Identification of the exuviae of larva from Teinopalpus aureus Mell, 1923 using the complete mitochondrial genome [version 1; peer review: 2 approved with reservations]

Teinopalpus aureus Mell (Lepidoptera: Papilionidae) is distributed throughout China, Vietnam and Laos, and is listed as a Class I species in China. To identify whether the exuviae of larva is belonging to Teinopalpus aureus Mell, 1923 or not, and to compare the gene structure and genetic differences among the known populations, ten mitogenomes of T. aureus from the exuviae of larva collected in the Michelia maudiae were sequenced. This method of sequencing the mitogenomes of exuviae of larva can give us the chance to monitor the conservation of rare butterflies. Ten mitogenomes of T. aureus showed typical gene arrangements and contained 13 protein-coding genes (PCGs), two ribosomal RNA genes (12S rRNA and 16S rRNA), 22 transfer RNA (tRNA) genes, and a non-coding control region (D-loop). The two haplotypes with one base different in T. aureus were found. We also conducted phylogenetic analyses including all different populations of T. aureus to assess the phylogenetic relationship of T. aureus . The lengths of the 12S rRNA and 16S rRNA genes from both haplotypes were 776 base pairs (bp) and 1,334 bp, respectively. The genetic distance of the ten samples was calculated as 0-0.000065 on the basis of the whole mitogenomes. T. aureus found in Taishun, Zhejiang province, China had a close phylogenetic relationship with the clade of T. aureus found in Pingshan, Jiangxi province, China, which was supported by neighbour-joining analysis.


Introduction
Because of its beautiful appearance and relative rarity in China, Teinopalpus aureus Mell, 1923 (Lepidoptera: Papilionidae) distributed in China, Vietnam and Laos is listed as a Class I species in China (Morita 1998;Masui 1999;Xing et al. 2019;Huang et al. 2015), which is one of three highest classifications for protection of endangered insect species in China.If we can identify the larva of T. aureus and obtain the mitochondrial genome using the exuviae of larva, we can determine its habitat sites and compare the genetic diversity of T. aureus using non-damage sampling to protect it in the larva stage.
No ethical approval was obtained for this study as we used wild exuviate gathered from the Wuyanling National Nature Reserve.

Methods
Ten exuviae of larva of T. aureus (No.ZJWYL20200601TP01-ZJWYL20200601TP10) were collected from wild host plants, Michelia maudiae, in Zhejiang Wuyanling National Nature Reserve, Taishun, Zhejiang province, China and stored in 100% absolute ethyl alcohol in the Natural Museum of Wuyanling National Nature Reserve, China.
A total of ten genomic DNAs from exuviae of larva (No.ZJWYL20200601TP01-ZJWYL20200601TP10) were extracted from partial exuviae using QIAGEN DNeasy Blood and Tissue Kit (QIAGEN, Valencia, CA) according to the manufacturer instructions, and stored at -20 °C refrigerator in Key Lab of Wildlife Biotechnology, Conservation and Utilization of Zhejiang Province.
The mitochondrial genome was obtained by the Sanger method.Ten mt genomes were amplified by polymerase chain reaction (PCR) by using the normal primers (Zhang et al. 2018;Guan et al. 2021;Xu et al. 2021;Yu et al. 2021) and six specific primers according to the reported mitochondrial genomes of T. aureus (Huang et al. 2015).This study used both normal polymerase chain reaction (PCR) and long-and-accurate PCR (LA PCR) methods with Takara Taq or Takara LA Taq DNA polymerase (Takara, Dalian, China).Normal PCR (or Long PCR) was performed in a 50 μL reaction mixture consisting of 32.5 μL (or 26.5 μL) of sterilized distilled water; 5 μL MgCl 2 , 25 mM; 5 μL 10 Â PCR Buffer (or 5 μL 10 Â LA PCR Buffer); 4 μL (or 8 μL) dNTP, 2.5 mM; 1 μL (or 2 μL) of each primer, 5 μmol; 1 μL DNA template; 0.5 μL Takara Taq enzyme (or Takara LA Taq enzyme) DNA polymerase (Takara Biomedical, Japan).Fragments were amplified using Takara Taq enzyme via normal PCR or Takara LA Tag enzyme: initial denaturation for 3 min at 95 °C, followed by 35 cycles of 40 s at 95 °C, 1 min or 3 min at 46-58 °C, and 50-90 s at 72 °C, and a subsequent 10 min final extension step at 72 °C.All PCR reactions were used in Applied Biosystems Veriti instrument (Singapore).PCR products were purified using the Axygen agarose-out kit (Axygen, Hangzhou, China), and sequenced using ABI 3730 system by primer walking with two-directions.
To further discuss the phylogenetic relationship of T. aureus found in Zhejiang Wuyanling National Nature Reserve, a total of 13 mitogenomes were analyzed, including 11 mt genomes of T. aureus downloaded from NCBI (Qin et al. 2012;Huang et al. 2015;Zou et al. 2021) and two mt genomes of T. imperialis (Huang et al. 2016) as outgroups (Figure 1).To align the all complete mt genomes, we used Clustal W in Mega 7.0 (Kumar et al. 2016).We constructed a Neighbour-Joining phylogenetic tree with the parameters as below: bootstrap replications (1000), substitution model (Kimura 2-parameter model), Rates among sites (Gamma distributed), other parameters used default parameters.

