Complete mitochondrial genome of Acanthochitona defilippii (Polyplacophora: Chitonida) from South Korea

Abstract

The chiton (Polyplacophora) occupies a significant position in molluscan evolutionary history as one of the most primitive groups within the phylum Mollusca. Acanthochitona defilippii (Tapparone-Canefri 1874) (Chitonida: Acanthochitonidae) is a commonly found intertidal chiton species in South Korea. In this study, we characterized the complete mitochondrial genome of A. defilippii (14,999 bp long), comprising 13 protein-coding genes (PCGs), 22 transfer RNA genes, two ribosomal RNA genes, and an A + T rich region (166 bp). The base composition is as follows: 31.82% for A, 11.63% for C, 16.69% for G, and 39.86% for T. We reconstructed a maximum likelihood (ML) tree to elucidate phylogenetic relationships among the eight chitonid families using the nucleotide sequences of all PCGs. The ML tree revealed that A. defilippii clustered with Acanthochitona avicula (BP 100) within the family Acanthochitonidae. Acanthochitonidae formed a sister group with Mopaliidae. The results could provide a valuable understanding the phylogenetic relationships of chitonid species.

Introduction

The genus Acanthochitona (Polyplacophora: Chitonida: Acanthochitonidae) consists of 83 species and is widely distributed in various regions, including the Southern Hemisphere and the northeastern Atlantic. Among them, the chiton Acanthochitona defilippii (Tapparone-Canefri 1874) is widely distributed in the intertidal zone across Japan, China, and the Indo-Pacific region, and it is among the most common intertidal species in South Korea (Hong and Van Belle 1990). They are small to medium-sized creatures with elongated oval bodies covered in eight overlapping, articulated plates. Their palates are small, and they have a wide, fleshy girdle with nine pairs of suture bundles and various short spicules, featuring spines that range in color from dark brown to green and white (Yeh et al. 2005) (Figure 1). A. defilippii is known for maintaining its past life history and ecological characteristics for approximately 300 myr, rendering chitons as living fossils and holding a significant position in molluscan evolutionary history as the most primitive group (Scherholz et al. 2013). Despite their importance in the evolutionary aspect, few studies reported with respect to the phylogenetic relationships of the species. In recent years, mitochondrial genome (mitogenome) data have emerged as a powerful tool across various scientific disciplines, including molecular phylogenetics and population genetics (Lee et al. 2012; Kim et al. 2019; Choi, Choi, et al. 2021; Choi and Hwang 2021; Choi, Yeo, et al. 2021; Park et al. 2021; Akintola et al. 2022; Choi and Hwang 2023). Until now, in the genus Acanthochitona, there have been published only two complete mitogenomes from Acanthochiona rubrolineata and Acanthochitona aviula. In this study, we aimed to provide a foundational basis for the molecular investigation of A. defilippii by characterizing a complete mitochondrial genome and elucidating its phylogenetic position. It is the third Acanthochitona mitogenome to be completely characterized.

Figure 1.

A Photograph of Acanthochitona defilippii attached on a rock surface. The photo was taken by the author (I Hyang Kim).

Materials and methods

The specimen of A. defilippii was collected from Seogwipo, Jeju Island, South Korea (33°14′24″N, 126°32′44″E) and preserved in absolute ethanol. It was deposited under the voucher number LEGOM040550 at Kyungpook National University, Daegu, South Korea (Prof. Ui Wook Hwang, uwhwang@knu.ac.kr). Genomic DNA was extracted from the foot of the specimen using a DNeasy Blood and Tissue kit (Qiagen, Hilden, Germany). The quality and quantity of DNA were checked using Nanodrop4000 (Thermo Fisher Scientific, United States). For genome sequencing, Illumina TruSeq library was constructed with an average insert size of 350 bp using TruSeq DNA Nano 350 bp kit (Illumina, United states). After library preparation, the sequencing was performed using the Illumina NovaSeq 6000 platform with the production of paired-end reads length of 151 bp in DNA Link Inc. (Seoul, South Korea). The detailed information of depth and coverage for sequencing is displayed as a plot in Supplementary Figure 1. The sequences were assembled using NOVOPlasty 4.3.5. (Dierckxsens et al. 2017) with the mitogenome of Acanthochitona avicula (Irisarri et al. 2020) as a reference genome. 22 tRNA genes were predicted using tRNAscan-SE (Chan and Lowe 2019) and ARWEN (Laslett and Canbäck 2008). The 13 protein-coding genes (PCGs) and two rRNA genes were searched using EMBOSS Transeq (Madeira et al. 2022) and the MITOS web server (http://mitos.bioinf.uni-leipzig.de/) (Bernt et al. 2013). After annotation, the circular mitogenome of A. defilippii was visualized using Proksee (Grant et al. 2023). The phylogenetic tree was reconstructed using the maximum likelihood (ML) method through the IQ-TREE web server (http://iqtree.cibiv.univie.ac.at/) (Trifinopoulos et al. 2016). For the sequence alignment set, mitogenome data from 22 polyplacophoran specimens were retrieved from the NCBI GenBank database. Scutopus ventrolineatus was used as an outgroup in the phylogenetic analysis.

Results

We successfully sequenced the complete mitogenome of A. defilippii, which is 14,999 bp in length (Figure 2). It has been deposited in the GenBank database (accession number: PP419021). The overall base composition of the mitogenome was 31.82% for A, 11.63% for C, 16.69% for G, and 39.86% for T, with a GC content of 28.33%. Gene annotation revealed that it encodes 13 PCGs, 22 tRNAs, and two rRNAs. All PCGs start with a typical ‘ATG’ codon. As for the stop codon, four PCGs (ND1, ND2, ND4, and ND5) are used a ‘TAG’ stop codon and eight PCGs (ATP6, ATP8, COX1, COX2, COX3, CytB, ND3, ND4L) end with a ‘TAA’ stop codon. The remaining one, ND6, has a stop codon that ends with T–. The A + T rich region is 166 bp in length, located between tRNA-Glu and COX3. The 12S rRNA and the 16S rRNA genes are 854 bp and 1273 bp long, respectively, and separated by tRNA-Val. All tRNAs, except for tRNA-Ser2, have the typical cloverleaf structure ranging from 58–68 bp in length.

