Figure S1: Multiple protein alignment of RLuc-like predicted proteins from A puzzling homology: a brittle star using a putative cnidarian-type luciferase for bioluminescence

Bioluminescence relies on the oxidation of a luciferin substrate catalysed by a luciferase enzyme. Luciferins and luciferases are generic terms used to describe a large variety of substrates and enzymes. Whereas luciferins can be shared by phylogenetically distant organisms which feed on organisms producing them, luciferases have been thought to be lineage-specific enzymes. Numerous light emission systems would then have co-emerged independently along the tree of life resulting in a plethora of non-homologous luciferases. Here, we identify for the first time a candidate luciferase of a luminous echinoderm, the ophiuroid <i>Amphiura filiformis</i>. Phylogenomic analyses identified the brittle star predicted luciferase as homologous to the luciferase of the sea pansy <i>Renilla</i> (Cnidaria), contradicting with the traditional viewpoint according to which luciferases would generally be of convergent origins. The similarity between the <i>Renilla</i> and <i>Amphiura</i> luciferases allowed us to detect the latter using anti-<i>Renilla</i> luciferase antibodies. Luciferase expression was specifically localized in the spines which were demonstrated to be the bioluminescent organs <i>in vivo</i>. However, enzymes homologous to the <i>Renilla</i> luciferase but unable to trigger light emission were also identified in non-luminous echinoderms and metazoans. Our findings strongly indicate that those enzymes, belonging to the haloalkane dehalogenase family, might then have been convergently co-opted into luciferases in cnidarians and echinoderms. In these two benthic suspension-feeding species, similar ecological pressures would constitute strong selective forces for the functional shift of these enzymes and the emergence of bioluminescence