Complete mitochondrial genome of the clearwing moth Synanthedon bicingulata (Lepidoptera: Sesiidae)

Abstract

The clearwing moth, Synanthedon bicingulata Staudinger, 1887 (Lepidoptera: Sesiidae), is a serious pest that infests cherry trees. This species, which is distributed in South Korea, was once regarded as S. hector found in Japan, but renamed as S. bicingulata based on morphology. Molecular data for this species are not available. Hence, we sequenced the 16,255-bp long complete mitochondrial genome (mitogenome) of S. bicingulata. Phylogenetic analyses in the Cossoidea superfamily supported monophyly of the Synanthedon genus and Synanthedonini tribe. This newly sequenced mitogenome of S. bicingulata will be useful for studies in a diverse field of evolutionary study.

Introduction

Synanthedon bicingulata Staudinger, 1887 (Lepidoptera: Sesiidae) is a major pest, which in its larval stage, causes wood-boring damage to trees of the Prunus genus, such as plum, peach, and cherry. S. bicingulata is a major cause for concern in South Korea because cherry trees account for the highest proportion of planted tree species in the natural landscape (Korea Forest Service 2023).

S. bicingulata, which is distributed in South Korea, China, and Russia, has been confused with its congener S. hector Butler, 1878 that is distributed in Japan because of the superficial similarities between them (Matsumura 1931). This was later revised, and the species was renamed S. bicingulata based on the morphological characteristics of the adults, including genital features, as well as those of the larvae and pupae (Arita et al. 2004; Lee et al. 2004). However, no sequencing information for S. bicingulata is available. Moreover, a very limited number of cox1 sequences for the tribe Synanthedonini, in which S. bicingulata is included, and the mitochondrial genomes (mitogenomes) of only four of approximately 287 Synanthedon species (Pühringer and Kallies 2004; Zheng et al. 2022) are available as public data to date.

In this study, we sequenced the complete mitogenome of S. bicingulata and scrutinized the major features of the genome to illustrate the evolutionary characteristics of the species. We also compared the baseline genomic characteristics and performed phylogenetic analysis in the context of the Cossoidea superfamily, to which S. bicingulata belongs. The information regarding the newly sequenced complete mitogenome of S. bicingulata will be useful for species identification and phylogenetic study of the Synanthedon genus and Synanthedonini tribe, which include a substantial number of species.

Materials and methods

In April 2023, male adult clearwing moths (S. bicingulata) were collected from Suncheon-si, South Korea (34°58′20.9″ N, 127°23′34.4″ E) using a species-specific sex pheromone trap (GreenAgrotech, Gyeongsan, South Korea). Several larvae feeding underneath the bark of a cherry tree that was leaking dark brown gum were also collected (Figure 1). The adults were identified by one of the authors (Iksoo Kim) based on the morphological characteristics, including the vivid yellow stripes that are found distally on the fourth and fifth abdominal tergites (Lee et al. 2004).

Figure 1.

Images of Synanthedon bicingulata Staudinger, 1887. (A) Male adult. (B) Larva caught from underneath the bark of a cherry tree. The photos were taken by the author, Woo Jin Kim.

Total DNA was extracted from the hind leg of the male adult insects using the Wizard™ Genomic DNA Purification Kit (Promega, Madison, WI). This voucher specimen and leftover DNA were deposited at the Jeollanam-do Forest Research Institute, Jeollanam-do, South Korea under the accession number SC(II)-0426 (Woo Jin Kim, littleboots@korea.kr).

The complete mitogenome was amplified into three long overlapping fragments (LFs). Subsequently, 26 short overlapping fragments were amplified using the LFs as templates (Figure S1). The primers reported by Kim et al. (2012) and two newly designed primers were utilized (Table S1). Sequencing was conducted using Sanger’s sequencing method. The full-length mitogenome was assembled using the SeqMan software from the DNASTAR package (SeqMan NGen^®, version 13.0, DNASTAR, Madison, WI) by aligning overlapping sequences of adjacent fragments. Individual genes and the A + T-rich region were annotated by aligning homologous sequences of known full-length lepidopteran mitogenomes using MAFFT, version 7 (Katoh and Standley 2013). The nucleotide sequences of protein-coding genes (PCGs) were translated based on the invertebrate mitochondrial DNA genetic code to check for any unconventional sequences, possibly from nuclear-embedded mitochondrial DNA sequences. tRNA genes were identified using tRNAscan-SE, version 2.0 (Lowe and Eddy 1997). The sequence data were deposited in the GenBank database under the accession number PP622747.

A total of 12 mitogenomes in the Cossoidea superfamily were downloaded from GenBank, and the baseline information of S. bicingulata and the Cossoidea members was analyzed. Phylogenetic analysis was conducted using the nucleotide sequences of 13 PCGs and two rRNAs of the Cossoidea members (13,592 bp including gaps) with the inclusion of an outgroup, Leguminivora glycinivorella, belonging to the Tortricoidea superfamily. For this, Bayesian inference (Ronquist et al. 2012) and IQ-TREE (Nguyen et al. 2015), integrated into PhyloSuite, version 1.2.3 (Xiang et al. 2023) were used. An optimal partitioning scheme (nine partitions) and substitution model were determined using PartitionFinder 2 with the Greedy algorithm (Lanfear et al. 2017).

