Next-generation sequencing yields the complete mitochondrial genome of Rhodoprasina callantha (Lepidoptera: Sphingidae) and its evolutionary status.

In this study, we sequenced and analyzed the complete mitochondrial genome of Rhodoprasina callantha Jordan, 1929. The complete mitochondrial genome sequence of R. callantha was 15,343 bp in size and encoded 13 protein-coding genes (PCGs), two ribosomal RNA genes (rRNA), 22 transfer RNA genes (tRNA) and one control region (CR). The nucleotide composition of mitogenome was highly biased to A and T. Most protein-coding genes (PCGs) initiate with the standard start codon of ATN and terminate with the typical stop codon TAA/TAG. The phylogeny of Sphingidae based on nucleotide sequences of PCGs recovered the monophyly of subfamilies of Sphingidae with high support values. Langinae was the first subfamily diverged in Sphingidae, which was in accordance with previous study. R. callantha was the member of subfamily Smerinthinae, and closely related to the genus Marumba.

Hawkmoths belong to the family Sphingidae, order Lepidoptera, most of which are well-known flower visitors and significant pollinators, and most adults have well-developed proboscises (Krpač et al. 2019). When a hawkmoth drinks from a flower, the proboscis picks up pollen, which can be spread to flowers farther than 29 km away as the moth travels along its feeding route. Their larvae have cylindrical, medium to large sized bodies, generally with a single caudate scolus, and are known as significant agricultural pests (Nagamine et al. 2019). Among them, Rhodoprasina callantha Jordan, 1929 is an endemic species in Asia. It is distributed from northeastern India and Nepal across Bhutan, northern Myanmar/Burma, southwestern China (Yunnan), northern Thailand, northern Laos to northern and central-southern Vietnam. And it is a member of ‘Cypa group’ of which the relationship is still unclear. Up to date, the mitogenome of R. callantha remains unknown and the related molecular research of R. callantha is still sparse. Therefore, we documented the first complete mitogenome of R. callantha and reconstructed the phylogenetic tree of Sphingidae including R. callantha, in order to provide comprehensive data for this species and also to elucidate its phylogenetic position within the family Sphingidae.

The specimens of Rhodoprasina callantha Jordan, 1929 (Lepidoptera: Sphingidae) were collected from Menghai, Yunnan, China (21°41′53″N, 100°03′24″E) on 17 April 2021, and were 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. YN20210417. Animal sampling was performed according to the protocols approved by the Institutional Animal Care and Use Committee of Anhui Normal University (Grant number AHNU-ET2021032). Mitochondrial DNA was extracted from the leg of each male adult specimen. After cluster generation, the library preparations were sequenced on an Illumina platform and 150 bp paired-end reads were generated. The library was prepared from DNA of this species only. Raw data were retrieved and quantified by FastQC (Andrews 2020). Totally, 10 Gb data was sequenced and 3 Gb was randomly generated to assemble with mean coverage of 2000X. NovoPlasty and mitoz was used to assemble and annotate the mitogenome (Dierckxsens et al. 2017; Meng et al. 2019). Protein-coding genes (PCGs) were identified according to open reading frames of Theretra japonica (MG655620). The tRNAs were indicated by Mitos Web Server (Bernt et al. 2013). Geneious was used to verify the results of assembly and annotation (Kearse et al. 2012).

The genome sequence data that support the findings of this study are openly available in the GenBank of NCBI (https://www.ncbi.nlm.nih.gov/) under the accession NO. MZ343573. The length of the mitogenome of R. callantha was 15,343 bp. The mitogenome was circular and double-stranded. It was composed of 37 genes, including 13 PCGs (protein-coding genes), 22 tRNAs, two rRNAs and one control region, which were usually found in animal mitogenomes (Cameron 2014). The gene order is identical to the referenced sphingid Ampelophaga rubiginosa Bremer & Grey, 1853 and also consistent with other taxon in Sphingidae. The majority strand (J-strand) encoded 23 genes (9 PCGs and 14 tRNAs), and the minority strand (N-strand) encoded 14 genes (4 PCGs, 8 tRNAs and 2 rRNAs). The overall base composition of the mitogenome had a high AT content of 80.8%. Among the PCGs of R. callantha, 9 PCGs (NAD2, COX1, COX2, ATP8, ATP6, COX3, NAD3, NAD6, CYTB) were encoded by the J strand, while the rest four were encoded on the N strand. Most PCGs started with ATG or ATT and stop with TAA. However, the COX1 gene started with CGA and three genes (COX1, COX2, NAD5) used the incomplete stop codon T. The usage of start and stop codon were similar to most insect mitogenomes (Crozier and Crozier 1993; Korkmaz et al. 2015).

All 22 tRNA genes that were usually found in the mitogenomes of insects, resided in that of R. callantha. The length of tRNA genes ranged from 62 bp (trnS1) to 71 bp (trnK and trnV), and A + T content ranged from 73.1% (trnL2) to 92.4% (trnE). The length of two rRNA genes were 1293 bp (rrnL) and 770 bp (rrnS), and the A + T content were 83.7% (rrnL) and 85.3% (rrnS), respectively. The A + T-rich region of R. callantha was 340 bp and located between the rrnS and trnM. The A + T content of this region was 93.2%.

To validate the phylogenetic position of R. callantha, we selected the mitochondrial DNA sequences of 36 Lepidoptera species (34 Sphingidae as ingroup and two as outgroup). Biston panterinaria (Bremer et Grey, 1853) (GenBank accession number KU325533) and Phthonandria atrilineata (Butler, 1881) (GenBank accession number EU569764) from Geometridae which were confirmed as the most closely related to the superfamily Bombycoidea, were selected as outgroup (Yang et al. 2009). Every nucleotide sequences of PCGs were aligned by MAFFT (Katoh et al. 2005). Then the aligned sequences were concatenated into a dataset. The alignment of Sphingidae was deposited in the Science Data Bank, DOI 10.57760/sciencedb.01748. We analyzed the concatenated dataset of PCGs using the maximum-likelihood (ML) on the W-IQ-Tree web server method to reconstruct the phylogenetic relationship of R. callantha with other species of Sphingidae under the best-fit model: GTR + F + I + G4 chosen according to BIC selected by W-IQ-Tree web server (Trifinopoulos et al. 2016). The results recovered the monophyly of subfamilies of Sphingidae with high supporting values (Figure 1). Langinae was the first subfamily diverged in Sphingidae, which was in accordance with the results of Wang et al. (2021). R. callantha was the member of subfamily Smerinthinae, and closely related to the genus Marumba which was agreed with the morphological features (Kitching 2022).

Figure 1.

Maximum likelihood tree of Sphingidae based on the nucleotide sequences of 13 protein-coding genes dataset. The ultrafast bootstrap approximation (UFBoot) value was shown on the nodes (values below 75 were not shown).

Authors contribution

Yin-Feng Meng, Guo-Tao Lv, Xu Wang, Yi-Xin Huang and Yong-Ling Wu were involved in the conception and design, analysis and interpretation of the data; Xu Wang and Yi-Xin Huang collected insect; Yin-Feng Meng and Yong-Ling Wu drafted the paper and revised it critically for intellectual content. Yong-Ling Wu gave final approval of the version to be published. All authors agree to be accountable for all aspects of the work.