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. 2026 Feb 1;11(3):324–328. doi: 10.1080/23802359.2026.2620168

Complete mitochondrial genome and phylogenetic analysis of the palecheek parrotfish Chlorurus japanensis (Bloch, 1789)

Qingyang Wu a,b, Lan Qiu b, Kai He a,✉, Zhiwei Zhang b,✉
PMCID: PMC12865835  PMID: 41641284

Abstract

We report the first complete mitochondrial genome of the palecheek parrotfish, Chlorurus japanensis. The 16,694 bp circular genome contains the standard vertebrate gene complement: 13 protein-coding genes, 22 transfer RNAs, two ribosomal RNAs, and one non-coding control region (D-loop). As is typical, most genes are located on the H-strand, with only the nd6 gene and eight tRNAs encoded on the L-strand. A tRNA gene rearrangement pattern conserved among parrotfishes was also identified. Phylogenetic analysis placed C. japanensis within the tribe Scarini, where it formed a well-supported clade with other Chlorurus species. This phylogenetic relationship reflects their shared ecological specialization in coral reef environments.

Keywords: Chlorurus japanensis, mitochondrial genome, phylogenetic analysis, Scaeidae

Introduction

Parrotfishes (Labriformes: Scaridae) are a diverse group of approximately 100 species across 10 genera (Comeros-Raynal et al. 2012; Rocha et al. 2012), inhabiting tropical and subtropical coastal oceans from the southeast Atlantic to Indo-Pacific regions (Bellwood 1994; Parrish 1993). They are distinguished by their dental plates, which are fused into a prominent beak used to scrape or excavate substrates like coral skeletons and limestone (Monod et al. 1994). Through this ecological specialization, parrotfishes exert a profound influence on their habitats, including controlling algal growth, mediating bioerosion, and promoting reef resilience (Bonaldo et al. 2014; Russ et al. 2015).

Chlorurus japanensis (Bloch, 1789), the palecheek parrotfish, is a reef-associated species distributed throughout the Western Pacific (Nicholson and Clements 2021). As a scraping parrotfish, it forages for benthic algae on limestone and dead coral substrates using its coalesced dental plates (Bonaldo et al. 2014; Clements et al. 2016). The species exhibits protogynous hermaphroditism (Kuwamura et al. 2020) and displays pronounced ontogenetic and environmental color variation (Randall and Choat 2008; Randall and Nelson 1979).

The mitogenome provides valuable insights into species identification, phylogenesis, and ecological adaption. A few studies have attempted to addressed these questions in parrotfishes, and most of them relied on mitochondrial or nuclear gene fragments, primarily due to the lack of complete mitogenome (Gao et al. 2023; Smith et al. 2008). In this study, we characterize the first complete mitochondrial genome for C. japanensis, and use it to estimate its phylogenetic position within parrotfishes.

Materials and methods

Sample collection and genomic DNA extraction

A live parrotfish was collected from Manbu Ansha near the Zhongsha islands, South China Sea [15°55′N, 114°29′E]. The fish was identified as female C. japanensis by Dr. Lan Qiu based on the taxonomic characteristics as described (Randall and Nelson 1979). Morphologically, the individual measured 27.60 cm in total length and exhibited two rows of cheek scales. A total of 13 pectoral fin rays were counted. The specimen displayed the typical characteristic of having teeth not covered by the lips, and was predominantly dark brown for most of the body, with a red-orange caudal fin (Figure 1). The fish was cold shocked and sacrificed, and muscle samples were dissected and cleaned with autoclaved artificial seawater. Samples were then dried with tissue paper and immediately flash-frozen in liquid nitrogen and stored at −80 °C. The samples were deposited in the Southern Marine Science and Engineering Guangdong Laboratory (Guangzhou) with the voucher number ZS-MB-2022-0042 (contact person: Xuanjin Luo, email: luoxuanjin@gmlab.ac.cn).

Figure 1.

Figure 1.

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Photograph of C. japanensis used in this work. The picture was provided by Dr. Lan Qiu. The individual showed typical characteristic of C. japanensis with teeth not covered by the lips. The fish was identified as female based on the entirely dark brown body color and red-orange caudal fin.