Results

The 16,255-bp long complete mitogenome of S. bicingulata contains two rRNAs, 22 tRNAs, 13 PCGs, and an A + T-rich region (Figure 2). cox1 starts with the CGA codon and atp8, with the TTG codon. The remaining PCGs start with typical ATN codons. cox1, nad5, and nad4 have the incomplete stop codon T, whereas the remaining PCGs terminate with TAA (Table 1). The S. bicingulata mitogenome has a rearranged gene block (trnQ-trnS₂-trnM-trnI [underlined gene names indicate a counter-clockwise transcriptional direction]) between the A + T-rich region and nad2, differing from the typical arrangement of trnM-trnI-trnQ found in a majority of the lepidopterans (Figure S2). The A/T content was highest in the A + T-rich region (94.1%), followed by that in 16S rRNA (85.1%), 12S rRNA (85.0%), 22 tRNAs (82.4%), whole genome (79.7%), and 13 PCGs (76.7%) (Table S2).

Figure 2.

Circular map of the mitochondrial genome of Synanthedon bicingulata obtained using the GenomeVx tool (http://wolfe.ucd.ie/GenomeVx/). tRNA abbreviations follow the IUPAC-IUB one-letter code. trnL₁, trnL₂, trnS₁, and trnS₂, denote tRNA^Leu(CUN), tRNA^Leu(UUR), tRNA^Ser(AGN), and tRNA^Ser(UCN), respectively. Gene names outside the circular map indicate transcription in a clockwise direction, excluding the A + T-rich region, while those inside the map indicate transcription in a counter-clockwise direction.

Table 1.

Summary of Synanthedon bicingulata mitochondrial genome.

Gene	Nucleotide number	Size (bp)	Anticodon	Codon		O/S
				Start	Stop
trnQ	1–67	67	TTG	–	–
trnS2	114–178	65	TGA	–	–	−46
trnM	181–247	67	CAT	–	–	−2
trnI	257–320	64	GAT	–	–	−9
nad2	351–1343	993	–	ATA	TAA	−30
trnW	1345–1411	67	TCA	–	–	−1
trnC	1404–1478	75	GCA	–	–	+8
trnY	1479–1542	64	GTA	–	–
cox1	1546–3076	1531	–	CGA	T–tRNA	−3
trnL2	3077–3140	64	TAA	–	–
cox2	3141–3821	681	–	ATA	TAA
trnK	3823–3893	71	CTT	–	–	−1
trnD	3894–3960	67	GTC	–	–
atp8	3961–4119	159	–	TTG	TAA
atp6	4113–4790	678	–	ATG	TAA	+7
cox3	4796–5590	795	–	ATG	TAA	−5
trnG	5595–5658	64	TCC	–	–	−4
nad3	5659–6012	354	–	ATT	TAA
trnA	6013–6076	64	TGC	–	–
trnR	6082–6144	63	TCG	–	–	−5
trnN	6151–6215	65	GTT	–	–	−6
trnS1	6214–6272	59	GCT	–	–	+2
trnE	6279–6343	65	TTC	–	–	−6
trnF	6344–6410	67	GAA	–	–
nad5	6411–8142	1732	–	ATT	T–tRNA
trnH	8143–8209	67	GTG	–	–
nad4	8210–9551	1342	–	ATG	T–tRNA
nad4l	9552–9836	285	–	ATG	TAA
trnT	9842–9906	65	TGT	–	–	−5
trnP	9906–9971	66	TGG	–	–	+1
nad6	10,018–10,503	486	–	ATA	TAA	−46
cytb	10,511–11,662	1152	–	ATG	TAA	−7
nad1	11,700–12,635	936	–	ATG	TAA	−37
trnL 1	12,637–12,704	68	TAG	–	–	−1
16S rRNA	12,735–14,022	1288	–	–	–	−30
trnV	14,671–14,737	67	TAC	–	–	−648
12S rRNA	14,738–15,496	759	–	–	–
A + T-rich region	15,497–16,255	759	–	–	–

Gene names that are not underlined indicate a clockwise transcriptional direction, whereas those that are underlined indicate a counter-clockwise transcriptional direction. tRNAs abbreviations follow the IUPAC-IUB one-letter code. trnL₁, trnL₂, trnS₁, and trnS₂ denote tRNA^Leu(CUN), tRNA^Leu(UUR), tRNA^Ser(AGN), and tRNA^Ser(UCN), respectively. O/S, number of overlapping (+)/intergenic spacer (−) sequences.

The S. bicingulata mitogenome contains unusually long intergenic spacer sequences at the 16S rRNA and trnV junction (648 bp; Table 1), comprising seven tandemly repeated sequences that are near-even at 60–64 bp, encompassed at each end by non-repeat sequences (Figure S3).

