The Complete Mitochondrial Genome of Eucrate alcocki (Decapoda: Brachyura: Euryplacidae) Provides New Insights Into Heterotrematan Crab Phylogeny

TL;DR

This study sequenced the mitochondrial genome of Eucrate alcocki, a marine crab, to better understand its evolutionary relationships and genetic uniqueness.

Contribution

The study provides the first complete mitochondrial genome of Eucrate alcocki and reveals a unique gene arrangement and phylogenetic position.

Findings

The mitochondrial genome of E. alcocki is 15,720 bp with 72.2% AT content and a unique gene rearrangement.

Phylogenetic analysis shows E. alcocki forms a distinct clade with Entricoplax vestita, separate from related species.

The findings suggest potential paraphyly within the genus Eucrate and highlight the value of genomic data for crab evolution.

Abstract

Background: This study determined the complete mitochondrial genome sequence of the marine crab to elucidate its phylogenetic position within Heterotremata, specifically the superfamily Goneplacoidea, and to explore the biological significance of its genetic composition and arrangement. Methods: The complete mitochondrial genome of Eucrate alcocki was sequenced using the Illumina platform and de novo assembled. Genome annotation and structural analysis were performed using MITOS2 and PhyloSuite. Phylogenetic relationships were reconstructed based on 13 protein-coding genes from 59 heterotrematan species using both Bayesian inference and maximum likelihood methods. Results: The mitochondrial genome of E. alcocki is a circular molecule of 15,720 bp with 72.2% AT content and a unique F-H-ND5 → H-F-ND5 gene rearrangement. Phylogenetic analysis robustly places E. alcocki in a distinct clade with Entricoplax vestita (BI = 1.00, ML = 100%), separate from the congeneric species Eucrate crenata and E. solaris, suggesting potential paraphyly within the genus Eucrate. Conclusions: This discovery provides preliminary evidence suggesting existing crab classification systems and molecular evidence for further understanding the evolutionary history of crabs. Our findings demonstrate that genomic characteristics hold significant value in revealing evolutionary pathways and can serve as a foundation for more comprehensive taxonomic and evolutionary research in the future.

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1. Introduction

The mitochondrial genome (mitogenome) is characterized by its compact architecture, strict maternal inheritance, elevated substitution rate, and conserved gene order, and has emerged as the marker of choice for molecular taxonomy, phylogenetic reconstruction, and population genetic surveys of metazoans [1,2,3]. A typical metazoan mitogenome is a circular double-stranded DNA molecule of approximately 15–20 kb that encodes 13 PCGs, 22 tRNAs and 2 rRNAs, and contains a single non-coding control region that governs replication and transcription initiation [4,5,6]. Driven by high-throughput sequencing, complete mitogenomes are increasingly resolving deep phylogenetic relationships within Crustacea, especially Brachyura. Nucleotide-composition bias, codon-usage profiles, and gene-rearrangement signatures collectively furnish robust molecular characters for reconstructing crab lineages [7,8,9].

Within Eubrachyura, it has traditionally been divided into two major clades: Heterotremata and Thoracotremata. Among these, the internal systematics of Heterotremata are the most complex, with its family-level and superfamily-level classifications undergoing multiple revisions over the past decades and remaining unsettled [10]. Although the accumulation of mitochondrial genomic data in recent years has progressively refined our understanding of the Eubrachyura phylogenetic framework, several contentious nodes persist within Heterotremata. These include the monophyly of certain superfamilies, the sequence of divergence among different clades, and discrepancies between morphological and molecular evidence [11,12]. One of the primary reasons for this uncertainty is the insufficient sampling of mitogenomes within Heterotremata, particularly the lack of data from certain key lineages, which limits the in-depth analysis of their internal evolutionary relationships.

