Complete mitochondrial genome of Austrapoda dentatus Oberthür, 1879 (Lepidoptera: Limacodidae)

Abstract

The phylogenetic relationships within Limacodidae remain unresolved, highlighting the need for expanded genomic resources. This study presents the first complete mitochondrial genome of Austrapoda dentatus, 15,354 bp in length and comprising 13 protein-coding genes, 22 tRNAs, two rRNAs, and an A+T rich region. The gene arrangement follows the typical Lepidopteran pattern. Phylogenetic analysis based on the 13 protein-coding genes supports A. dentatus and Apoda limacodes as sister taxa within the Apoda clade. These findings aid in understanding Limacodidae phylogeny and emphasize the importance of securing broader mitochondrial genome sequences across the family.

Introduction

The slug moth family Limacodidae is a diverse group comprising approximately 1,800 species across 310 genera (Cai and Yin 2024). These moths are distributed across various regions, with a particular concentration in tropical and subtropical areas (Epstein 1996; Solovyev 2014). Several species within this family are considered economically significant pests, primarily targeting trees and shrubs (Holloway 1986; Conant et al. 2002; Wu and Fang 2023). However, despite extensive research, the higher classification of Limacodidae remains a subject of debate (Zaspel et al. 2016; Lin et al. 2019). A recent large-scale study by Liang et al. (2024) provided new insights into the phylogeny of Limacodidae. Using approximately 171,000 bp of nuclear and mitochondrial DNA sequences from 126 species representing 67 genera and five morphologically defined groups, they identified six major clades within Limacodidae. Despite these advancements, additional molecular data from a broader range of taxa are still required to resolve taxonomic uncertainties and clarify phylogenetic relationships among these six lineages.

Austrapoda dentatus (Oberthür, 1879) is widely distributed across Korea, China, Japan, and southeastern Siberia (Shin 2001). The adult moth has a forewing length of 11–12 mm, with brown forewings featuring black patterns and a distinctive white spot at the base of each wing (Shin 2001). The larvae are found on various tree species, particularly chestnut, oak, cherry, apricot, and pear trees (Shin 2001; Solovyev 2008; Kim and Kim 2022). The species is generally referred to as A. dentatus, though some checklists list it under Heringodes.

To date, complete mitochondrial genome (mitogenome) sequences have been reported for only 16 species across 13 genera within the family Limacodidae, with no available data for any species of Austrapoda (as of 31 January 2025). In this study, we present the first complete mitogenome sequence of A. dentatus, characterizing its genomic features and comparing them with those of other Limacodidae species. Additionally, a phylogenetic analysis was conducted to determine the placement of Austrapoda within Limacodidae based on mitogenomic data. The newly sequenced A. dentatus mitogenome will contribute to species identification and enhance our understanding of phylogenetic relationships within the family.

Materials and methods

In July 2013, an adult male A. dentatus was collected using an insect net in Gwangui-myeon, Gurye-gun, Jeollanam-do, South Korea (35°16′36″ N, 127°28′41″ E). Species identification was carried out by S.-S. Kim, a specialist in Lepidoptera morphology, based on comprehensive examination of external morphological features, particularly wing shape, and coloration. The adult of A. dentatus displays an overall brown coloration, with noticeably darker thorax and forewings. The forewings are relatively quadrangular and exhibit a blackish-brown ground color with distinct black markings. A semicircular spot with white edging is located near the costal margin, while a broad, inverted V-shaped band stretches across the wing—darkened proximally and bordered distally with white, forming a distinctive pattern. A prominent white spot is present at the wing base, and the apex is distinctly darkened, which serves as a key diagnostic feature of the species (Figure 1). These characteristics are in full agreement with the original description and diagnostic traits outlined by Solovyev (2008).

Figure 1.

Adult male of Austrapoda dentatus Oberthür, 1879. Photographs of the adult specimen were taken by the author, Sung-Soo Kim, using a Nikon D300 camera equipped with a Micro Nikkor 105 mm VR (vibration reduction) lens. The male genitalia were photographed using the built-in digital camera of a Leica S9i stereomicroscope. (A) dorsal view, (B) ventral view of the adult male, (C) male genitalia, and (D) aedeagus.