Phylogenetic analysis showed that the Synanthedon genus, Synanthedonini tribe, Sesiinae subfamily, and Sesiidae family, to which S. bicingulata belongs, are monophyletic groups with the highest nodal supports (Figure 3). S. bicingulata was placed as the sister to the group containing S. anderenaeformis and S. myopaeformis, but nodal support for this relationship was extremely low. Currently, the Synanthedon genus comprises 287 species (Pühringer and Kallies 2004; Zheng et al. 2022). Thus, the limited number of species included in this study may be the reason for this low support. Further studies scrutinizing an extended taxon diversity are required for better understanding.

Figure 3.

Phylogeny of 13 mitochondrial genomes of members of the Sesiidae and Cossidae families in the Cossoidea superfamily, including Synanthedon bicingulata, derived using maximum-likelihood and Bayesian’s inference methods. The numbers at each node are Shimodaira–Hasegawa-like approximate likelihood ratio test (SH-aLRT)/Ultrafast bootstrap (UFBoot)/Bayesian’s posterior probabilities (BPPs). The scale bar indicates the number of substitutions per site. Leguminivora glycinivorella (MZ506769; unpublished) belonging to the Tortricoidea superfamily, was used as an outgroup. The following sequences were used: Synanthedon bicingulata (PP622747; this study), Synanthedon vespiformis (OU906976; Boyes and Lees 2022), Synanthedon formicaeformis (OX243984; Langdon and Fagan 2023), Synanthedon andrenaeformis (OW387807; unpublished), Synanthedon myopaeformis (OX122944; unpublished), Bembecia ichneumoniformis (OU342551; Boyes 2023), Sesia bembeciformis (OX031055; Boyes and Langdon 2023), Sesia siningensis (MN708363; Yan et al. 2019), Eogystia hippophaecolus (KC831443; Gong et al. 2014), Chalcidica minea (KX364097; Li et al. 2018), Endoxyla cinereus (OK644702; unpublished), Zeuzera multistrigata (KX364098; Li et al. 2018), Zeuzera multistrigata (MF491642; Kim et al. 2017), and Leguminivora glycinivorella (MZ506769; unpublished).

Discussion and conclusions

The length and A + T content of the whole genome, genes, and the A + T-rich region are well within the range found in other members of the Cossoidea superfamily (Table S2). The A + T content of a majority of the Cossoidea members is higher in 12S rRNA than that in 16S rRNA, but Zeuzera species in the Zeuzerinae subfamily and the current S. bicingulata have higher A + T content in 16S rRNA (Table S2).

The rearranged gene block found in S. bicingulata mitogenome (trnQ-trnS₂-trnM-trnI) differs from the typical trnM-trnI-trnQ arrangement at the same junction, which is regarded as a derived character in lepidopterans, with a few exceptions (Figure S2; Cao et al. 2012; Timmermans et al. 2014). The translocation of trnQ and trnS₂ could be the prime events involved in this new arrangement. Considering all six species, including S. bicingulata in the Synanthedonini tribe, have the same rearrangement at this junction, it could be a shared-derived character for this tribe. However, further studies are required to confirm this hypothesis.

The 648-bp long intergenic spacer sequences containing seven near-even tandem repeat sequences between 16S rRNA and trnV are unique (Figure S3) because other species in the Cossoidea superfamily have a maximum of 38-bp long intergenic spacer sequences at the same junction without any repeat sequence (Endoxyla cinereus; GenBank accession number OK644702; unpublished). This region could potentially be a prime target site for species identification. Previous phylogenetic studies involving Synanthedon and Synanthedonini did not include S. bicingulata (Liang et al. 2022; Cognato et al. 2023). Thus, the current S. bicingulata mitogenome will be useful for future phylogenetic analysis of Synanthedon and Synanthedonini. Moreover, the S. bicingulata mitogenome sequences, particularly the 648-bp long intergenic spacer sequences, will be useful for species identification.

Funding Statement

This research was funded by the Ministry of Agriculture, Food, and Rural Affairs [Grant Number 321001-03].

Author contributions

Woo Jin Kim and Iksoo Kim designed the study. Woo Jin Kim collected the samples. Seung Hyun Lee, Jeong Sun Park, and Woo Jin Kim conducted the experiments and analyzed the data. Woo Jin Kim and Iksoo Kim prepared the manuscript draft. All the authors participated in the discussion and revision of the manuscript. All authors read, revised, and approved the final manuscript.

Ethical approval

This research does not involve experiments that require ethical review. The study species is not endangered or collected in nature reserves; hence, specific permissions were not required. All acquisition and sequencing were carried out in strict compliance with the relevant local laws and laboratory regulations to preserve wild resources.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

The genome sequence data that support the findings of this study are openly available in GenBank of NCBI (https://www.ncbi.nlm.nih.gov) under the accession number PP622747.