Mitochondrial genome sequencing and annotation

Muscle-derived genomic DNA was extracted using the TIANamp Genomic DNA Kit (TIANGEN, Beijing, China). DNA concentration and quality was assessed using a NanoDrop One (Thermo Fisher Scientific, Waltham, MA, USA). The sequencing libraries were prepared using the Rapid Plus DNA Lib Prep Kit (Illumina, San Diego, CA, USA) and sequenced using the DNB-SEQ-T7 (BGI, Shenzhen, China) platform with 150 bp paired-end reads. A total of 35.75 G raw data were generated and clean paired-end reads were obtained using Fastp v0.23.2 (Chen 2023). Mitogenome assembly and annotation were performed using MitoZ v3.6 (Meng et al. 2019) with default parameters. The results were validated using MitoFish DB Ver. 3.85 (Zhu et al. 2023). The coverage depth (Figure S1) was calculated with BedTools (Quinlan and Hall 2010) and the ggplot2 package was used for visualized illustration (Wickham 2011). Strand asymmetry in nucleotide composition was evaluated by calculating AT-skew [(A − T)/(A + T)] and GC-skew [(G − C)/(G + C)].

Phylogenetic analysis

Phylogenetic analysis was based on complete circular mitochondrial genome. The mitogenomes of a total of 19 species within Scaridae are available in the GenBank database and were included for phylogenetic relationship evaluation. We estimated the phylogenetic relationship of C. japanensis with the other 19 Scaridae species using a concatenated dataset of 13 protein-coding genes (PCGs), with a Labridae species Halichoeres margaritaceus as the outgroup. Extracted PCGs were aligned using MAFFT v7.313 (Katoh and Standley 2013). After that, PartitionFinder2 was used to determine the best evolutionary model and the best-fit model was GTR+I + G (Lanfear et al. 2017). We then used IQ-TREE v2.2.0 (Wei et al. 2024) to estimate the maximum likelihood (ML) tree, allowing the software to determine the best-fit partitioning scheme and substitution models. Branch support was evaluated with 1000 ultrafast bootstrap replicates.

Results

The mitochondrial genome of C. japanensis is 16,694 bp in length and the nucleotides are composed of 27.7% adenine (A), 25.8% thymine (T), 29.8% cytosine (C) and 16.8% guanine (G) (GenBank accession: PV743143). The skewness analysis shows that the AT skewness value is positive (0.036) and the GC skewness value is negative (−0.279). A total of 37 genes and one control region was annotated, including two rRNA genes, 13 PCGs, 22 tRNA genes, and one D-loop region. The nd6 gene and eight tRNA genes (tRNAGlu, tRNAPro, tRNAGln, tRNAAla, tRNAAsn, tRNACys, tRNATyr, and tRNASer) are encoded on the light chain, while the remaining genes are encoded on the heavy chain (Figure 1).

The 13 protein-coding genes (PCGs) spanned a total of 11,435 bp (Figure 2). The shortest gene was atp8 (168 bp) and the longest was nd5 (1839 bp). The majority of PCGs initiated with the ATG codon, except for cox1 (GTG) and atp6 (TTG). Complete termination codons included TAA (for atp8, nd1, nd4, nd4l, and nd5), TAG (for nd6), and AGG (for cox1). The other six genes (cox2, atp6, cox3, nd3, nd2, and cytb) featured incomplete termination codons (Table S1).

Figure 2.

Figure 2.

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The mitogenome map of C. japanensis. Genes are marked with different colors: green for NADH dehydrogenase (nd1-nd6 and nd4l), yellow for cytochrome c oxidase (cox1-cox3 and cytb), yellow-green for ATP synthase (atp6 and atp8), red for ribosomal RNA (12s rRNA and 16s rRNA) and blue for transfer RNA. The D-loop region is shown as gray. The outer ring shows the genes location, and the inner ring shows the GC content. The grey arrows indicate the orientation of gene transcription.

Maximum likelihood (ML) analysis resolved Scarini group as a monophyletic clade comprising seven species of the genus Scaurus and one species of the genus Bolbometopon. The analysis further indicated that C. japanensis, C. microrhinos, C. sordidus, and C. spilurus collectively formed the most highly supported branch (bootstrap value = 100). However, the internal relationships among C. microrhinos, C. sordidus, and C. spilurus are poorly resolved within this clade (bootstrap values < 70) (Figure 3).