The superfamily Goneplacoidea belongs to Heterotremata and represents a significant lineage characterized by diverse morphological features and a complex evolutionary history [13,14]. The composition of families within this superfamily has undergone frequent revisions in recent years, while molecular phylogenetic evidence remains relatively scarce, leading to persistent uncertainty regarding its systematic position [15,16]. Euryplacidae is a core member of Goneplacoidea, primarily comprising deep-sea benthic crabs (50–500 m depth) distributed in tropical to subtropical continental shelves, playing important roles in benthic food webs as scavengers and predators [17,18]. The genus Eucrate within the family Euryplacidae was established by De Haan in 1835. Members of this genus exhibit a high degree of morphological similarity, making accurate species identification in traditional taxonomy challenging based on a limited number of morphological characteristics, necessitating molecular data to resolve cryptic diversity and establish robust phylogenetic relationships [19]. E. alcocki is a key representative species within this genus, yet no reports of its complete mitogenome have been published to date [20]. The scarcity of published Goneplacoidea mitogenomes (currently limited to three species) constrains robust phylogenetic inference regarding its systematic position within Heterotremata.

To date, only a few Goneplacoidea species (such as E. crenata) have completed mitogenome sequencing [21,22]. This study presents the first whole-mitogenome sequencing and annotation of E. alcocki, systematically analyzing its genomic structure, base composition, codon bias, non-coding region characteristics, and gene arrangement patterns. It also compares this genome with previously reported Brachyura mitogenomes. After constructing a phylogenetic tree based on PCGs, we examined the systematic position of Euryplacidae and Goneplacoidea within Eubrachyura: Heterotremata and evaluated the evolutionary relationships between this lineage and other major superfamilies within Heterotremata. This study not only provides the first mitogenome data for E. alcocki but also offers new molecular evidence for refining the phylogenetic relationships within the superfamily Goneplacoidea and the clade Eubrachyura: Heterotremata.

2. Materials and Methods

2.1. Sample Collection and DNA Extraction

A specimen of E. alcocki was collected at 20–30 m depth from subtidal sandy-mud bottoms from Zhoushan, Zhejiang Province, China (29°53′49.601″ N, 122°18′18.615″ E) in August 2024. Muscle tissue was preserved in 95% ethanol at −20 °C following morphological identification by taxonomic experts at Zhejiang Ocean University. Genomic DNA was extracted using a modified salting-out method [23].

2.2. Mitochondrial DNA Sequencing and Assembly

Mitogenome sequencing was performed by BeijingTsingke Biotech Co., Ltd. Company (Beijing, China) using the Illumina HiSeq™ platform, yielding approximately 10 GB of paired-end sequencing data. First, the extracted genomic DNA is tested for concentration and integrity to ensure it meets library construction requirements. Subsequently, DNA was randomly fragmented using ultrasonication to produce fragments of approximately 300–500 bp. The resulting fragments were purified for use in constructing sequencing libraries. The library construction process includes DNA end repair, 3′ A-tailing, adapter ligation, and recovery of target fragments via agarose gel electrophoresis. After PCR amplification, the library undergoes quality control testing to ensure that fragment distribution and concentration meet sequencing standards. After sequencing completion, quality control is performed on the raw reads, including removal of adapter contamination, filtering of low-quality reads, reads with high N content, and sequences that are too short, thereby yielding high-quality clean data. Raw paired-end sequencing data (150 cycles + 150 cycles) were processed using fastp (v0.23.2) for quality control and filtering. The main parameters were: -q 20 -u 40 -n 10 -f 15 -F 15 --detect_adapter_for_pe -z 4 --thread 16. Prior to filtering, a total of 80,832,994 reads (12.124949 Gb) were obtained; after filtering, 78,045,252 reads (10.200289 Gb) were retained. Post-filtering Q20/Q30 values were 97.91%/93.58%, with mean read lengths of R1 = 130 bp and R2 = 131 bp. The duplication rate was 7.79%. Based on this, the mitogenome was de novo assembled using the GetOrganelle software [24]. Mitochondrial genome assembly was performed using GetOrganelle (v1.7.7.1), with dependency versions Bowtie2 2.3.5.1, SPAdes 3.13.1, and BLAST 2.12.0. The GetOrganelle parameters were: -R 15 -k 21,45,65,85,105 -F animal_mt -o animal_mt_out -t 32. The mean k-mer coverage was 280.7×, and the mean base coverage depth was 1374×. Through multiple rounds of parameter adjustment and iterative optimization, a structurally complete and sufficiently covered mitogenome assembly result was ultimately obtained.

