Morphological characterization, mitochondrial genome assembly, and phylogenetic reconstruction of Branchiostoma malayanum within Branchiostomatidae

Branchiostoma malayanum is a poorly studied amphioxus species within the family Branchiostomatidae, with limited genomic resources available to support its taxonomic and evolutionary characterization. In this study, we present the first complete mitochondrial genome of B. malayanum and assess its phylogenetic position through a combination of morphological characterization, mitogenomic analysis, and comparative genetic divergence. The assembled mitogenome is 15,154 base pairs in length, encoding 13 protein-coding genes, 22 tRNAs, and 2 rRNAs, with conserved gene order and a strong AT bias (63.46%). Codon usage analysis revealed compositional asymmetry and functional constraints typical of cephalochordates. Morphological measurements showed a mixture of conserved segmental traits and variable external features influenced by developmental allometry. Comparative analysis of four mitochondrial markers (COX1, Cytb, 12 S rRNA, 16 S rRNA) demonstrated substantial intergeneric divergence among Branchiostoma, Asymmetron, and Epigonichthys, and supported the distinctiveness of B. malayanum from its congeners. Phylogenetic reconstruction based on complete mitogenomes positioned B. malayanum within a derived clade of Branchiostoma, closely related to B. belcheri and B. japonicum. These findings confirm the independent evolutionary status of B. malayanum and highlight the value of mitogenomic data in resolving cephalochordate diversity and systematics.