Molecular Evolution and Gene Diversity of Dermatan Sulfate Sulfotransferases in Ascidians

ABSTRACT Ascidians (Chordata, Tunicata) are model organisms for studying molecules, particularly sulfated glycosaminoglycans (GAGs), due to their phylogenetic proximity to vertebrates and unique GAG variants. Over‐sulfated dermatan sulfate (DS) contributes to proteoglycan diversity and cellular processes in metazoans. Research has identified highly sulfated DS in six ascidian species across two orders: Phlebobranchia ( Phallusia nigra and Ciona intestinalis ) and Stolidobranchia ( Halocynthia pyriformis , Styela plicata , Herdmania pallida , and Halocynthia roretzi ). These polymers share a common disaccharide backbone structure [→4 Iduronic Acid (2‐SO₄) β ‐1→3 N ‐Acetylgalactosamine β ‐1→], but show distinct sulfation patterns at N ‐acetylgalactosamine residues, suggesting differences in biosynthetic pathways. To investigate DS sulfation patterns, we analyzed sulfotransferase (SULT) gene families through genomic analysis. We sequenced draft genomes of S. plicata (Stolidobranchia), which produces 2,4‐disulfated DS, and P. nigra (Phlebobranchia), which produces 2,6‐disulfated DS. We conducted comparative genomic analysis of 19 additional ascidian species using C. intestinalis as a reference for its SULT repertoire. Phylogenetic analyses revealed 154 novel putative dermatan SULT genes across three enzyme families: uronyl 2‐ O ‐SULTs, and carbohydrate 4‐ O ‐ and 6‐ O ‐SULTs. Analysis of mitochondrial cytochrome c oxidase subunit I (COI) sequences provided insights into evolutionary relationships among taxa. Results indicate that Phlebobranchia represents a basal lineage within Ascidiacea, while Stolidobranchia comprises more recently diverged species. The DS 6‐ O ‐SULTs gene family originated recently in ascidian evolution, with diversification potentially influenced by lineage‐specific selective pressure. These findings advance our understanding of SULT evolution in tunicates and provide a foundation for future functional studies. SULT genes represent targets for expression profiling and biotechnological applications, including enzyme cloning and characterization in DS biosynthesis. This study provides a genetic framework for understanding and harnessing the biomedical potential of unique sulfated polysaccharides in ascidians.