2.3. Mitogenome Annotation and Sequence Analysis

Genome annotation was conducted using the online server MITOS2 (MITOS Web Server) [25] under the invertebrate mitochondrial genetic code (NCBI Genetic Code 5). The MITOS2 annotations were further manually curated, with particular attention to tRNA anticodon loops and the positions of start/stop codons. Start and stop codons were determined following previously published studies [26]. After final checks and corrections in Sequin, the complete mitogenome sequence was submitted to NCBI GenBank under accession number PV990112.1.

A circular mitogenome map was generated using Proksee (https://proksee.ca) [27]. PhyloSuite v2.0 was used to calculate nucleotide composition (A, T, C, and G), the nucleotide composition of rRNAs and tRNAs, amino acid usage frequencies, and relative synonymous codon usage (RSCU) values for PCGs. AT skew and GC skew were calculated as AT skew = (A − T)/(A + T) and GC skew = (G − C)/(G + C) [28].

2.4. Methods for Phylogenetic Analysis

Based on the complete mitogenome sequences of 59 species, a phylogenetic tree of Heterotremata was successfully constructed. This tree structure not only incorporates the newly sequenced mitogenome of E. alcocki but also integrates whole-genome sequence data from 56 species across nine superfamilies within Heterotremata obtained from the NCBI database (Potamoidea, Majoidea, Parthenopoidea, Goneplacoidea, Bythograeoidea, Dorippoidea, Leucosioidea, Calappoidea, Portunoidea) (Table 1). Additionally, to enhance the precision of the phylogenetic tree, Pinnaxodes major (NC_063605.1) and Amusiotheres obtusidentatus (NC_063600.1) were classified as outgroups. Protein-coding genes (PCGs) were identified using PhyloSuite v2.0 [29,30], aligned with MAFFTv7.490 [31], and trimmed with Gblocks0.91b [32]. ModelFinder v3.0.1 [33] was used to determine optimal partitioning schemes and substitution models (GTR + F + I + G4). Phylogenetic analyses were conducted using MrBayes v3.2.7a [34] (BI, 2 million generations, 25% burn-in) and IQ-TREE 2.1.3 [35]. BIC was used to select GTR + F + R6 analysis, and branch support was evaluated using 1000 ultra-fast bootstrap repetitions. Finally, the phylogenetic tree was professionally edited and enhanced using the online platform TVBOT [36] to enable clearer visualization and analysis of phylogenetic relationships.

3. Results

3.1. Basic Structural Features of the Mitogenome

The mitogenome of E. alcocki obtained in this study is a circular double-stranded DNA molecule 15,720 bp in length (GenBank: PV990112). It comprises 37 genes, including 13 PCGs, 22 tRNA genes, and 2 rRNA genes, along with a non-coding regulatory region (Figure 1, Table 2). The overall structure is compact, consistent with most crustacean mitogenomes. The genome is 17 bp longer than that of the congeneric E. crenata, with differences primarily attributable to length variation in the control region [22]. In terms of gene strand distribution, a total of 15 genes are located on the light strand (L-strand), including four PCGs (ND1, ND5, ND4, ND4L), 16S rRNA and 12S rRNA, and nine tRNA genes. The remaining 22 genes are located on the heavy strand (H-strand). Base composition analysis revealed that the genome contained 36.0% A, 36.2% T, 10.9% G, and 16.9% C, with an AT content of 72.2%. The AT-skew value of −0.002 indicates a relatively balanced ratio between A and T, while the GC-skew value of −0.218 reveals a significantly higher proportion of C compared to G (Table 3). A total of 9 gene overlap regions were detected within the genome, with a combined length of 22 bp and overlap lengths ranging from 1 to 7 bp. Notably, the longest overlap (7 bp) occurs between ND4 and ND4L, a conserved feature in decapod mitogenomes (Table 2). These short overlapping regions reflect the highly compact coding pattern of the mitogenome. Additionally, the genome contains 15 intergenic regions with a total length of 1025 bp. The longest spacer region is located between 12S rRNA and Ile(I) (776 bp), corresponding to a portion of the mitochondrial control region. Other longer intergenic spacers include ND5/ND4 (46 bp), Leu(L2)/COII (25 bp), and ND1/Leu(L1) (36 bp) (Table 2).

