https://orcid.org/0000-0002-9478-359X
Figures
Abstract
Sea cucumbers, as typical representatives of deep-sea benthic animals, possess significant scientific and economic importance. This investigation examined the genomic structural characteristics and genetic evolutionary relationships of Holothuria atra through the analysis of its complete mitochondrial genome. The findings indicated that the total length of the mitochondrial genome of the H. atra specimen was 15,788 bp, encompassing seven NADH genes, three cox genes, two ATP genes, and one cob gene. Additionally, two rRNAs and 22 tRNAs were identified. The entire sequence contained a total of 18 non-coding regions and seven overlapping gene regions, with the combined A + T content reaching 59.2%. The lengths of the 22 tRNAs ranged from 62 to 72 base pairs, and their cloverleaf secondary structures were predicted. Regarding codon usage, the PCGs within the mitochondrial genome of H. atra utilized 61 codons, encoding information for 20 amino acids. The most abundantly encoded amino acid in the mitochondrial genome of H. atra is leucine (Leu), representing 16.58%, while cysteine (Cys) is the least represented, accounting for 1.03%. Codons with higher usage frequencies include AGA (Ser 1), CUA (Leu 1), and CCA (Pro), whereas those with comparatively lower frequencies are GCG (Ala), CCG (Pro), and AGG (Ser 1). Evolutionary analysis revealed that H. atra is most closely related to Holothuria polii. By comparing the mitochondrial genomic sequences of 19 species within the class Holothuroidea, it was observed that eight mitochondrial sequences are shared among these species. This study provides valuable data supporting genetic evolutionary research and the development and utilization of H. atra resources within the Kiribati region.
Citation: Wang J, Ge J, Liao M, Li B, Rong X, Wang Y, et al. (2026) The complete mitochondrial genome of Holothuria atra (Holothuriida: Holothuriidae: Holothuria) and its structural characteristics. PLoS One 21(4): e0347494. https://doi.org/10.1371/journal.pone.0347494
Editor: Tzong-Yueh Chen, National Cheng Kung University, TAIWAN
Received: September 8, 2025; Accepted: April 1, 2026; Published: April 17, 2026
Copyright: © 2026 Wang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: The complete mitochondrial genome sequence data that support the findings of this study are available in GenBank of NCBI database (https://www.ncbi.nlm.nih.gov/) under the accession number PV998923.
Funding: This research was financially supported by the National Key R&D Program of China (2022YFD2400105, PI: J.G.), Central Public-interest Scientific Institution Basal Research Fund, CAFS (2023TD29, PI: X.R.), Central Public-interest Scientific Institution Basal Research Fund, YSFRI, CAFS (20603022025016, PI: A.C.) and Research Fund of State Key Laboratory of Mariculture Biobreeding and Sustainable Goods (BRESG-JB202508, PI: M.L.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
1. Introduction
Sea cucumbers are marine animals classified within the class Holothuroidea of the phylum Echinodermata, with approximately 1,400 species identified globally [1]. These organisms are significant not only as marine food sources but also as valuable medicinal resources [2]. The nutritional and therapeutic advantages of sea cucumbers stem from their rich content of bioactive substances, which include carotenoids, vitamins, fatty acids, gelatin, amino acids, bioactive peptides, minerals, collagen, chondroitin sulfate, and fucoidan, among others [3,4]. Holothuria atra, a member of the phylum Echinodermata, Holothuroidea, and Holothuria, is an edible species predominantly found in coral reef environments and sandy substrates characterized by calm waters, abundant seagrass, and high organic matter content. This species is widely distributed throughout the Indian-West Pacific region [5,6].
Mitochondrial DNA (mtDNA) is characterized by strict maternal inheritance and a simple structure, small molecular weight, lack of tissue specificity, and a rapid evolutionary rate. It is widely utilized in studies concerning species origin, genetic differentiation, phylogenetic relationships both within and between species, and species identification [7]. Analyzing mtDNA not only serves as a routine method for investigating genetic diversity in species populations but has also emerged as a significant focus of research in evolutionary biology, genomics, and bioinformatics. The complete mitochondrial genome provides valuable information on individual genes and encompasses broad genomic features that are essential for studying genome evolution, phylogenetic relationships, and species identification [8–10]. Moreover, characteristics such as ribosomal RNA (rRNA), transfer RNA (tRNA), gene arrangement, and protein-coding genes (PCGs) in animal mitochondrial genomes are critical topics in the exploration of gene function and genetic variation [11,12].
