Characterization of the Complete Mitochondrial Genome of Tachypleus tridentatus and Mitochondrial Phylogenetic Analysis of Extant Limulidae

The tri-spine horseshoe crab, Tachypleus tridentatus, is an evolutionarily ancient marine chelicerate of considerable ecological and conservation importance in the Indo-West Pacific. Although several mitochondrial genomes have been reported, comprehensive comparative mitogenomic analyses encompassing all extant limulid genera remain limited. In this study, we sequenced and characterized the complete mitochondrial genome of an F1 Tachypleus tridentatus individual derived from wild-caught broodstock from the Beibu Gulf, northern South China Sea, and subsequently maintained at an aquaculture facility, using high-throughput sequencing, and performed an integrated comparative mitogenomic analysis across the three extant genera of Limulidae. The circular mitochondrial genome was 15,006 bp in length and contained the typical set of 37 mitochondrial genes, including 13 protein-coding genes (PCGs), 22 transfer RNA genes, and two ribosomal RNA genes. The newly sequenced Tachypleus tridentatus mitogenome showed highly conserved genome organization and nucleotide composition relative to previously published conspecific mitogenomes, confirming established mitochondrial characteristics of the species. The principal contribution of this study is the addition of a complete Beibu Gulf mitogenome and its integration with 12 additional complete mitochondrial genomes representing all extant limulid species, together with four mitochondrial markers, K2P genetic-distance analyses, and independent BI and ML phylogenetic reconstruction. The concordance among these mitochondrial datasets provides an integrated assessment of genetic divergence and mitochondrial phylogenetic relationships within Limulidae and expands the geographic representation of complete mitochondrial genomic resources for Tachypleus tridentatus.