The first complete mitochondrial genome of the hawkmoth Marumba saishiuana (Lepidoptera: Sphingidae) and insights into its phylogenetic position.

In this study, we sequenced and analyzed the complete mitogenome of Marumba saishiuana Okamoto, 1924. The complete mitogenome sequence of M. saishiuana is circular, 15,662 bp in size and encodes 13 protein-coding genes (PCGs), two ribosomal RNA genes (rRNAs), 22 transfer RNA genes (tRNAs), and a control region (CR). Nucleotide composition is highly biased toward A + T nucleotides (81.2%). Most of 13 PCGs initiate with the standard start codon of ATN, except cox1, which starts with CGA. Phylogenetic analyses were performed using nucleotide sequences. A total of 32 Smerinthinae species were selected. The topology based on mitogenome showed that M. saishiuana, M. gaschkewitschii, and M. sperchius formed a clade, and this indicated that M. saishiuana was a member of genus Marumba. Polyptychus trilineatus was the most closely related to genus Marumba on the phylogenetic tree reconstructed by mitogenomes.

Hawkmoths (Sphingidae) are a family of moths comprising of more than 1460 species in 206 genera (van Nieukerken et al. 2011), among which Marumba saishiuana Okamoto, 1924 (Lepidoptera: Sphingidae) is widely distributed in Southeast Asia. Currently, the complete mitogenome sequences of Sphingidae are very limited. The mitogenome sequence of M. saishiuana so far remains unknown. Therefore, we sequenced the complete mitochondrial genome of M. saishiuana to provide more comprehensive data for this species and reconstructed the phylogenetic relationship of Smerinthinae to validate the phylogenetic position of M. saishiuana.

Marumba saishiuana was collected from the Dabie Mountain, Lu'an City, Anhui Province, China (31°13′08″N, 116°20′19″E) in May 2021 and deposited in the Entomological Museum, College of Life Sciences, Anhui Normal University (https://www.ahnu.edu.cn/, YX, Huang, huangyx@ahnu.edu.cn) under the accession no. DB20210524. All animal-related experiments were performed according to the protocols approved by the Institutional Animal Care and Use Committee of Anhui Normal University (Grant number AHNU-ET2021032). A whole genome shotgun (WGS) strategy was used with sequencing on the Illumina platform. The raw paired reads were quality-trimmed and assembled into the complete circular mitogenome in Novoplasty 2.7.2 using the mitogenome of Ampelophaga rubiginosa Bremer & Grey, 1853 as a reference (Nicolas et al. 2017).

The complete mitogenome of M. saishiuana (GenBank accession number MZ593602) was 15,662 bp and consisted of two ribosomal RNA genes (rRNAs), 22 transfer RNA genes (tRNAs), 13 protein-coding genes (PCGs), and one major non-coding region (the A + T-rich region). The overall base composition of the mitogenome was calculated to be A: 40.4%, T: 40.8%, C: 11.5%, and G: 7.3%. Its nucleotide composition is highly biased toward A + T nucleotides (81.2%). The majority strand (J-strand) encodes 23 genes (nine PCGs and 14 tRNAs), and the minority strand (N-strand) encodes 14 genes (four PCGs, eight tRNAs, and two rRNAs). Most of 13 PCGs start with ATN, except cox1, which starts with CGA. Having cox1 genes that start with CGA is common among Lepidopterans, especially for all the species in Sphingidae (Wang et al. 2021; Chen et al. 2022). All of the 13 PCGs stop with TAA or TAG codons or incomplete T, which is similar to most of other insect mitogenomes (Crozier and Crozier 1993; Korkmaz et al. 2015). All 22 tRNA genes usually found in the mitogenomes of insects are present in M. saishiuana. The nucleotide length of tRNA genes ranges from the shortest of 64 bp (trnC and trnI) to the longest of 71 bp (trnK and trnW), and A + T content ranges from 70.4% (trnK) to 91.4% (trnE).

To validate the phylogenetic position of M. saishiuana based on mitogenome, we selected the mitochondrial genome sequences of 32 species which were all Smerinthinae species currently deposited in the GenBank. The subfamily Langiinae was selected as outgroup. Nucleotide sequences were aligned by MUSCLE nested within MEGA X (Sudhir et al. 2018). Alignments of individual genes were then concatenated as a combined matrix with DAMBE 5.3.74 (Xia 2013). The maximum-likelihood (ML) tree was reconstructed under the W-IQ-Tree web server to validate the relationship among M. saishiuana and other sphingids under the best substitution models for each partition selected by W-IQ-Tree web server (Chernomor et al. 2016; Trifinopoulos et al. 2016; Kalyaanamoorthy et al. 2017; Minh et al. 2020). An ultrafast bootstrap (UFB) of 1000 replications was used in this analysis to assess branch supports (Hoang et al. 2018). The results based on mitogenome showed that M. saishiuana, M. gaschkewitschii, and M. sperchius formed a clade, and this indicated that M. saishiuana was a member of genus Marumba. Polyptychus trilineatus was the most closely related to genus Marumba on the phylogenetic tree reconstructed by mitogenomes (Figure 1).

Figure 1.

Phylogenetic relationships within Smerinthinae based on the nucleotide sequences were performed using ML methods. The following records were used: Adhemarius dariensis MK784108, Adhemarius dentoni MK804148, Ambulyx dohertyi MK804150, Ambulyx substrigilis MK804151, Amplypterus mansoni MK804152, Amplypterus panopus MK804153, Barbourion lemaii MK804154, Batocnema coquerelii MK804155, Clanis bilineata MK804156, Leucophlebia lineata MK804158, Orecta lycidas MK804159, Protambulyx astygonus MK804160, Protambulyx eurycles MK804161, Protambulyx ockendeni MK804162, Protambulyx strigilis MK804163, and Trogolegnum pseudambulyx MK804164 (Timmermans et al. 2019); Ambulyx liturata MT712132, Clanis deucalion MT712135, Langia zenzeroides MT922035, Marumba gaschkewitschii MT712137, and Marumba sperchius MT712138 (Wang et al. 2021); Kentrochrysalis streckeri MZ593600 (Huang et al. 2022); Smerinthus planus MZ593604 (Meng, Chen, et al. 2022); Ambulyx tobii MZ593597 (Meng, Lv, et al. 2022); Laothoe amurensis MZ593601 (Sun et al. 2022); Marumba saishiuana MZ593602 (This study); Ambulyx japonica MZ593596, Ambulyx liturata OK011996, Cypoides chinensis MZ593598, Parum colligata MG888667, Polyptychus trilineatus OK011999, Rhodoprasina callantha MZ343573, and Smerinthus caecus MZ593603 (unpublished).