Complete mitochondrial genome of the recently discovered multivoltine Graphium (Pazala) confucius Hu, Duan & Cotton, 2018 (Lepidoptera: Papilionidae).

Graphium (Pazala) confucius Hu, Duan & Cotton, 2018 is a recently discovered, wide ranging, multivoltine swordtail butterfly in China and Vietnam. The present study reports the complete mitochondrial genome of this butterfly, which is the fifth mitochondrial genome record for subgenus Pazala Moore, 1888. The mitochondrial genome of G. (P.) confucius is circular and 15,212 bp in length, and consists of 37 genes, including 13 PCGs, 22 tRNAs, and two rRNAs. The Bayesian phylogenetic tree containing the focal species and 33 other Papilioninae members clusters G. (P.) confucius with other Pazala taxa inside tribe Leptocircini, which agrees with its taxonomic position. The findings of this study added data to the complex subgenus Pazala and are beneficial to future understanding and conservation planning of butterfly diversity.

Phylogenetic diversity has been increasingly accepted as a tool in conservation planning (Lu and Bullock 2021). To achieve such meta-analysis, the accumulation of molecular data, especially those of the regionally endemic, recently discovered, and little-known species are crucial (Wang et al. 2020). Graphium (Pazala) confucius Hu, Duan & Cotton, 2018 is among several recently discovered species of the subgenus Pazala Moore, 1888 (Lepidoptera: Papilionidae), a complex Sino-Himalayan group of butterflies (Racheli and Cotton 2009). G. (P.) confucius occupies a wide distribution range from Southwest to Central China, as well as North Vietnam (Hu et al. 2018); and it is also the only known multivoltine Pazala species with three generations per year (Zhang et al. 2018). The mitochondrial genome of G. (P.) confucius reported herein would add new data to facilitate future understanding and conservation planning for butterflies.

The butterfly specimen used in this study was collected from Da Moyu (25.066411°N, 102.589607°E, 2200 m), Kunming, Yunnan, China. The specimen was deposited in the Zoological Museum (insect collection) of Yunnan University, Kunming, China (specimen number: YNU-LEP-PAP-2021GC01, contact person: Shao-Ji Hu). Genomic DNA was extracted from the thoracic muscle tissue of a single male adult using the TianGen TIANamp Genome DNA Kit (TianGen Biotech Co., Ltd., Beijing, China). The PCR amplification was performed using in a 25 μl system containing 2.5 μl 10× PCR buffer, 2 μL MgCl2 (25 mM), 2 μl dNTPs (2.5 mM each), 0.5 μl each of forward and reverse primers (20 μM; Table S1), 0.25 μl Taq DNA polymerase (TaKaRa Biotechnology Co., Ltd., Dalian, China), and 1 μl genomic DNA. The PCR thermal profile consisted of an initially denaturation at 95 °C for 3 min; followed by 30 cycles of 94 °C denaturation for 60 s, 50 °C annealing for 60 s, and 72 °C extension for 90 s; then an external extension at 72 °C for 5 min. All PCR products were sequenced on an ABI 3730xl automatic sequencer (Applied Biosystems, CA, USA). Resultant gene fragments were assembled using DNAStar (https://www.dnastar.com/) with G. (P.) parus (MT198821), G. (P.) mullah chungianus (MW549197), and G. (P.) eurous asakurae (MW549198) as the reference genomes (Duan et al. 2020; Hu et al. 2021). Transfer RNA genes (tRNAs) and ribosomal RNA genes (rRNAs) were predicted using the web based MITOS (http://mitos.bioinf.uni-leipzig.de/index.py) (Bernt et al. 2013), while all PCGs were determined using the Alignments | CDS feature under BLASTn of NCBI (https://blast.ncbi.nlm.nih.gov/).

The complete mitochondrial genome of G. (P.) confucius is circular and 15,212 bp in length (GenBank accession number: OK136253). The base composition is 39.84% for A, 39.28% for T, 8.13% for G, and 12.75% for C. This mitochondrial genome contains 37 genes, including 13 PCGs, 22 tRNAs, and two rRNAs, plus a non-coding control region. The plus (+) strand encodes nine PCGs (nad2, cox1, cox2, atp8, atp6, cox3, nad3, nad6, and cob), while the minus (−) strand encodes four PCGs (nad5, nad4, nad4l, and nad1).The gene arrangement and character of this genome fit those of ditrysian Lepidoptera mitochondrial genomes (Cao et al. 2012; Chen et al. 2020; Wang et al. 2019).

To validate this mitochondrial genome, a Bayesian phylogenetic tree was reconstructed by PhyloSuite 1.2.2 (Zhang et al. 2020) using the 37 genes (13 PCGs, 22 tRNAs, and two rRNAs) for 1,000,000 generations, with the GTR + F + I + G model selected by ModelFinder (Kalyaanamoorthy et al. 2017). Thirty-three species of Papilioninae with available mitochondrial genomes were used as ingroups and Parnassius apollo Linnaeus, 1758 (Parnassiinae; KF746065) (Chen et al. 2014) was chosen as the outgroup. The result shows that G. (P.) confucius clusters with G. (P.) parus first and then with other Pazala species. All Pazala taxa are related to other Graphium species within Leptocircini, forming a monophyletic clade, supported by the maximal support values (Figure 1).

Figure 1.

The Bayesian phylogenetic tree for Graphium (Pazala) confucius Hu, Duan & Cotton, 2018 (marked with bold font) and other Papilioninae taxa. Node labels represent support values.

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