3.2. PCGs Codon Usage

Based on the annotation results presented in Table 1, all PCGs within the mitogenome of E. alcocki are initiated by the canonical mitochondrial start codon ATN. Among these, ATG is the most prevalent initiation codon, utilized by genes including COI, COII, COIII, ND4, ND4L, ND5, and ATP8. The codon ATT serves as the start signal for ND1, ND2, ND3, and ND6, whereas ATA is employed as the initiation codon for ATP6. Regarding stop codons, PCGs employ three types: complete stop codons TAA (ATP6, ND3, ND4L, ND1) and TAG (ATP8, ND4, ND2), as well as single-base T-type incomplete stop codons found in COI, COII, COIII, ND5, ND6, and Cytb.

3.3. Amino Acid Content and Frequency of Use

The amino acid composition of the E. alcocki mitogenome exhibits a notably uneven distribution (Figure 2A). Among these, leucine, phenylalanine, isoleucine, and serine are the most abundant, constituting the primary amino acid types in the encoded proteins; conversely, cysteine, tryptophan, and arginine account for the lowest proportions. Overall, the more abundant amino acids generally correspond to high-frequency codons ending in A/U. Analysis of codon usage preferences (Table 4, Figure 2B) revealed that E. alcocki exhibits a marked preference for codons terminating with A or U. The most frequently used codons include UUA (Leu), UCU (Ser1), CCU (Pro), GGA (Gly), and AUU (Ile). Conversely, codons ending in G or C occur less frequently, such as CUG (Leu), AGC (Ser), GCG (Ala), and CGC (Arg). Among aromatic amino acids, phenylalanine is primarily encoded by UUU; tryptophan is primarily encoded by UGA. Regarding acidic amino acids, aspartic acid is most frequently used with the GAU codon, while glutamic acid primarily employs the GAA codon. Overall, the amino acid composition and codon usage of the E. alcocki mitogenome exhibit a typical strong AT bias, with a notably high frequency of A/U-terminating codons. This aligns with the 72.2% AT content observed in the species’ mitogenome.

3.4. Transfer RNA and Ribosomal RNA

The mitogenome of E. alcocki encodes a total of 22 tRNA genes. Among these, eight tRNA genes—Pro (P), Leu (L1), His (H), Val (V), Gln (Q), Cys (C), Tyr (Y), and Phe (F)—are encoded by the light chain (L chain), while the remaining 14 tRNAs are encoded by the heavy chain. These tRNA genes range in length from 63 bp (Asp (D)) to 73 bp (Val (V)), with a total length of 1496 bp (Table 2). The AT content is 75.4%, and the AT-skew and GC-skew values are 0.034 and 0.109, respectively (Table 3). Predictions of the secondary structures of 22 tRNAs revealed that most tRNAs fold into typical cloverleaf structures. However, Ser (S1) from E. alcocki failed to form a DHC stem, and the TψC loop was absent in Asp (D), Thr (T), and Tyr (Y). Additionally, several base mismatches—C-T, A-C, T-T, and A-A—were identified within the tRNA secondary structure. Specifically, C-T mismatches occurred in Lys (K), A-C mismatches in Met (M), T-T mismatches in His (H) and Asp (D), and A-A mismatches in Cys (C) (Figure 3).

Both ribosomal RNA genes in the E. alcocki mitogenome are located on the light chain. The 16S rRNA gene spans 1292 bp, while the 12S rRNA gene spans 822 bp. The AT content is 78.4%, with an AT-Skew of 0.022 and a GC-Skew of 0.287 (Table 3). The GC-Skew values for both rRNA genes are greater than zero, consistent with the pattern that GC-Skew values for light chain-encoding genes are positive.

3.5. Gene Rearrangement

To further reveal the structural changes undergone by the mitogenomes of Goneplacoidea during evolution, this study systematically compared the gene arrangements of the sequenced species and related reported species using the gene order of the predicted decapod ancestor as a reference (Figure 4). The hypothetical ancestral decapod mitochondrial gene order is based on the consensus arrangement of Limulus polyphemus (Xiphosura) and previously published ancestral decapod patterns [37]. The results indicate that multiple types of gene rearrangement events exist within Goneplacoidea, with significant differences observed among different lineages. These primarily comprise the following three representative rearrangement patterns.

