protenor is identical to known Papilionidae mitogenomes, belonging to the gene order tRNA Met -tRNA Ile -tR-NA Gln . However, the arrangement is different from the ancestral arrangement of tRNA Ile -tRNA Gln -tRNA Met such as found in the species Thitarodes renzhiensis and Ahamus yunnanensis in the Hepialoidea (Cao et al. 2012).

Protein-Coding Genes
The PCG regions of the P. protenor mitogenome, containing 13 PCGs, were consistent with those of other known Papilionidae mitogenomes. All the PCGs except for COI start with a canonical start codon, ATN (ATP6, COIII, ND4, ND4L, CytB, and ND1 with ATG; ATP8 and ND5 with ATT; and ND2 and ND3 with ATC), whereas the COI gene is tentatively designated by the CGA codon.
With respect to the stop codon, 11 of the PCGs terminate with the complete stop codon TAA or TAG whereas COI and COII are terminated with a single T nucleotide. These incomplete stop codons are common in metazoan mitogenomes (Wolstenholme 1992) and are corrected via post-transcriptional polyadenylation during the mRNA maturation process (Ojala et al. 1981). The RSCU value of the P. protenor mitogenome is summarized in Table 3 and Fig. 2. Excluding the initiation and stop codons, the 13 PCGs were 11,121 bp in length, encoding 3,707 amino acid residues. The codons CUG, CCG, ACG, and AGC were not present in these PCGs. The codons UUA (12.2), AUU (11.3), UUU (9.3), AAU (6.8), and AUA (7.3) were the five most frequently used codons in the P. protenor mitogenome, accounting for 40.1% of the total usage. RSCU analysis also demonstrated that the RSCU of the NNU and NNA  codons are >1, indicating that the third positions of U/A exhibit a high frequency of codon usage in the P. protenor mitogenome. In addition, our analysis also showed that tRNA Leu(UUR) (12.7%), tRNA Ile (12.1%), tRNA Phe (10.2%), tRNA Met (7.7%), and tRNA Asn (7.3%) were the most frequent amino acids in the P. protenor mitochondrial proteins, accounting for 50.0% of the total. The observed frequencies are similar to those detected in some other Papilionidae species.

Intergenic Spacers and Overlapping Sequences
The P. protenor mitogenome contains 118 bp of intergenic spacer regions in total, spread over 14 noncoding regions and ranging from 1 to 53 bp in length. Most of the spacers are shorter than 10 bp; only three spacers are more than 10 bp. The three intergenic spacers are located between tRNA Gln and ND2 (53 bp), tRNA Ser(UCN) and ND1 (16 bp), and ND5 and tRNA His (15 bp), respectively. The tRNA-Gln -ND2 has a high AT content (86.8%) and has been reported in other lepidopteran mitogenomes but has not been found in non-lepidopteran insects to date. Therefore, this spacer is regarded as a constitutive synapomorphic feature of lepidopteran mitogenomes (Cameron and Whiting 2008). The tRNA Ser(UCN) -ND1 intergenic spacer is inserted along with an ATACTAA motif that is found in all sequenced Papilionidae butterflies. The motif has been reported as the possible binding site for the transcription termination peptide (mtTERM protein) (Taanman 1999).
The mitogenome of P. protenor has a total overlap of 35 bp between genes in 13 locations, ranging from 1 to 8 bp. The longest overlapping sequence (8 bp, AAGCCTTA) is located between tRNA Trp and tRNA Cys . The second longest overlapping sequence (7 bp, ATGATAA) is located between ATP8 and ATP6. The overlapping sequence has been found in most of the lepidopteran insect mitogenomes and is reported to possibly contribute to form the structure of a hairpin loop required for posttranslational modifications (Fenn et al. 2007). The third longest overlapping sequence (6 bp) is located between tRNA Phe and ND5; all other overlapping sequences are shorter than 5 bp.