Figure 3.

Figure 3.

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Phylogenetic trees using ML analyses based on 13 PCGs. The number on each node represent support value. The following sequences were used: FJ227899 (unpublished), FJ449707 (unpublished), FJ619271 (unpublished), OP056958 (unpublished), FJ595020 (unpublished), OP035141 (unpublished), AP006567 (Mabuchi et al. 2004), AP017568 (Mabuchi 2016), PV212379 (unpublished), PV743143 (this study), PV742864 (unpublished), KY235362 (Chiang et al. 2017), OP056868 (unpublished), OP056842 (unpublished), OP035290 (unpublished), OP056874 (unpublished), OP056872 (unpublished), OP056812 (unpublished), OP035255 (unpublished), OP056940 (unpublished) and PQ738625 (Huang et al. 2025).

Discussion and conclusion

This study reports the first complete mitochondrial genome of C. japanensis. Its mitochondrial genome has a comparable structure to the other 19 reported Scaridae species (Gao et al. 2023). C. japanensis also exhibits positive AT-skew and negative GC-skew (Gissi et al. 2008). Besides, a typical gene rearrangement of tRNA genes happened in parrotfish involved tRNAMet, tRNAIle, and tRNAGln was also evidenced in the mitogenome of C. japanensis (Mabuchi et al. 2004). Notably, the cox1 gene utilizes the non-canonical stop codon AGG, a variation that has also been observed in the mitochondrial genomes of some teleost and human (Saurer et al. 2023). A total of six PCGs demonstrate incomplete stop codons (Table S1). This is relatively common in fish mitochondrial genomes (Li et al. 2025) and is suggested to be completed by addition of polyadenylate tail during RNA processing (Satoh et al. 2016).

In this study, maximum likelihood (ML) reconstruction based on mitochondrial genomes revealed that our C. japanensis sample clustered within the Chlorurus genus as a basal lineage (Figure 3). However, the internal relationships within Chlorurus genus are poorly resolved with low nodal support. This may result from reticulation, historical hybridization or gene flow, indicating the potential limitation of mitochondrial genome data alone for phylogenetic study (Edwards et al. 2016; Sanderson et al. 2023). Future studies using more nuclear gene data and denser taxonomic sampling are needed to resolve the low supported phylogenetic relationships in this study. In conclusion, the mitochondrial genomes assembled in this study provide a valuable genomic resource for further investigations into the ecological roles and adaptive strategies of these species in their natural habitats.

Supplementary Material

Figure_S1.tif
TMDN_A_2620168_SM1896.tif (76.8MB, tif)

Acknowledgments

LQ collected and identified the sample; KH and ZZ conceived and designed the experiments; Data analysis and the initial drafting of the manuscript were performed by QW. KH and ZZ revised the manuscript. ZZ obtained funding and provided research resources. All authors participated in editing and finalizing the manuscript, and approved the final version for publication.

Funding Statement

This work was supported by Science and Technology Department of Guangdong Province (No: 2024A1515013196) and Guangzhou Nansha District Science and Technology Bureau Project (No: GML2024BZ0909).

Ethical approval

Sample collection in this study complied with the standards of the Chinese Association for Laboratory Animal Science and the Institutional Animal Care and Use Committee (IACUC). All the experimental procedures involving animals were approved by the Ethics Committee and Animal Welfare Committee of the Southern Marine Science and Engineering Guangdong Laboratory (Guangzhou). The data collection of this work was conducted with the permission of the Southern Marine Science and Engineering Guangdong Laboratory (Guangzhou) and Guangzhou University, and complied with national or international guidelines and legislation.

Disclosure statement

No potential conflict of interest was reported by the authors.

Data availability statement

The mitogenome is deposited in GenBank (accession PV743143). The associated BioProject, Bio-Sample and SRA number are PRJNA1271671, SAMN48884729 and SRR33823335, respectively.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure_S1.tif
TMDN_A_2620168_SM1896.tif (76.8MB, tif)

Data Availability Statement

The mitogenome is deposited in GenBank (accession PV743143). The associated BioProject, Bio-Sample and SRA number are PRJNA1271671, SAMN48884729 and SRR33823335, respectively.

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