Complete Mitochondrial Genome of Decametra tigrina (A.H. Clark, 1907) (Crinoidea, Comatulida, Colobometridae) and Phylogenetic Analysis

Abstract

The mitochondrial genome of Decametra tigrina (A.H. Clark, 1907) was fully sequenced and characterized. This circular, double-stranded genome spans 15,794 bp and encompasses 13 protein-coding genes (PCGs), 22 tRNA genes, and two rRNA genes. The arrangement of genes remains conserved, matching those of Cenometra bella and the MW405444 (submitted to GenBank as Oligometra serripinna). The base composition consists of 24.6% A, 47.6% T, 16.2% G, and 11.6% C. Most PCGs use ‘ATG’ as the initiation codon, with NADH4L and NADH5 initiating with ‘GTG’. Each PCG terminates with a complete stop codon (‘TAA’ or ‘TAG’). Twelve tRNA genes, three NADH genes, and two rRNA genes are located on the L strand, while the remaining genes reside on the H strand. Phylogenetic analysis utilizing all 13 PCGs demonstrated that D. tigrina forms a clade with MW405444 (submitted to GenBank as O. serripinna, reassessed here as D. tigrina) and Florometra sp. (MN883538). Decametra tigrina forms a sister group with C. bella; together, they cluster with Stephanometra indica (Mariametridae) and Zygometra comata (Zygometridae) in the ML tree.

1. Introduction

The genus Decametra A. H. Clark, 1911 belongs to Colobometridae A. H. Clark, 1909 within Comatulida A. H. Clark, 1908, class Crinoidea Miller, 1821, phylum Echinodermata Klein, 1778 [1]. Seventeen valid species are currently recognized in Decametra [1], and among these only D. tigrina (A. H. Clark, 1907) has been reported from Korean waters [2]. Decametra tigrina is characterized by a discoidal centrodorsal, a comparatively broad and flat polar area, and densely arranged cirrus sockets in a single or partially double marginal row. The species bears relatively long cirri, numbering 26 to 30, each with roughly 28 segments and ending in long, curved, sharply pointed claws [2]. Its distribution includes the southern coast and Jeju Island of South Korea, southern Japan, and the Philippines [2]. The species is associated with habitats influenced by warm currents and appears to be extending its range northward along the Korean coast, possibly in response to rising ocean temperatures. To date, only partial molecular data from Decametra have been available, including mitochondrial gene fragments and nuclear rRNA markers, and no complete mitochondrial genome has been deposited for the genus in public databases.

Mitochondrial genomes provide comparable characters for crinoids, such as gene content, gene order, strand assignment, base composition, codon usage, and tRNA secondary structures. These features allow tests of conserved patterns and lineage specific deviations at family and superfamily levels. However, the absence of a complete mitogenome for Decametra has limited direct comparisons within Colobometridae and across related families. Here, we sequence and annotate the complete mitochondrial genome of D. tigrina, assess gene order and compositional features, summarize codon usage and tRNA secondary structures, and infer a maximum likelihood phylogeny from all 13 protein coding genes. We use these data to evaluate the placement of D. tigrina relative to closely allied genera and to examine whether current family or superfamily limits align with the resulting topology. This genome fills a taxonomic gap for Decametra and provides a reference for future phylogenomic and evolutionary work on comatulids.

2. Materials and Methods

2.1. Collection and Storage of Decametra tigrina

A specimen of D. tigrina was collected by netting at a depth of 10 m in the adjacent water of Jwiseom Island in Busan (35°01′20.9″ N, 128°58′21.1″ E) on 13 March 2025 (Figure 1). It was immediately preserved in ethyl alcohol (>95%) after collection. The preserved specimen was subsequently deposited at the Marine Echinoderms Resources Bank of Korea (MERBK) (https://www.mbris.kr (accessed on 20 October 2025), contact: Taekjun Lee, leetj@syu.ac.kr) at Sahmyook University and assigned the voucher number MERBK-C0066.

2.2. DNA Extraction and Genome Sequencing

Genomic DNA was isolated from gonad tissue using the DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany), in accordance with the manufacturer’s protocol. Targeted primers were employed to amplify partial regions of the mitochondrial COI and 16S rRNA genes, which were then used for map-to-reference assembly in Geneious Prime (v.2024.0.7). To enhance DNA yield, the REPLI-g Mitochondrial DNA Kit (Qiagen, Hilden, Germany) was applied for additional amplification of the genomic DNA. The genome analysis teams at the National Instrumentation Centre for Environmental Management of Seoul National University (NICEM), Korea, conducted next-generation sequencing (NGS) analysis. A genomic library with an average insert size of 300 bp constructed with the KAPA Hyper Prep Kit (Kapa Biosystems, Woburn, MA, USA) was subjected to paired-end sequencing using the Illumina HiSeq 2500 platform (Illumina, San Diego, CA, USA) (pair-end 2 × 151 bp). Raw reads were deposited in the NCBI Short Read Archive (BioSample: SAMN48400990; BioProject: PRJNA1260676; SRA: SRR33479964).