(①) Inversion of the F–ND5–H gene block. In the Decapoda ancestral genome, this segment was arranged as F–ND5–H. In E. alcocki, E. vestita, E. insignis, E. crenata, and E. solaris, this order consistently changed to H–F–ND5, indicating that this inversion is a shared derived character within the genus Eucrate. In G. rhomboides, however, this gene block appears as ND5–F–H. Multiple independent inversion events with opposite orientations have occurred within Goneplacoidea, providing strong phylogenetic information (Figure 4B). (②) Large-scale inversion events accompanied by coding strand switching. In G. rhomboides, a substantial gene cluster spanning from L2 to Y underwent a significant inversion, with COI serving as one of the inversion breakpoints. This inversion not only resulted in the complete transfer of genes originally located on the heavy chain (H-strand) to the light chain (L-strand), but also reversed their internal arrangement entirely, forming a typical large-scale strand inversion structure. This rearrangement feature is absent in the Eucrate clade, making it a key genomic marker for distinguishing the genus Goneplax (Figure 4C). (③) Transposition of the ND1–L1 region. The ancestral sequence ND1–L1–16S–V–12S retains its contiguous arrangement, a structure unchanged in E. alcocki, E. vestita, and E. insignis. However, in G. rhomboides, this segment shifted forward as a whole; in E. crenata and E. solaris, ND1 and L1 significantly shifted backward to follow 12S, forming a new combination: 16S–V–12S–I–ND1–L1. The ND1–L1 region is presumed to be a hotspot for frequent rearrangements during the evolution of Goneplacoidea (Figure 4D).

3.6. Phylogenetic Analysis

The phylogenetic tree of BI and ML methods constructed based on 13 PCGs (Figure 5) exhibits a fundamentally identical topology, with all key nodes receiving high support. The phylogenetic tree shows Potamoidea occupying a basal divergent position within Heterotremata, followed by the sequential divergence of Majoidea and Parthenopoidea form a topologically stable monophyletic clade. This clade occupies a higher position than the combined clade (Goneplacoidea + Bythograeoidea), establishing a clear sister relationship with it. The overall structure thus presents a hierarchical framework: (Parthenopoidea + (Goneplacoidea + Bythograeoidea)).

Goneplacoidea and Bythograeoidea first clustered into a highly supported paraphyletic clade. Within Goneplacoidea, two adjacent monophyletic clades emerged: one comprising E. crenata and E. solaris, and the other consisting of E. vestita, the sequenced species E. alcocki, E. insignis, and G. rhomboides. All nodes within the latter clade received full support with BI = 1.00 and ML = 100, indicating that E. alcocki possesses a stable and clearly defined systematic position within this monophyletic unit. Bythograeoidea emerges as the sister clade to Goneplacoidea, and together they form the subordinate union cluster under Parthenopoidea. Within this framework, E. alcocki did not cluster with the traditional Euryplacidae species E. crenata and E. solaris, but instead stably placed itself in a neighboring clade containing E. vestita, E. insignis, and G. rhomboides. This result indicates that species within the genus Eucrate do not form a single linear lineage at the family level, but rather diverge into at least two systemically recognizable branches. Among these, the branch containing E. alcocki exhibits closer systematic relationships with the Goneplacidae clade.

In the lower structure of the phylogenetic tree, (Dorippoidea + Leucosioidea) and (Calappoidea + Portunoidea) form two parallel clusters of comparable differentiation levels, exhibiting stable topology and well-defined boundaries. Within Calappoidea, slight differences in node ordering were observed between BI and ML analyses (Figure 5B). Topological discrepancies between BI and ML analyses primarily involve weakly supported nodes (BS < 70, PP < 0.85), reflecting different model assumptions and prior specifications in Bayesian inference. Importantly, the sister relationship between E. alcocki and E. vestita and the separation from the E. crenata + E. solaris clade are recovered by both methods with full support (BI = 1.00, ML = 100%), suggesting these conclusions are robust to analytical methodology. The ML support for the relevant nodes reached 100%, confirming their monophyly and sister group relationship with Portunoidea.