Total DNA was extracted from the hind leg using the Wizard^™ Genomic DNA Purification Kit (Promega, Madison, WI). The voucher specimen and remaining DNA were deposited at Chonnam National University, Gwangju, South Korea, under the accession number CNU5715 (Iksoo Kim, ikkim81@chonnam.ac.kr). The complete mitogenome was amplified in three long overlapping fragments (LFs), from which 26 short overlapping fragments (SFs) were further amplified (Supplementary Figure S1; Supplementary Table S1) using the primer pairs described by Kim et al. (2012). DNA sequencing was performed using the Sanger method (Macrogen, Seoul). The full mitogenome was assembled by aligning the 26 short fragments with ∼100–200 bp of overlapping sequences between the ends of adjoining fragments, with the SeqMan software from the DNASTAR package (SeqMan NGen^®, version 13.0, DNASTAR, Madison, WI). Gene annotation, including the A + T-rich region, was conducted by aligning the assembled sequence with full-length mitogenomes of other lepidopterans using MAFFT version 7 (Katoh and Standley 2013). To ensure structural and functional integrity, all nucleotide sequences of protein-coding genes (PCGs) were translated using the invertebrate mitochondrial genetic code, confirming the absence of internal stop codon. Each tRNA gene and corresponding anticodon was identified using tRNAscan-SE, version 2.0 (Lowe and Eddy 1997).

For phylogenetic analysis, all currently available complete mitogenomes of 15 Limacodidae species were obtained from GenBank database (accessed on 27 February 2025). The nucleotide sequences of 13 PCGs (11,316 bp, including gaps) were used for phylogenetic reconstruction. Two Zygaenidae species, Erasmia pulchella Hope, 1840, and Histia rhodope Fabricius, 1775, were included as outgroups, as they belong to the Zygaenoidea superfamily, which also includes A. dentatus. A phylogenetic tree was constructed using IQ-TREE (Nguyen et al. 2015), implemented in PhyloSuite, version 1.2.3 (Xiang et al. 2023). PartitionFinder 2, with the Greedy algorithm (Lanfear et al. 2017), was used to determine the optimal partitioning scheme (seven partitions), and the GTR+I + G model was selected as the best-fit substitution model for all partitions.

Results

The complete mitochondrial genome of A. dentatus is 15,354 bp in length and contains the typical gene set found in lepidopterans: two rRNA genes, 22 tRNA genes, 13 PCGs, and an A + T-rich region (Figure 2). The COX1 gene initiates with the CGA codon, whereas the remaining PCGs begin with the standard ATN start codon. The COX2 gene ends with an incomplete stop codon (T), while all other PCGs end with TAA. The typical trnM-trnI-trnQ gene arrangement (underline indicating gene inversion), common in most lepidopterans, is present between the A + T-rich region and ND2 (Figure 2). The overall A + T content of the A. dentatus mitogenome is 80.93%, with individual components exhibiting the following A + T compositions: srRNA (85.64%), lrRNA (84.82%), tRNAs (82.02%), and PCGs (79.11%) (Supplementary Table S2).

Figure 2.

Circular map of the mitochondrial genome of Austrapoda dentatus generated using the GenomeVx tool (http://wolfe.ucd.ie/GenomeVx/). tRNA genes are labeled following 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.

Available complete mitogenome sequences of Limacodidae, including A. dentatus were clustered into three groups, each forming a strong monophyletic group (Figure 3). A. dentatus was placed within the Apoda clade, where it formed a sister group relationship with Apoda limacodes Hufnagel, 1766, with high nodal support (SH-aLRT = 99.9, UFBoot = 100; Figure 3).

Figure 3.

