Complete mitochondrial genome data and phylogenetic analysis of Papilio macilentus Janson, 1877 (Lepidoptera: Papilionoidea: Papilionidae)

Abstract In the present study, the complete mitochondrial genome (mitogenome) of the Papilio macilentus (Lepidoptera: Papilionoidea: Papilionidae) was sequenced by next-generation sequencing method. The mitochondrial genome is a circular DNA molecule of 15,264 bp in size with 80.7% AT content, including 37 genes (13 protein-coding genes, 2 rRNA genes, and 22 tRNA genes), and a long non-coding region (Control region). All protein-coding genes are initiated by ATN codons, and terminated with TAA, TAG, or single T. All tRNAs can be folded into common clover leaf secondary structure, except trn-S1. Phylogenetic analyses based on 13 protein-coding genes and 2 rRNA genes using maximum likelihood and Bayesian inference confirmed that P. macilentus and Papilio memnon are clustered into a clade, and revealed the relationships between Papilionini, Troidini, Teinopaippini and Leptocircini.


Introduction
Papilionidae has attracted more attention due to extensive morphological diversity and their colorful wing patterns, and have been widely studied regarding ecological adaption, phylogeny, genetics, and evolution (He et al. 2022).Papilio macilentus Janson, 1877 belongs to Papilionidae, and is mainly distributed in China, Korea, Japan, and Eastern Siberia, more than one or two generations a year in China, larvae mainly feed on Zanthoxylum and Ruta, and adults are visible from April to August in Anhui province, China (Wu 2001).As a member of the ornamental butterfly species, P. macilentus is morphologically similar to Papilio bianor, but P. macilentus shows more narrow hind wings, and the outer margin area is different in red spots (Figure 1).Relatively little attention has been paid to P. macilentus, possibly because the population of the P. macilentus is significantly smaller in nature.In order to fill in the gap and clarify the mitochondrial genome (mitogenome) characteristics of P. macilentus, we have sequenced and annotated the complete mitogenome of P. macilentus by next-generation sequencing method, which would be useful for species identification, phylogenetic analysis and conservation of this species.

Materials and methods
In this study, total genome DNA was extracted from the male adult leg of P. macilentus which was collected from Chuzhou City (118.295 E,32.271 N), Anhui Province, China in April 2017.The total DNA was extracted using a DNeasy# Tissue Kit (Qiagen, Hilden, Germany).The genome was sequenced by the next-generation sequencing method with the Illumina Hiseq 2500 platform.The mitogenome was assembled using Geneious Primer v.2021.2.2 (Kearse et al. 2012) and annotated with MITOS server (Bernt et al. 2013) and NCBI BLAST (https://blast.ncbi.nlm.nih.gov).The specimen and genome DNA were deposited at −80 � C in the Animal Collection of Chuzhou University with the voucher number CHZU-LPP-0254 (Yan Dong,dongyan_bio@126.com).To investigate the phylogenetic status of P. macilentus in Papiliononae, 46 currently available complete mitogenomes of Papiliononae retrieved from GenBank were used in phylogenetic analysis, two Parnassiinae insects (Sericinus montela, HQ259122 and Zerynthia polyxena, MK507888) were used as outgroup.The phylogenetic tree was reconstructed with maximum likelihood (ML) and Bayesian inference (BI) methods based on the 13 protein-coding genes (PCGs) and 2 rRNAs by IQ-TREE v2.1.3(Minh et al. 2020) and PhyloBayes MPI 1.8c (Lartillot et al. 2013), respectively.The number of bootstrap replicates was set to 1000 with automatic model prediction in ML analyses.The site-heterogeneous mixture model (CAT þ GTR) was implemented in BI analyses.The consensus tree was computed with a burn-in of 25% of sampled values.Each gene was aligned using MAFFT 7 (Katoh K & Standley, 2013) and trimmed with trimAl 1.4.1 (Capella-Guti� errez et al. 2009), then concatenated individual genes using MEGA X (Kumar et al. 2018).