2.3. Mitochondrial Genome Annotation and Analysis

Raw sequence data were initially processed in Geneious Prime v.2024.0.7, which included adapter trimming and reference-guided assembly. Gene annotation was conducted using the MITOS2 pipeline implemented on Galaxy.org (accessed on 23 September 2025) [3,4,5]. Protein-coding genes were identified using MITOS2, while transfer RNAs (tRNAs) were predicted using tRNAscan-SE 2.0 [6,7]. Finally, a circular graphical representation of the mitogenome was produced using OGDRAW v1.3.1 [8]. Sequencing depth across the assembled mitogenome was assessed by mapping the trimmed reads back to PV742399 in Geneious Prime, and the coverage profile is provided in Supplementary Figure S1.

2.4. Phylogenetic Analysis

The analysis incorporated 22 complete mitochondrial genomes from Comatulida and a single isocrinid used as an outgroup (Metacrinus rotundus), all sourced from NCBI (

Table 1. Detailed information of 22 crinoids mitochondrial complete genome sequences used for comparative and phylogenetic analysis in this study.

Order	Family	Species	Accession Number in GenBank	Reference
Comatulida	Antedonidae	Antedon mediterranea	AM404181	[13]
		Florometra serratissima	AF049132	[14]
		Florometra sp.	MT302206	[15]
		Poliometra prolixa	OP177937	[16]
	Colobometridae	Cenometra bella	OK509084	[17]
		Decametra tigrina	PV742399	This study
		Florometra sp.	MN883538	[18]
		Oligometra serripinna	MW405444	Unpublished
	Comatulidae	Anneissia pinguis	MW008594	[19]
		Anneissia intermedia	MW376476	[20]
		Capillaster sp.	OP546034	[21]
		Comanthus parvicirrus	MW526392	[21]
		Comenthus sp.	OM272942	[21]
		Comaster schlegelii	MW526391	[22]
		Comatella nigra	OM321037	[21]
		Comatella stelligera	OM313186	[21]
		Phanogenia gracilis	DQ068952	[22]
	Mariametridae	Stephanometra indica	MF966246	[23]
	Pentametrocrinidae	Thaumatocrinus sp.	OQ207656	[21]
	Ptilometridae	Ptilometra sp.	OP546035	[21]
	Tropiometridae	Tropiometra macrodiscus	ON381167	[21]
	Zygometridae	Zygometra comata	ON585667	[21]
Isocrinida	Isocrinidae	Metacrinus rotundus	OM964491	[21]

). The optimal nucleotide substitution models for the dataset comprising 13 protein-coding genes (PCGs) were determined using jModelTest v.2.1.1 [9,10] for each individual PCG. Maximum likelihood phylogenetic inference was conducted using RAxML GUI v.2.0 [11], applying the TrN+G model to ATPase8, TrN+I+G for NADH2, and the GTR+I+G model for the remaining PCGs. To assess branch reliability, bootstrap resampling was performed using the rapid bootstrapping option set to 1000 replicates. Visualization of the resulting phylogenetic tree was achieved with FigTree v.1.4.4 [12].

3. Results

3.1. Morphological Characteristics of Decametra tigrina

The identification of the examined specimen (voucher no. MERBK-C0066) as Decametra tigrina was based on the diagnostic morphological characters described in Shin [2]. Key diagnostic features observed in our specimen include a discoidal centrodorsal with a broad, flat polar area, and 26 cirri composed of 26 segments, terminating in long, curved, and sharply pointed claws (Figure 1). These morphological characters are consistent with the description of D. tigrina provided by Shin [2]. Furthermore, we re-examined the voucher specimen associated with MW405444, which had been originally submitted to GenBank as Oligometra serripinna, and reidentified it as D. tigrina based on the diagnostic characters defined by Shin [2]. All cirrals were broader than long, and indistinct dorsal spines were visible on the distal segments. Ten arms were present, and pinnule P2 was noticeably longer and stouter than P1 or P3. Collectively, these characters agree with the diagnostic description of D. tigrina and distinguish it from related congeners (e.g., Oligometra serripinna with stouter cirri composed of 20–24 segments).

