Abstract
The diverse array of codon reassignments demonstrate that the genetic code is not universal in nature. Exploring mechanisms underlying codon reassignment is critical for understanding the evolution of the genetic code during translation. Hemichordata, comprising worm-like Enteropneusta and colonial filter-feeding Pterobranchia, is the sister taxon of echinoderms and is more distantly related to chordates. However, only a few hemichordate mitochondrial genomes have been sequenced, hindering our understanding of mitochondrial genome evolution within Deuterostomia. In this study, we sequenced four mitochondrial genomes and two transcriptomes, including representatives of both major hemichordate lineages and analyzed together with public available data. Contrary to the current understanding of the mitochondrial genetic code in hemichordates, our comparative analyses suggest that UAA encodes Tyr instead of a "Stop" codon in the pterobranch lineage Cephalodiscidae. We also predict that AAA encodes Lys in pterobranch and enteropneust mitochondrial genomes, contradicting the previous assumption that hemichordates share the same genetic code with echinoderms for which AAA encodes Asn. Thus, we propose a new mitochondrial genetic code for Cephalodiscus and a revised code for enteropneusts. Moreover, our phylogenetic analyses are largely consistent with previous phylogenomic studies. The only exception is the phylogenetic position of the enteropneust Stereobalanus, whose placement as sister to all other described enteropneusts. With broader taxonomic sampling, we provide evidence that evolution of mitochondrial gene order and genetic codes in Hemichordata are more dynamic than previously thought and these findings provide insights into mitochondrial genome evolution within this clade.