Extensive Mitogenomic Remodeling Delineates the Family-Level Split in Velvet Worms

Background: Velvet worms (Onychophora) occupy a pivotal phylogenetic position for deciphering the evolution of Panarthropoda, yet their exact placement within this clade remains debated. Furthermore, early studies in some onychophoran species revealed extensive gene rearrangements and the truncation or even loss of canonical transfer RNAs (tRNAs), features uncommon in other panarthropods. However, due to sparse representation, the pervasiveness and evolutionary significance of these genomic peculiarities across the phylum remain poorly understood. Methods: We sequenced and assembled three novel mitogenomes representing both extant onychophoran families (Epiperipatus barbadensis Peripatidae; Euperipatoides rowelli and Phallocephale tallagandensis Peripatopsidae) and conducted comparative analyses with five published species. Results: Onychophoran mitogenomes displayed high A+T content (mean 77.32%) but revealed a family-level divergence in GC skew. All genomes contained the standard 13 protein-coding genes (PCGs) and two ribosomal RNAs, yet tRNA counts varied significantly (ranging from 13 to 22), reflecting lineage-specific tRNA loss. Ancestral state reconstruction uncovered deep architectural divergence: Peripatopsidae retains the ancestral onychophoran gene arrangement, whereas Peripatidae exhibits a stable but derived gene order. Despite this architectural plasticity, synonymous codon usage patterns remained strictly conserved across the phylum, with all but one PCG evolving under strong purifying selection. Maximum likelihood phylogenetic reconstruction based on PCGs strongly supported Onychophora as the sister group to Arthropoda within Panarthropoda. Conclusions: Our findings provide robust molecular evidence supporting the Antennopoda hypothesis over the Tactopoda hypothesis for Panarthropoda phylogeny. Furthermore, we demonstrate extensive mitogenomic remodeling between the two extant onychophoran families, including divergent GC-skew patterns, tRNA contents, and gene arrangements.