In recent years, the complete mitochondrial genomes of several sea cucumber species, including Bohadschia argus, Holothuria polii, and Benthodytes marianensis, have been sequenced and assembled [13–15]. Furthermore, the complete genome sequencing and assembly of Apostichopus japonicus, Chiridota heheva, and Stichopus monotuberculatus have been accomplished, which elucidates the functions of various genes and the underlying genetic mechanisms [16–18]. However, research on H. atra has primarily concentrated on its nutritional components and bioactive substances, with limited studies addressing its mitochondrial genome or population genetic diversity [19,20]. Therefore, this study utilized high-throughput sequencing technology to assemble the mitochondrial genome of H. atra and conducted analyses of its base composition, codon usage patterns, gene arrangement, phylogenetic relationships, and population genetic diversity. These findings provide valuable scientific data to support genetic research and conservation efforts for H. atra populations in the Kiribati region.
2. Materials and methods
2.1 Sample source
A total of 30 Wild H. atra specimens were collected from the sea area of Kiribati (1°22’ N, 173°8’ E). The sampling was conducted with the permission of The Fisheries and Marine Resources Development Department of The Republic of Kiribati. The length of the collected individuals was 24.32 ± 6.12 cm, the width was 6.66 ± 0.63 cm, and the weight was 182.50 ± 102.58 g. The sex ratio of distinguishable individuals was 11 females to 2 males. Following their capture from the natural habitat, the specimens were transported to the laboratory, where longitudinal muscle tissues were meticulously dissected and preserved in absolute ethanol for further analysis.
2.2 DNA sequencing and genome assembly
Total genomic DNA was extracted from the longitudinal muscle tissue using the DNeasy tissue kit (Qiagen, Beijing, China), adhering to the manufacturer’s protocols. Following DNA isolation, 1 μg of purified DNA was fragmented to 500 bp using the Covaris M220 system, which was employed to construct short-insert libraries as per the manufacturer’s instructions (TruSeq™ Nano DNA Sample Prep Kit, Illumina) and subsequently sequenced on an Illumina NovaSeq 6000 platform (BIOZERON Co., Ltd, Shanghai, China) with 150 bp paired-end reads. Before assembly, raw reads were filtered using Trimmomatic 0.39 (http://www.usadellab.org/cms/index.php?page=trimmomatic). This filtering step aimed to eliminate reads with adaptors, those exhibiting a quality score below 20 (Q < 20), reads containing uncalled bases (“N” characters) at a percentage equal to or greater than 10%, and duplicated sequences. The mitochondrial genome was reconstructed through a combination of de novo and reference-guided assemblies, employing the following three steps. First, the filtered reads were assembled into contigs using MitoZ v2.3, and potential mitochondrial contigs were extracted by aligning against the NCBI mitochondrial genome database. Second, the potential mitochondrial contigs were aligned to the reference mitogenomes using BLAST v2.8.1, with aligned contigs (>Q80% query coverage) being ordered and connected manually according to the reference mitogenomes. GetOrganelle v1.7.5 (https://github.com/Kinggerm/GetOrganelle) was utilized for mitochondrial genome assembly. Finally, MUMmer 3.23 was employed to verify the circularity of these contigs. Through the aforementioned assembly steps, we successfully obtained a circular representation of the H. atra mitogenome.
2.3 Gene annotation
The mitochondrial genes were annotated using the online tool MITOS2 (http://mitos.bioinf.uni-leipzig.de/index.py). For the prediction of PCGs, tRNA genes, and rRNA genes, the mitochondrial selection “codon Table 9 of Echinoderms and flatworms” (S1 Table) was utilized. The positions of each coding gene were determined through BLAST searches against reference mitochondrial genes. Manual corrections for start and stop codons were conducted using SnapGene Viewer. The circular mitochondrial genome map of H. atra was generated using CGview (http://stothard.afns.ualberta.ca/cgview_server/).