Results
In this research, about 6.4 Gb of clean data were obtained and produced a final mitogenome for P. macilentus with an average sequencing depth of 1,374� (Figure S1).After checking and annotating, the complete mitogenome of P. macilentus is 15,264 bp (Figure 2), which is comparable to the sizes of previously documented mitogenomes of Papiliononae species, ranging from 14,642 bp of Graphium antiphates (MN013005) to 16,094 bp of Papilio maraho (FJ810212; Wu et al. 2010;Liu et al. 2020), the gene order of the P. macilentus mitogenome is identical to other commonly sequenced Papilionidae species.The mitogenome of P. macilentus nucleotide composition was A (39.7%), T (41.0%),G (7.4%), C (11.9%), and A þ T content (80.7%).The complete mitogenome was composed of 37 typical mitochondrial genes (13 PCGs, 22 transfer RNA (tRNA) genes, two ribosomal RNA (rRNA) genes), and one large non-coding region (Control region).The total length of the 13PCGs is 11,216 bp, and these encode 3,727 amino acids, accounting for 73.5% of the complete mitogenome.All the PCGs are initially encoded by ATN and terminated coding with TAA, TAG, or single T. The base composition of the 13 PCGs is 79.4A þ T (A ¼ 33.6%, T ¼ 45.8%, C ¼ 10.1%, and G ¼ 10.5%).
All of the tRNAs were identified by ARWEN version 1.2 software (Laslett and Canb€ ack 2008), all tRNAs could form a typical cloverleaf structure except trn-S1, in which the dihydrouridine arm formed a loop.The lengths of the tRNAs ranged from 60 bp (trrn-S1) to 71 bp (trn-K).rRNA genes are identified by comparison with Papilio memnon (MN013052).The 16S rRNA gene is 1,322 bp in size and is located between trn-L1 and trn-V; the 12S rRNA gene is 772 bp in length and is located after trn-V.The control region is located between 12S rRNA and trn-I with 457 bp.
The resulting tree was represented and edited using FigTree v1.4.1 (Figures S2 and S3).The two topologies are highly similar (Figure 3).Both trees support the monophyly of each tribe of Papiliononae with high bootstrap among almost all nodes.Papilionini and Teinopaippini were sister groups and then clustered with Troidini and eventually converged with Leptocircini.The result also indicated that P. macilentus has the closest relationship with P. memnon and is clustered within Papilionini clade.

Discussion and conclusion
In this study, the composition and structure of P. macilentus mitogenome are predicted and analyzed, and the features are consistent with those of other Papilio insects, including the general genome size, AT content, and gene arrangement (Zuo et al. 2018).ML and BI analysis produced two consensus structures on tribe level, with the topology (((Papilionini þ Teinopaippini)þTroidini)þLeptocircini), and this topology was affirmed by Miller's research (1987) but in contrast to the later hypothesis, which believed that the Papilionini and Troidini formed a sister relationship, and then clustered with Teinopaippini (Simonsen et al. 2011).The monophyly of the tribe Papilionini was recovered in this research, the sister relationship of Papilio and Meandrusa has also been confirmed in previous studies (Yan et al. 2022).The phylogenetic status of Papilio demoleus in Papilionini and Iphiclides podalirius in Leptocircini is unclear.Otherwise, the relationships among Graphium parus, G. confucius, and G. timur were inconsistent by ML and BI analysis.These inconsistent analysis results may be due to insufficient sample size and the limited information sites of mitochondrial genes.This research will provide basic data for phylogenetic analysis and population genetic diversity protection of Papilionidae in the future.

Figure 1 .
Figure 1.The adult specimen of Papilio macilentus.Photographed and processed by Yun-Fei Wu.

Figure 2 .
Figure 2. Circular map of the Papilio macilentus mitochondrial genome.