Figure 2.

Circular map of the complete mitochondrial genome of acanthochitona defilippii. The total length of the complete mitochondrial genome is 14,999 bp. Genes located on the outer side (ATP6, ATP8, COX1–3, ND2, and ND3) are on the heavy strand, while those on the inner side (CytB, ND1, ND4, ND4L, and ND6) are on the light strand. The inner circle illustrates the GC-skew, representing the variation from the overall average GC content across the entire sequence. The complete mitochondrial genome map was visualized using the Proksee program (Grant et al. 2023).

Phylogenetic relationships among 23 polyplacophoran species were investigated, including 19 species of Chitonida, 1 of Callochitonida, and 3 of Lepidopleurida (Figure 3). Most of the clades in the ML tree were supported with high bootstrapping values ranging from BP 70 to BP 100. Chitonida is largely divided into two groups, and within that group, Acanthochitonina and Chitonina belongs to each suborder. A. defilippii clustered with A. avicula within the family Acanthochitonidae with a high confidence value (BP 100) within the suborder Acanthochitonina. Nuttallochiton mirandus, belonging to the Mopaliidae, appeared as a sister taxon of the two Acanthochitona species (BP 100). Interestingly, the family Mopaliidae, belonging to the suborder Acanthochitonina, was not supported as monophyletic, which was divided into three different genetic lineages.

Figure 3.

A Maximum-likelihood tree reconstructed with nucleotide sequences of 13 mitochondrial PCGs from 23 chitonid species. The phylogenetic dataset consists of Chitonida (19 species), Callochitonida (1 species), and Lepidopleurida (3 species). The gray square box represents the examined species, A. defilippii in this strand. Scutopus ventrolineatus (caudofoveata) was set as an outgroup species. The number next to or below the species name indicated the accession number in GenBank. The numbers above each branch indicate the bootstrap support value. The following sequences were used: Acanthochitona avicula NC047426 (Irisarri et al. 2020), Acanthopleura echinate MN864062 (Irisarri et al. 2020), Acanthopleura loochooana OM047184 (unpublished), Acanthopleura vaillanti OQ355692 (Alnashiri et al. 2024), Callochiton steinenii MN864061 (Irisarri et al. 2020), Chiton albolineatus NC047425 (Irisarri et al. 2020), Cryptochiton stelleri NC026850 (Irisarri et al. 2014), Cyanoplax caverna NC026848 (Irisarri et al. 2014), Dendrochiton gothicus NC047424 (Irisarri et al. 2020), Hanleyella oldroydi NC047423 (Irisarri et al. 2020), Katharina tunicata NC001636 (Boore and Brown 1994), Lepidozona coreanica NC046935 (Sun et al. 2023), Leptochiton nexus NC047422 (Irisarri et al. 2020), Mopalia retifera NC068065 (unpublished), Nierstraszella lineata NC047421 (Irisarri et al. 2020), Nuttallina californica NC026849 (Irisarri et al. 2014), Nuttallochiton mirandus MN864053 (Irisarri et al. 2020), Plaxiphora albida MN864050 (Irisarri et al. 2020), Scutopus ventrolineatus NC025284 (Osca et al. 2014), Sypharochiton pelliserpentis NC024174 (Veale et al. 2016), Sypharochiton sinclairi NC024173 (Veale et al. 2016), Tonicia forbesii MN864054 (Irisarri et al. 2020) and Tonicina zschaui MN864051 (Irisarri et al. 2020).

Discussion and conclusion

We characterized the complete mitogenome of A. defilippii and the arrangement of the 13 PCGs in order and strand position was identical to that of A. avicula. The ML phylogeny based on the 13 PCGs in this study (Figure 3) places A. defilippii within the family of Acanthochitonidae (BP 100). Acanthochitonidae was clustered with Mopaliidae, but the monophyly of Mopaliidae was not supported, which the taxon was divided into three different groups. The phylogenetic relationship was also reported in Irisarri et al. (2020). There requires further examination for the phylogenetic relationship of the family Mopaliidae with a reexamination of taxonomic status based on an integrative approach based in morphological and molecular characters. The mitogenome information of A. defilippii reported here could contribute to a comprehensive understanding of acanthochitonid phylogeny and chitonid evolutionary history.

Funding Statement

This work was supported partly by a grant to UWH from the National Institute of Biological Resources (NIBR) funded by the Ministry of Environment (MOE), South Korea [NIBR202333201], and partly by a grant to EHC from the National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT [2021R1C1C2012882].

Authors contributions

UWH and EHC designed the study. IHK and UWH wrote the manuscript. IHK, CRS, GK, BP and KBK carried out the sampling, molecular experiments, and data analyses. All authors revised the manuscript and agreed to be responsible for all aspects of the work.

Ethical approval

The material involved in this article does not involve any ethical conflicts. This species is not endangered according to the CITES catalog or IUCN Red List, and the sample was not collected from a natural reserve, so the collection did not require any specific permissions or licenses.

Data availability statement

The data that support the findings of this study are openly available in GenBank of NCBI at https://www.ncbi.nlm.nih.gov/, under the accession number PP419021. The associated BioProject, Bio-Sample, and SRA numbers are PRJNA1085534, SAMN40304573, SRR28268872 respectively.

Disclosure statement

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.