2.4 Mitochondrial genomic structure analysis
The base composition statistics of 13 PCGs, two rRNAs, and 22 tRNAs were conducted using MEGA 7.0 software [21]. Base usage bias was determined with the equation (1) and equation (2) [22]:
MITOS2 and Vinna(http://rna.tbi.univie.ac.at/forna/) were used for tRNA secondary structure prediction and visual image construction. CodonW v1.4.2 (https://codonw.sourceforge.net/) was used to analyze the codon usage of mitochondrial PCGs by calculating Relative synonymous codon usage (RSCU) values.
2.5 Genetic diversity analysis
The 16S rDNA and COⅠ genes were amplified using a Taq PCR master mix kit and two primer sets: 16Sar (5′-CGCCTGTTTATCAAAAACAT −3′) and 16Sbr (5′-CTCCGGTTTGAACTCAGATCA −3′) [23], as well as COⅠ ef (5′-ATAATGATAGGAGGRTTTGG −3′) and COⅠ er (5′-GCTCGTGTRTCTACRTCCAT −3′) [24]. Thermocycling conditions were 95 °C for 60 s; 35 cycles at 95 ℃ for 30 s, 52 °℃ for 30 s, and 72 ℃ for 1 min; and a final extension at 72 °C for 10 min. The amplified products were subjected to bidirectional sequencing after being qualified by 1.5% agarose gel electrophoresis. The variation in the COⅠ region and the 16S rRNA region was analyzed using DnaSP 5.0. This analysis included the number of haplotypes (H), haplotype diversity (Hd), number of polymorphic sites (S), average number of nucleotide differences (k), nucleotide diversity (Pi), and distances within groups.
2.6 Phylogenetic analysis
Maximum likelihood phylogenetics were constructed using MEGA 7.0 to determine the phylogenetic relationships among species. Tree confidence levels were assessed through 1,000 bootstrap random replicates and data resampling, while distances between all haplotypes were calculated using the General Time Reversible model. As in the Mega software, the General Time Reversible model is one of the options in the ML phylogenetic analysis, and it belongs to the category of the ML phylogenetic analysis. To conduct a more scientific evolutionary analysis of the species in the Holothuroidea, we selected all the publicly available species of the Holothuroidea from NCBI. Each species uses its complete genome to construct a phylogenetic tree for systematic evolution. All mitochondrial genomic reference sequences utilized for comparison were sourced from the NCBI database: Holothuria hilla (MN1630001.1), Holothuria leucospilota (NC_046849.1), Holothuria pervicax (NC_045853.1), Holothuria spinifera (NC_046508.1), Holothuria edulis (NC_051928.1), Holothuria polii (LR694133.1), Holothuria scabra (NC_027086.1), Holothuria forskali (NC_013884.1), Holothuria sanctori (OZ199603.1), Bohadschia argus (NC_061402.1), Holothuria fuscogilva (MZ305460.1), Actinopyga echinites (MN793975.1), Actinopyga Lecanora (MW248463.1), Apostichopus japonicus (NC_012616.1), Cucumaria miniata (AY182376.1), Stichopus horrens (MN128376.1), Stichopus chloronotus (MZ052220.1), Colochirus quadrangularis (MW218895.1), Euapta godeffroyi (LC704718.1), Cercodemas anceps (NC_054245.1), Thelenota ananas (NC_059759.1).
2.7 Mitochondrial gene order and rearrangements analysis
Eighteen species of Holothuroidea (Holothuridae: A. echinites, A. lecanora, B. argus, H. scabra, H. fuscogilva, H. hilla, H. pervicax, H. leucospilota, H. edulis, H. forskali; Stichopodidae: A. japonicus, S. chloronotus, S. horrens, T. ananas; Cucumariidae: C. quadrangularis, C. anceps, C. miniata; Synaptidae: E. godeffroyi) were selected from the NCBI database, alongside one species from this study, all of which have comprehensive annotations of their mitochondrial gene compositions. A comparative analysis of the mitochondrial gene arrangement order was performed. The species were categorized based on their taxonomic classification and gene arrangement order at the family level, and a comparative diagram illustrating the mitochondrial gene arrangement orders for the selected species was constructed.
