Annona_procb_ESM_File S1_tree_alignment.fas from Evolution of the nitric oxide synthase family in vertebrates and novel insights in gill development

Nitric oxide (NO) is an ancestral key signalling molecule essential for life and has enormous versatility in biological systems, including cardiovascular homeostasis, neurotransmission and immunity. Although our knowledge of NO synthases (Nos), the enzymes that synthesize NO <i>in vivo</i>, is substantial, the origin of a large and diversified repertoire of <i>nos</i> gene orthologues in fishes with respect to tetrapods remains a puzzle. The recent identification of <i>nos3</i> in the ray-finned fish spotted gar, which was considered lost in this lineage, changed this perspective. This finding prompted us to explore <i>nos</i> gene evolution, surveying vertebrate species representing key evolutionary nodes. This study provides noteworthy findings: first, <i>nos2</i> experienced several lineage-specific gene duplications and losses. Second, <i>nos3</i> was found to be lost independently in two different teleost lineages, Elopomorpha and Clupeocephala. Third, the expression of at least one <i>nos</i> paralogue in the gills of developing shark, bichir, sturgeon, and gar, but not in lamprey, suggests that <i>nos</i> expression in this organ may have arisen in the last common ancestor of gnathostomes. These results provide a framework for continuing research on <i>nos</i> genes’ roles, highlighting subfunctionalization and reciprocal loss of function that occurred in different lineages during vertebrate genome duplications.