3.2. Gene Arrangement and Characteristics of Decametra tigrina

The mitochondrial genome of D. tigrina is circular and double-stranded (Figure 2). The annotated mitochondrial genome sequences have been registered in GenBank under accession number PV742399. The complete mitochondrial genome is 15,794 bp in length. The mitochondrial genome of D. tigrina contains 13 protein-coding genes (PCGs), 22 tRNA genes, and two rRNA genes (Figure 2,

Table 2. The arrangement of Decametra tigrina mitochondrial complete genome.

Name	Site	Length (bp)	Strand	Start Codon	Stop Codon	Anti-Codon
COI	1–1557	1557	H	ATG	TAA
tRNA-Arg	1558–1625	68	H			UCG
NADH4L	1626–1922	297	H	GTG	TAA
COII	1924–2613	690	H	ATG	TAG
tRNA-Lys	2616–2685	70	H			CUU
ATPase8	2686–2859	174	H	ATG	TAA
ATPase6	2853–3542	690	H	ATG	TAA
COIII	3552–4334	783	H	ATG	TAA
tRNA-Ser	4333–4403	71	L			UGA
NADH3	4407–4760	354	H	ATG	TAA
NADH4	4773–6164	1392	H	ATG	TAA
tRNA-His	6155–6223	69	H			GUG
tRNA-Ser	6233–6300	68	H			GCU
NADH5	6301–8181	1881	H	GTG	TAA
NADH6	8182–8676	495	L	ATG	TAA
CytB	8751–9902	1152	H	ATG	TAG
tRNA-Pro	9893–9961	69	H			UGG
tRNA-Gln	9967–10,038	72	L			UUG
tRNA-Asn	10,042–10,114	73	H			GUU
tRNA-Leu	10,117–10,187	71	H			UAG
tRNA-Ala	10,189–10,259	71	L			UGC
tRNA-Trp	10,262–10,329	68	H			UCA
tRNA-Cys	10,330–10,396	67	H			GCA
tRNA-Val	10,401–10,470	70	L			UAC
tRNA-Met	10,474–10,548	75	H			CAU
tRNA-Asp	10,552–10,621	70	L			GUC
tRNA-Thr	10,851–10,922	72	L			UGU
tRNA-Glu	10,924–10,991	68	L			UUC
12S rRNA	10,992–11,832	841	L
tRNA-Phe	11,833–11,905	73	L			GAA
tRNA-Leu	11,906–11,977	72	L			UAA
tRNA-Gly	11,978–12,046	69	L			UCC
16S rRNA	12,047–13,565	1519	L
tRNA-Tyr	13,566–13,636	71	L			GUA
NADH2	13,635–14,675	1041	L	ATG	TAA
tRNA-Ile	14,676–14,746	71	L			GAU
NADH1	14,751–15,719	969	L	ATG	TAA

). No gene rearrangements, insertions, or deletions were detected in PV742399 relative to other available Colobometridae mitogenome records, and its gene arrangement mirrors that of two additional Colobometridae species (Cenometra bella and MW405444: submitted to GenBank as O. serripinna). The nucleotide composition in the mitochondrial genome of D. tigrina was A (24.6%), T (47.6%), G (16.2%), and C (11.6%). Three NADH genes (NADH 1, 2, and 6), two rRNA genes, and 12 tRNA genes in D. tigrina are located on the L strand, while the other mitochondrial genes are transcribed from the H strand (Figure 2). The total length of 13 protein-coding genes is 11,475 bp. Individual tRNA genes range in size from 67 bp (tRNA-Cys) to 75 bp (tRNA-Met). Both NADH4L and NADH5 initiate with the ‘GTG’ codon, in contrast to the other 11 PCGs which initiate with ‘ATG’. Prior research on echinoderm mitochondrial code has indicated that ‘GTG’ may function as a start codon, although there are some reservations [24]. All PCGs terminate with the canonical stop codons ‘TAA’ or ‘TAG’ (Table 2).

3.3. Phylogenetic Analysis of Comatulids Including Decametra tigrina

The maximum likelihood (ML) tree inferred from the 13 mitochondrial protein-coding genes clearly positioned D. tigrina (PV742399), newly sequenced in this study, within a distinct and well-supported clade (bootstrap 100%). This clade includes MN883538 (submitted to GenBank as Florometra sp.) and MW405444 (submitted to GenBank as O. serripinna), showing effectively no sequence divergence (very short branches) among PV742399–MN883538–MW405444. The voucher associated with MW405444 was re-examined and the specimen was reassessed as D. tigrina (see Section 3.1). The voucher for MN883538 (specimen ID 201904-Crinoid014) is reported to be deposited at the Research Institute of Basic Sciences, Incheon National University (Incheon, Republic of Korea) [18]. However, we were unable to access the specimen and obtain curator-provided photographs for re-examination in this study. Importantly, the other Florometra sp. record (MT302206) is not part of this near-identical group in our ML tree (Figure 3), but instead forms a clade with Antedon mediterranea (bootstrap 51%), with F. serratissima (AF049132) as sister to that pair.