3. Results
3.1 Mitochondrial genome organization
After sequencing with the Illumina NovaSeq 6000, the raw sequencing data volume obtained was 5,066.3 Mb. Following quality control and trimming, 4,946.9 Mb of clean data was acquired, with a Q20 score of 97.70%. Data assembly yielded 15,788 bp of genomic data. The annotation results for the mitochondrial genome of H. atra are presented in Table 1 and Fig 1. The mitochondrial genome sequence and its annotation information have been deposited in GenBank under accession number PV998923. The genes within the genome are closely arranged, with a partial base overlap observed between them. This genome contains seven genes encoding different subunits of NADH dehydrogenase, three genes for different subunits of cytochrome C oxidase, two genes for ATP synthase, one gene for cytochrome B, two rRNAs, and 22 tRNAs. Among the 13 PCGs identified, 12 are situated on the positive strand of the mitochondrial genome, while only one gene, nad6, is positioned on the negative strand. Of the 22 tRNAs, five are located on the negative strand: trnS2, trnQ, trnA, trnV, and trnD. The remaining 17 tRNAs are found on the positive strand. Notably, there are variations in the base composition of different mitochondrial genomes (Table 2). The AT content across various genes ranges from 27.9% to 67.1%, with the highest percentage, 67.1%, observed in trnT and the lowest, 20.6%, in atp8. The overall AT content of the mitochondrial genome is 59.2%. The AT skew across different genes varies from −0.530 to 0.333, with nad6 exhibiting the lowest value of −0.530 and atp8 displaying the highest value of 0.333. The AT skew for the entire mitochondrial genome is 0.154. The GC skew across different genes ranges from −0.524 to 0.400, with atp8 showing the lowest value of −0.524, nad6 presenting the highest value of 0.333, and the overall GC skew of the mitochondrial genome at −0.268.
3.2 Spacers and overlaps
Several adjacent genes exhibited overlapping nucleotides and intergenic spacers within the H. atra mitogenome. A total of 18 intergenic spacers, ranging from 1 to 446 bp in size, were identified (Table 1). The largest intergenic spacer, measuring 446 bp, was located between the trnT and trnP genes. Additionally, seven overlaps were noted, with the longest measuring 7 bp, found between the atp8 and atp6 genes. The H. atra mitogenome displayed four distinct types of overlaps. The first type involved overlaps between genes located on the “ + ” and “-” strands, with three overlaps identified: 2 bp between trnS2 and cox3, 4 bp between trnP and trnQ, and 1 bp between trnL1 and trnA. The second type of overlap pertained to TA-termination codons, where a 2 bp overlap was observed in the termination codons of cox3. The third type was found within PCGs sequences, specifically a 7 bp overlap between atp6 and cox3. The fourth type of overlap lacked distinct characteristics.
3.3 Transfer RNA and ribosomal RNA genes
A total of 22 tRNAs, ranging from 62 to 72 bp in length, were identified within the mitogenome of H. atra (Table 1). The longest tRNA is trnL1, while trnC is the shortest. Notably, the H. atra mitogenome contains two copies each of trnL and trnS. All tRNAs exhibit the standard cloverleaf secondary structure; however, there are ten pairs of base mismatches, including six pairs on the amino acid acceptor arm, one pair on the TψC arm, and one pair on the anticodon arm (Fig 2). The H. atra mitogenome includes two rRNAs. The rrnS, which is 832 bp in length, is located between the trnF and trnE genes. The rrnL, measuring 1,393 bp, is situated between the nad2 and cox1 genes (Table 1).
3.4 Codon usage and sequence features of PCGs
The features of the mitochondrial PCGs are summarized in Table 1, which includes gene length, start and stop codons, and their respective strands. All mitochondrial PCGs initiate with an ATG codon. The termination codon TAA is frequently observed. TAG serves as a termination codon for the nad6 gene. The nad5 gene, located between trnS and nad6, is the longest among the 13 PCGs, measuring 1,836 bp. In contrast, the shortest gene, atp8, is situated between trnK and atp6, with a length of 165 bp.