Results
The two haplotypes with one base different in control region were found.The two obtained whole mt genome haplotypes were deposited in the National Center for Biotechnology Information with accession numbers OL449691 and OL449692.
The two mt genome haplotypes of T. aureus in majority-strand was 15,243 base pairs (bp) in length with negative AT-skew and GC-skew, which were -0.0025 and -0.2376, respectively, which showed typical lepidopterous gene structure and contained 13 protein-coding genes (PCGs), two ribosomal RNA genes (12S rRNA and 16S rRNA), 22 transfer RNA (tRNA) genes, and one control region (D-loop).One base T replaced C in control region between two haplotypes was found.The start codons of PCGs were ATG (in COX2, COX3, ATP6, ATP8, ND1, ND4, ND4L, and Cytb), ATT (in ND2, ND3, ND5, and ND6), and CGA (in COX1).The stop codons of PCGs were TAA (in ND2, ATP6, ATP8, COX3, ND1, ND5, ND6, and Cytb), TAG (in ND4L), and the incomplete stop codon T-(in COX1, COX2, ND3, and ND4).The gene arrangement was identical to the reported mt genome of T. aureus wuyiensis from Lee (Zou et al. 2021).The overall nucleotide composition of A, T, C and G in majority-strand was 39.8%, 40.0%, 12.5%, 7.7%, respectively.The lengths of the 12S rRNA and 16S rRNA genes from two haplotypes were 776 bp and 1,334 bp, respectively.The genetic distance of between two haplotypes and other reported population of T. aureus based on Kimura 2-parameter model was calculated as 0.00007-0.01013on the basis of the whole mitogenomes.According to genetic distance, we can identify that ten exuviae of larva is belonging to the exuviae from T. aureus.This method using the exuviae to identify species can help us to monitor and protect T. aureus.
In NJ tree, two haplotypes from Taishun, Zhejiang province and the sample from Wuyishan, Fujian province were formed one clade 1.Then this clade is closed to the clade 2 from Pingshan and Meihuashan.Clade In this study, the monophyly of T. aureus was well supported.The samples collected in Taishun, Zhejiang province can be identified as T. aureus wuyiensis because the genetic distance is below 0.00013 and the phylogenetic relationship of the samples collected in Taishun are clustered together with T. aureus wuyiensis.The new mitochondrial genomes obtained from the exuviae of larva of T. aureus can give us a further understanding of phylogenetic relationships of T. aureus to protect it.Reviewer Expertise: Genomics and bioinformatics I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.
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Figure 1 .
Figure 1.Phylogenetic tree of the relationships among 11 mt genomes of Teinopalpus aureus and two mt genomes of Teinopalpus imperialis based on the whole complete mitochondrial genomes.Numbers around the nodes are the the bootstrap values of NJ.The GenBank numbers and the abbreviations of location information of all species are shown in the figure.JXPS: Pingshan, Jiangxi province; ZJMHS: Meihuashan, Zhejiang province; WYL: Wuyanling, Zhejiang province; JXJLS: Jiulianshan, Jiangxi province; GXDYS: Dayaoshan, Guangxi Zhuang Autonomous Region; WZ: Zhuang Autonomous Region.