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 in vivo, is substantial, the origin of a large and diversified repertoire of nos gene orthologues in fishes with respect to tetrapods remains a puzzle. The recent identification of nos3 in the ray-finned fish spotted gar, which was considered lost in this lineage, changed this perspective. This finding prompted us to explore nos gene evolution, surveying vertebrate species representing key evolutionary nodes. This study provides noteworthy findings: first, nos2 experienced several lineage-specific gene duplications and losses. Second, nos3 was found to be lost independently in two different teleost lineages, Elopomorpha and Clupeocephala. Third, the expression of at least one nos paralogue in the gills of developing shark, bichir, sturgeon, and gar, but not in lamprey, suggests that nos expression in this organ may have arisen in the last common ancestor of gnathostomes. These results provide a framework for continuing research on nos genes' roles, highlighting subfunctionalization and reciprocal loss of function that occurred in different lineages during vertebrate genome duplications.

Andalusian Centre for Nanomedicine and Biotechnology Málaga Spain

Biology and Evolution of Marine Organisms Stazione Zoologica Anton Dohrn Napoli 80121 Italy

Department of Animal Biology Faculty of Sciences University of Málaga Spain

Department of Biological Sciences Nicholls State University Thibodaux LA 70301 USA

Department of Integrative Biology and Program in Ecology Evolution and Behavior Michigan State University East Lansing MI 48824 USA

Department of Neuroscience Spinal Cord and Brain Injury Research Center and Ambystoma Genetic Stock Center University of Kentucky Lexington KY USA

Department of Zoology Faculty of Science Charles University Prague Czech Republic

Division of Biology and Biological Engineering California Institute of Technology Pasadena CA 91125 USA

Evolutionary Morphology Laboratory RIKEN Cluster for Pioneering Research Chuo ku Kobe Hyogo 650 0047 Japan

Faculty of Marine Sciences University Institute of Environmental Studies and Natural Resources University of Las Palmas de Gran Canaria Canary Islands Spain

Institute of Neuroscience University of Oregon Eugene OR 97403 USA

Laboratory for Evolutionary Morphology RIKEN Center for Biosystems Dynamics Research Kobe 650 0047 Japan

South Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses Faculty of Fisheries and Protection of Waters University of South Bohemia in Ceske Budejovice Vodnany Czech Republic

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