From the perspective of amino acid composition, leucine (Leu) is the most abundantly encoded amino acid in the mitochondrial genome of H. atra, comprising 16.58% of the total. It is followed by isoleucine (Ile), which accounts for 10.60%. Conversely, cysteine (Cys) and lysine (Lys) are the least encoded amino acids, representing 1.03% and 1.48%, respectively (Fig 3). The mitochondrial genome of H. atra contains 61 codons that encode proteins corresponding to 20 amino acids. The frequency distribution chart of codons reveals that the most frequently used codons are AGA (Ser1), CUA (Leu1), and CCA (Pro), while those with relatively lower usage are GCG (Ala), CCG (Pro), and AGG (Ser1). Notably, the third position of the codon predominantly features the A base, with all relative usage frequency values of these codons exceeding 1, indicating a marked preference for this base (Fig 4).
3.5 Genetic diversity analysis
Through sequence assembly analysis, we obtained a 625 bp COⅠ gene fragment located in the 250 bp to 874 bp region of the mitochondrial genome and 784 bp COⅠ gene fragment located in the 15010 bp to 15493 bp region of the mitochondrial genome for genetic diversity analysis. The nucleotide genetic diversity parameters for 16S rRNA and COⅠ among the H. atra are presented in Table 3. Analysis of the 30 individual 16S rRNA sequences revealed 3 polymorphic loci and 4 distinct haplotypes. The 16S rRNA gene exhibited moderately high haplotype diversity (Hd = 0.697) but low nucleotide diversity (Pi = 0.0026) and low average nucleotide differences (k = 1.234) The genetic distance within this population was found to be 0.003. In contrast, the COⅠ gene showed moderate haplotype diversity (Hd = 0.384), moderate nucleotide diversity (Pi = 0.0072), and moderate nucleotide differences (k = 4.469). The analysis of 30 individual COⅠ sequences identified 13 polymorphic loci and 3 haplotypes. This phenomenon of numerous polymorphic loci is caused by the existence of shared mutations among multiple individuals. The genetic distance within this population was observed to be 0.007. The genetic diversity analysis revealed that the H. atra population collected from the coast of Kiribati in this study had a higher genetic diversity compared to the A. japonicus from the Sanriku coast and the H. edulis from Okinawa Island [25,26].
3.6 Phylogenetic relationships analysis
An evolutionary analysis of 22 species within the Holothuroidea (Fig 5) indicates that H. atra is most genetically similar to H. polii, subsequently clustering with other species in the Holothuriidae. In contrast, A. echinites from the Holothuriidae Actinopyga groups with A. lecanora, forming a distinct branch separate from other Holothuriidae species and those in the Stichopodidae. This finding suggests a significant genetic divergence from other Holothuriidae species. Furthermore, C. miniata from the Cucumariidae clusters with C. anceps, while A. japonicus clusters with S. chloronotus and S. horrens from the Stichopodidae. Collectively, these two families cluster together, exhibit a relatively close genetic relationship.
The position of H. atra in the tree is marked by a red circle.
3.7 Mitochondrial gene order and rearrangements
By comparing the mitochondrial genome arrangements of 19 different species within the Holothuroidea (Fig 6), it was observed that multiple species exhibited mitochondrial gene rearrangement. This phenomenon was evident not only among species at the order level but also at the family level. For instance, the mitochondrial gene arrangement of nine species in the Holothuriidae, including H. atra, differed from that of H. edulis and H. forskali, yet bore similarities to that of A. japonicus from the Stichopodidae. The observed gene rearrangements included alterations in the position of single gene (e.g., the translocation of trnM in S. chloronotus and S. horrens), shifts in the positions of multiple genes (e.g., in C. quadrangularis), and inversions of multi-gene sequences (e.g., in T. ananas, where the entire gene sequence was inverted except for cox1). In E. godeffroyi, only the cox1 gene maintained the same arrangement as in other species, while all other genes displayed completely altered orders.
There are regions with gene rearrangement. Short straight lines indicate a gene rearrangement, while long straight lines represent the overall positional changes of multiple genes within the linearly marked area;
The position undergoes changes before and after;
The position has undergone a reversed change in its existence; The meanings of different colors in the figure: green: protein-coding genes, violet: rRNA, blue: tRNA.