Ribosomal RNA Genes
The two rRNA genes, 12S rRNA and 16S rRNA, in the P. protenor mitogenome are located between tRNA Leu(CUN) and tRNA Val and between tRNA Val and the A+T-rich region, respectively. The length of the 16S rRNA gene is 1321 bp whereas that of 12S rRNA is 767 bp. The A+T contents of the 16S rRNA and 12S rRNA genes are 83.5% and 85.0%, respectively. Both of these values are well within the range reported for other lepidopteran insects (Salvato et al. 2008).

Transfer RNA Genes
The mitogenome of P. protenor contains 22 tRNA genes, ranging in length from 61 bp (tRNA Ser(AGN) ) to 71 bp (tRNA Lys ). These 22 tRNA genes are scattered throughout the genome. Among these, 14 genes are encoded on the H-strand and 8 on the L-strand. All tRNA genes had the typical cloverleaf secondary structures with respective anticodons, except for the tRNA Ser(AGN) gene, in which a simple loop was substituted for a dihydrouridine arm in the P. protenor mitogenomes. All tRNA genes had a common length of 7 bp for the aminoacyl stem and an invariable size of 7 bp for the anticodon loop. There are 14 base pair mismatches present in the tRNA secondary structures of P. protenor mitogenome, including eight wobble G-U pairs, one U-G pairs, two U-U pairs, and one C-A pairs. The predicted secondary structures of the tRNAs in the P. protenor mitogenome were shown in Fig. 3. The nucleotide composition of these 22 tRNA genes (1,446 bp in total length) is AT biased (81.3%) and presents negative AT-skew (−0.016). The AT content of the tRNAs is higher than that in the 13 concatenated PCGs but lower than that in the rRNA genes and A+T-rich region.

A+T-rich Region
The A+T-rich region of P. protenor is 458 bp in length and is located between the 12S rRNA and tRNA Met loci. This region contains the highest AT content (93.2%) of any region of the P. protenor Some conserved features of other lepidopteran mitogenomes have been found in the A+T-rich region of the P. protenor mitogenome. Among these, the origin of minority or light strand replication (Q N )  contains the motif ATAGA followed by a poly-T stretch downstream of the 12S rRNA. Although the length of the poly-T differs among species, the ATAGA motif is prevalent in lepidopteran mitogenomes Whiting 2008, Tang et al. 2014). The ploy-T has been considered to act as a transcription control site and the initiation site of replication (Zhang and Hewitt 1997). A poly-A commonly reported in lepidopteran mitogenomes was also observed upstream of the tRNA Met , representing a microsatellite-like (AT) 6 element preceded by the structural motif ATTTA (Fig. 4). In addition, two tandem 17 bp repeat elements are also found within the A+T-rich region of the P. protenor mitogenome.
In addition, the phylogenetic position of Parnassiini and Zerynthiini is highly debated. In our study, the tribe Parnassiini clustered with Zerynthiini as a sister group, and phylogenetic analysis revealed that Parnassiinae is a subfamily of Papilionidae, whereas Zerynthiini and Parnassiini can be regarded as two clades of the subfamily Parnassiinae. This result is consistent with those reported earlier based on morphological and molecular data (Ehrlich 1958, Igarashi 1984, Nazari et al. 2007, Huang et al. 2015. The phylogenetic results demonstrate strong support for the monophyly of the Parnassiinae subfamily, which is concordant with some previous studies (Miller 1987, Nazari et al. 2007), although other studies have shown the group to be nonmonophyletic Sperling 1999, Omoto et al. 2004).
Although our phylogenetic analyses, which is based on the mitochondrial DNA sequence of 13 PCGs, provide evidence for the phylogenetic position of Parnassiini and Zerynthiini, additional analyses involving sequence information from both mitogenomes and nuclear genomes of Parnassiinae members are required to determine the accurate phylogeny of Papilionidae.  5. The genealogical tree based on Bayesian inference for 36 complete or nearly complete mitogenome sequence of butterfly species plus two Nymphalidae sequences as the outgroup taxa; the scale bar indicates substitutions per site. Numbers above the branches represent Bayesian posterior probabilities and bootstrap branch support for maximum likelihood and maximum parsimony estimations, respectively (BPP/MLBS/MPBS). Nodes, which all support rates were 100%, were marked with an asterisk.