In the broader phylogenetic framework, this group is part of a larger clade composed mainly of species traditionally classified within Tropiometroidea, including Florometra indica, Cenometra bella, and Decametra indica. This topology places MW405444 (submitted as O. serripinna) within the same broader clade, indicating an inconsistency between the current database identification and the mitochondrial phylogeny.

At the family level, Antedonidae and Tropiometridae are not recovered as monophyletic groups in our tree. For Antedonidae, records labeled as Antedon mediterranea and Florometra serratissima occur on separated branches. A similar pattern is seen for Tropiometridae, where records currently assigned to this family are distributed across distant parts of the topology. These patterns align with earlier findings reported by Xu et al. [21] and with multilocus phylogenies incorporating nuclear loci (e.g., [25,26]), underscore the need to interpret morphology-based groupings in light of molecular phylogenetic evidence, while noting that misidentification in public databases cannot be excluded without curated vouchers.

4. Discussion

The complete mitogenome of D. tigrina fills a gap for the genus and enables direct comparison within Colobometridae. The genome spans 15,794 bp and contains 13 protein-coding genes, 22 tRNAs, and two rRNAs, without any detected rearrangement, insertion, or deletion. The base composition is strongly AT-biased at 72.2 percent. L-strand localization of several loci, including nad1, nad2, nad6, both rRNAs, and 12 tRNAs, accords with strand asymmetry commonly reported for echinoderms. Most protein-coding genes initiate with ATG. Two genes, nad4L and nad5, initiate with GTG, a start codon previously reported under the echinoderm mitochondrial code [27]. All protein-coding genes terminate with complete stop codons, either TAA or TAG. Taken together, these features are consistent with conserved replication and transcription dynamics combined with mutational bias, rather than with annotation artifacts, and they agree with expectations for crinoid mitogenomes.

The phylogeny based on all 13 protein-coding genes recovers a fully supported group (bootstrap 100%) that includes PV742399 (D. tigrina), MW405444 (submitted to GenBank as O. serripinna), and MN883538 (submitted as Florometra sp.). MW405444 corresponds to a voucher specimen that we re-examined and reassessed as D. tigrina using the diagnostic characters in the morphological description and taxonomic keys of Shin [2] (see Section 3.1). Accordingly, the GenBank organism assignment for MW405444 should be corrected to D. tigrina. The genus Florometra has been described as having a low discoidal centrodorsal, more numerous but shorter flattened cirri, and well-developed spinulose pinnules, which differs from Decametra [2,28]. As mitochondrial genomes represent a single nonrecombining locus, the close clustering of these three records may reflect recent divergence, retention of an ancestral haplotype, or introgression, and synonymy should not be inferred without nuclear evidence.

Signals at higher ranks match earlier findings [22]. Members assigned to Tropiometroidea and Antedonoidea do not form single clades in our tree, and the position of the MW405444 record (submitted as O. serripinna) near Decametra and MN883538 (submitted as Florometra sp.) highlights the importance of curated vouchers when interpreting mitochondrial phylogenies. At the family level, Antedonidae and Tropiometridae are not monophyletic in the phylogenetic tree. Taxa attributed to Antedonidae, such as Antedon mediterranea and Florometra serratissima, occur on separated branches, and a similar dispersion is observed for Tropiometridae. These patterns argue for reassessment of family- and superfamily-level limits with broader taxon coverage and multilocus datasets. We also note that our topology places Florometra sp. (MT302206) separately from F. serratissima (AF049132), in contrast to [18]; this difference may reflect limited taxon sampling and the single-locus nature of mitochondrial data, and should be re-evaluated using nuclear markers.

Two constraints frame the present inferences. Only one Decametra mitogenome was newly generated here, and publicly available records remain sparse for several relevant genera. Geographic representation is also incomplete, and MN883538 (submitted as Florometra sp.) is reported to have a deposited voucher (specimen ID 201904-Crinoid014; [18]), but it was not re-examined in this study. Future work that combines nuclear markers, such as UCEs or exon capture, with curated vouchers and denser sampling across Colobometridae and Mariametridae will allow explicit tests of monophyly.

In summary, D. tigrina exhibits conserved gene content typical of Colobometridae, a pronounced AT bias with strand asymmetry consistent with echinoderm patterns, and a strongly supported placement with MW405444 (submitted as Oligometra serripinna) and MN883538 (submitted as Florometra sp.). Together with the non-monophyly signals for Antedonidae and Tropiometridae, these results motivate an integrative revision of higher-level classification that combines mitochondrial and nuclear data with vetted morphology [22,24,27,28].