4. Discussion

This study presents the first complete mitogenome of E. alcocki and provides a systematic characterization of its genomic architecture. Specifically, we summarize its nucleotide composition, codon usage bias, and gene rearrangement patterns, and show that the overall strand-distribution pattern is consistent with the strand preference commonly reported in eubrachyuran crabs [38,39,40]. Results indicate that the E. alcocki mitogenome exhibits high structural and compositional similarity to other reported true crab species, displaying a typical AT-biased composition and compact gene arrangement [41,42]. This further validates the stability and applicability of mitogenomes in crustacean phylogenetic studies. However, in terms of the positions of light and heavy chains within the mitogenome and the AT and GC biases, E. alcocki exhibits significant differences compared to the previously published E. crenata within the same genus. Only 14 genes were identified on the heavy chain (H) of E. crenata, while an additional 23 genes were found on the light chain (L). The AT skew was 0.00166 and the GC skew was 0.218, with both skew values being positive [22].

For the single-base T-type incomplete stop codons found in PCGs (COI, COII, COIII, ND5, ND6, and Cytb), previous studies have revealed that incomplete stop codons are equally prevalent in the mitogenomes of metazoans. These codons are often repaired through post-transcriptional polyadenylation mechanisms [43,44]. In the co-suborder, the species Chiromantes neglectum, Parasesarma affine, and Episesarma lafondii also exhibit incomplete stop codons in the Cytb gene [45,46]. The deletion of the DHU arm in the tRNA-Ser1 gene is widely distributed across metazoan mitogenomes, exhibiting significant phylogenetic universality. Furthermore, to maintain secondary structure stability in metazoan mitochondria, base mismatches are permitted during tRNA folding, and this phenomenon of base mismatching is also prevalent [47].

Variations in mitochondrial gene sequences have been widely regarded as robust phylogenetic markers for tracing their deep evolutionary trajectories [48]. Multiple studies consistently reveal that the mitogenomes of metazoans exhibit high inertia at the gene sequence level. Rearrangement events not only occur with extremely low probability but also show random distribution in timing, constituting a “conservative and rare” evolutionary feature. Therefore, it can serve as direct evidence for evolutionary relationships between species [49,50]. In this study, the sequence of mitochondrial genes was consistent with phylogenetic relationships as determined by phylogenetic analysis. In the phylogenetic analysis, both the ML tree and BI tree constructed based on 13 PCGs revealed that the majority of superfamilies and families are monophyletic groups, consistent with the findings of most studies [51,52,53]. However, the superfamily Goneplacoidea failed to form a monophyletic group, with its internal members dispersed across two major clades. Among these, E. alcocki of the Euryplacidae and E. vestita of the Goneplacidae form one clade, which in turn constitutes a sister group to E. insignis and G. rhomboides. Meanwhile, E. crenata and E. solaris of the Euryplacidae form an independent clade. This result is consistent with the findings of Kong [54] on the family Goneplacidae, suggesting that the phylogenetic relationships between Goneplacidae and Euryplacidae are complex, potentially involving paraphyly or polyphyly. In contrast to the findings regarding the superfamily Goneplacoidea in the studies by Meng and Pang [21,22], the discrepancy may stem from the fact that both their papers employed phylogenetic analysis based solely on individual species within the Goneplacoidea superfamily. In Pang’s study, the ML tree based on 13 PCGs placed A. lunaris in family Matutidae closely related to E. crenata. However, in Figure 5, Matutidae, represented by three species exhibits strong internal monophyly, yet its clustering with Eucrate does not display the close neighbour relationship described by Pang et al. Additionally, a novel pattern emerged within the genus Eucrate, where E. alcocki diverged from (E. crenata + E. solaris) into distinct lineages. This discrepancy suggests that internal nodes within Eucrate may be sensitive to taxon coverage and dataset composition, necessitating further validation through denser sampling and nuclear gene data. The split of Eucrate into two well-supported lineages in our mitochondrial phylogeny highlights a taxonomic issue that deserves careful interpretation. Such apparent non-monophyly is a recurrent pattern in brachyuran systematics, because many genera and families were historically defined using a limited number of morphological characters that may be affected by convergence and homoplasy [55]. In Eucrate, the high degree of morphological similarity among congeners may reflect either shared ancestry or the repeated evolution of similar character states, which can obscure true relationships when morphology alone is used for diagnosis. At the same time, mito-nuclear discordance and sparse taxon sampling may also contribute to this pattern. Therefore, we treat the non-monophyly of Eucrate inferred here as a working hypothesis that should be evaluated with denser sampling of congeners and genome-wide nuclear datasets.