4. Discussion
In this study, we identified and analyzed the mitochondrial genome composition of H. atra. The total length of the mitogenome is 15,788 base pairs (bp), and its organization is comparable to that of the members of other Holothuriidae [27,28]. Mitochondrial PCGs in H. atra, like those in other sea cucumbers, typically comprise 13 PCGs, a relatively conserved feature, two rRNAs and 22 tRNAs molecules [24–31]. The AT content of 59.2% indicates a preference for AT base pairs, which is consistent with observations in other Holothuroidea and Echinoidea species [32,33]. The AT-skew value for H. atra, measured at 0.154, aligns with the positive values reported for other echinoderms. Most species within the Holothuriidae display positive AT-skew values, suggesting a higher frequency of adenine (A) compared to thymine (T). This phenomenon may be attributed to shared environmental stresses experienced during evolution, which could influence mitochondrial DNA transcription or replication [34,35]. In the PCGs of H. atra, the AT-skew values are primarily positive, with the exception of nad6, which shows a negative value of −0.530. Among the rRNA sequences analyzed, all AT-skew values are positive. Of the 22 tRNAs examined, only six—trnS2, trnQ, trnL1, trnA, trnV, and trnD exhibit negative AT-skew values, while the remaining 14 tRNAs show positive AT-skew values.
The mitochondrial genome of H. atra encodes 20 distinct amino acids, with Leu being the most abundant, comprising 16.58% of the total, while Cys is the least abundant at 1.03%. This codon usage bias is also evident in related species such as B. marianensis and Stichopus naso [13,36]. In terms of codon usage frequency, the most frequently utilized codons in the mitochondrial genome of H. atra are AGA (Ser1), CUA (Leu1), and CCA (Pro), whereas codons such as GCG (Ala), CCG (Pro), and AGG (Ser1) are utilized less frequently. All codons ending with adenine (A) exhibit RSCU values greater than 1, indicating a preference for A at the third codon position in the mitochondrial genome of H. atra. Likewise, species such as S. naso and B. argus also demonstrate relatively high frequencies of codons ending with A [14,36].
The analysis of rearrangement events within mitochondrial genomes is valuable for phylogeographic and phylogenetic studies, a fact substantiated by numerous studies on vertebrates. Generally, the arrangement of mitochondrial genomes remains relatively stable among vertebrates, including fish, amphibians, and most mammals [37–40]. In contrast, varying degrees of gene recombination are frequently observed in the mitochondrial genomes of invertebrates [28,41]. The same genetic arrangement implies the existence of a common ancestor. Therefore, the analysis of genetic rearrangement is of great significance for the evolutionary relationship analysis of echinoderms. In previous studies, Ma et al. compared the mitochondrial gene arrangements among 20 sea cucumber species, identifying two distinct arrangements [14]. Mu et al. analyzed the mitochondrial gene arrangements of 13 sea cucumber species, revealing seven distinct types [42]. In the present study, we identified eight different types of mitochondrial gene arrangements across 19 species from four families in the Holothuroidea. Notably, two distinct mitochondrial gene arrangements were observed in both the Holothuriidae and Stichopodidae, while three distinct arrangements were found in the Cucumariidae，this indicates that even among different species within the same family, there are significant differences in the arrangement of their genes.
5. Conclusion
In summary, this study presents a complete nucleotide sequence of the H. atra mitogenome. This research addresses a significant gap in the molecular biology studies of H. atra, enhances the mitochondrial genome database for this species, and provides molecular evidence and theoretical references for the classification, identification, and evaluation of germplasm resources of H. atra. Furthermore, it lays a foundational data basis for the conservation and sustainable utilization of H. atra resources in the Kiribati region.
Supporting information
S1 Table. Codon Table 9 of echinoderms and flatworms.
https://doi.org/10.1371/journal.pone.0347494.s001
(XLSX)
Acknowledgments
The authors would like to thank Yellow Sea Fisheries Research Institute and The Ministry of Fisheries and Marine Resources Development of the Republic of Kiribati for support of this study.
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