Maximum likelihood (ML) phylogenetic tree based on 15 mitochondrial genomes of limacodidae, including Austrapoda dentatus. Numbers at each node represent Shimodaira-Hasegawa-like approximate likelihood ratio test (SH-aLRT) and Ultrafast bootstrap (UFBoot) support values. The scale bar indicates the number of nucleotide substitutions per site. Erasmia pulchella and Histia rhodope, belonging to the Zygaenidae, were used as outgroups. The dataset includes the following mitochondrial genomes: Austrapoda dentatus (PV221946; this study), Chibiraga houshuaii (OR619569; Cai and Yin 2024), Iragoides fasciata (MK250437; Unpublished), Quasithosea sythoffi (MW813978; Unpublished), Thosea sinensis (MN661155; Jiang et al. 2023), Setora sinensis (OP160524; Jiang et al. 2024), Phlossa conjucta (OP132387; Jiang et al. 2024), Latoia hilarata (MK122617, Unpublished), Parasa consocia (KX108765, Liu et al. 2017), Parasa lepida (OP132386; Jiang et al. 2024), Phocoderma betis (OP919337; Unpublished), Monema flavescens (KU946971; Liu et al. 2016), Scopelodes kwangtungensis (OQ848600; Chu et al. 2025), Narosa fulgens (OP919326; Lin and Song 2024), Narosa nigrisigna (MH675969, Jiang et al. 2019), Apoda limacodes (OX291565, Broad et al. 2023), Erasmia pulchella (OQ134124; Zhang et al. 2024), and Histia rhodope (MF542357; Peng et al. 2017). The three major clades (Parasa, Phlossa, and Apoda) were defined by Liang et al. (2024).

Discussion and conclusions

The mitogenome of A. dentatus exhibits typical characteristics of Limacodidae in terms of size and A + T content across the whole mitogenome, PCGs, srRNA, lrRNA, tRNAs, and the A + T-rich region (Supplementary Table S2). For example, the overall A + T content of A. dentatus (80.93%) falls within the range observed in other Limacodidae species, such as Chibiraga houshuaii (79.83%) (Cai and Yin 2024) and Iragoides fasciata (82.03%) (Jiang et al. 2022). The gene order and orientation of the A. dentatus mitogenome are consistent with those of most lepidopterans (Jeong et al. 2021). However, this arrangement differs from the ancestral gene order found in the majority of insects and ancient lepidopteran groups (Boore 1999; Cao et al. 2012; Timmermans et al. 2014), as it features the trnM-trnI-trnQ arrangement at the A + T-rich region and ND2 junction (Figure 2) instead of the ancestral trnI-trnQ-trnM configuration. Phylogenetic analysis based on available mitogenome sequences placed A. dentatus within the Apoda clade, along with species from the genera Austrapoda and Narosa (Figure 3), supporting the findings of Liang et al. (2024). However, further genomic data are needed for the remaining clades (Cania, Phrixolepia, and Euphlyctinides), which are currently unrepresented in the mitogenome database. in consistent with three of the six clades (Parasa, Phlossa, and Apoda) proposed by Liang et al. (2024).

This study presents the first complete mitogenome of A. dentatus, making it the first representative of the genus Austrapoda. Despite the high species diversity within Limacodidae, research on its taxonomy and phylogeny remains limited. The mitogenome of A. dentatus provides a valuable foundation for further phylogenetic studies and taxonomic classification within Limacodidae and related taxa.

Funding Statement

This study was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science, and Technology (2015R1D1A3A03018119).

Acknowledgments

JSP, JSJ and IK designed the study. S-SK collected and identified the samples. JSP, JSJ and J-YP analyzed the data. JSP and IK drafted the manuscript. All authors contributed to discussions, manuscript revisions, and editing. All authors reviewed and approved the final manuscript.

Ethical approval

This study did not involve experiments requiring ethical approval. The species studied is not endangered and was not collected from nature reserves, and therefore no specific permits were required. All sample collection and sequencing procedures were conducted in strict compliance with local regulations and laboratory guidelines to ensure the preservation of wild resources.

Disclosure statement

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

Data availability statement

The genome sequence data supporting this study are available in NCBI GenBank database (https://www.ncbi.nlm.nih.gov) under accession number PV221946. Additionally, the corresponding chromatogram files are accessible via Mendeley Data at https://doi.org/10.17632/yxd96pnmbv.1.