Notably, the gene rearrangement observed in the F-ND5-H region of E. alcocki (F-H-ND5 → H-F-ND5) likely results from the Tandem Duplication–Random Loss (TDRL) model, the predominant mechanism driving mitochondrial gene order evolution in crustaceans [56]. Under this model, a tandem duplication of the ancestral gene block (F-ND5-H) occurred, followed by the stochastic deletion of redundant gene copies, resulting in the derived H-F-ND5 arrangement observed in the genus Eucrate. E. alcocki and E. vestita exhibit high genetic sequence similarity, with only minor shifts observed in the tRNA-H-F-ND5 region. This indicates a high degree of genomic structural conservation among Euryplacidae and certain Goneplacidae members. However, G. rhomboides exhibits distinct gene rearrangements and large-scale inversions, suggesting that different genomic reshaping events may have occurred within Goneplacoidea during evolution. Such rearrangements are not uncommon in crab mitogenomes and may be associated with species adapting to different ecological niches or experiencing evolutionary bottlenecks [57].

It is important to acknowledge that these conclusions are based solely on mitochondrial protein-coding genes, which represent a single linkage group subject to lineage sorting effects and saturation at deeper phylogenetic levels. Verification through nuclear markers (e.g., UCEs or transcriptomic data) is essential before formal taxonomic changes are implemented. Future phylogenomic studies utilizing Ultra-Conserved Elements (UCEs) or anchored hybrid enrichment markers are necessary to verify these mitogenomic hypotheses and resolve the evolutionary history of Goneplacoidea. Although this study provides new molecular evidence for the phylogenetic relationships within Goneplacoidea, issues of insufficient sample representation remain. Currently, the number of reported mitogenomes within the Goneplacoidea superfamily remains limited, particularly as only a few species from the Euryplacidae family have been included in analyses. This constraint hinders a thorough examination of the internal systematics within this family. Therefore, future research should expand sample collection to incorporate more key genera and species, thereby comprehensively elucidating the evolutionary history of Goneplacoidea and its systematic position within Heterotremata.

5. Conclusions

This study presents the first assembly and annotation of the complete mitogenome of E. alcocki. The mitogenome is a typical closed-circular animal molecule that has a total length of 15,720 base pairs and encodes 37 genes (13 PCGs, 22 tRNAs, and 2 rRNAs). Compared to the ancestral sequence of the Decapoda, E. alcocki exhibits changes in the gene cluster F-H-ND5, resulting in the H-F-ND5 arrangement. The phylogenetic tree based on 13 PCGs indicates that E. alcocki is closely related to E. vestita (BI = 1.00, ML = 100), while diverging from the E. crenata and E. solaris clade. We acknowledge that conclusions regarding the non-monophyly of Eucrate are based on only three congeneric species and should be interpreted with caution. Sparse sampling may bias topology due to long-branch attraction or missing intermediate lineages. This suggests potential paraphyly of the genus Eucrate, though this hypothesis requires verification with denser sampling of congeners and related genera within Euryplacidae and that the boundaries between Euryplacidae and Goneplacidae require re-evaluation. The newly generated mitogenome of E. alcocki expands mitogenomic resources for Euryplacidae by increasing congeneric representation within Eucrate and providing an additional reference for comparative and phylogenetic analyses of Goneplacoidea. With the accumulation of more mitogenome data, it is anticipated that the intricate network of relationships within Heterotremata will be further clarified in the future, laying a solid foundation for the taxonomic and evolutionary research of true crabs.