Replication-origin inversions accompany compositional skew and phylogenetic artifacts in fan worm mitochondrial...

Inversion of the origin of replication (ORI) in metazoan mitochondrial genomes (mitogenomes) can induce compositional biases misleading phylogenetic inferences. Fan worms (Sabellida, Annelida) exhibit atypical positive guanine-cytosine (GC) skew, a presumed ORI indicator, yet structural evidence remains elusive. We analyzed 38 complete Sabellida mitogenomes, including 20 newly sequenced, and identified inverted replication-associated motifs in the control region of species with positive GC skew species, providing structural evidence for ORI. Throughout Sabellida evolution, three major ORI events arose independently, accompanied by pronounced shifts in GC skew, synonymous codon usage, hydrophobic amino acid frequencies, and elevated nonsynonymous substitution rates in ORI-affected genes. Additionally, extensive gene rearrangement correlated with ORI events, elevated GC skew and substitution rates, but reduced adenine-thymine (AT) skew. ORI lineages show artificial clustering, confounding phylogeny. We propose that ORI events consistently co-occur with compositional skew, structural variations, and gene rearrangements. Coalescent-based methods integrating nuclear and mitochondrial loci are recommended to resolve ORI-affected phylogenies.