Phylogenetic Insights into the Evolutionary History of the RSPO Gene Family in Metazoa

<b>Background</b>: The <i>RSPO</i> gene family encodes secreted glycoproteins that are rich in cysteine, which generally serve as activators of the Wnt signaling pathway in animals. Four types of this family have been identified in a few model species. However, the evolution of the family remains unclear. <b>Methods</b>: In this study, we identified a total of 1496 RSPO homologs through an extensive survey of the <i>RSPO</i> genes in 430 animals. Gene family clustering and phylogenetic analysis identified four major subtypes of the family (RSPO1-RSPO4) and clarified their distribution of copy number in different species. <b>Results and Conclusions</b>: Members of the RSPO4 subfamily that were closest to ancestral forms existed in both Deuterostomes and Protostomates, and we speculate that representatives of this subfamily already existed in <i>Urbilatera</i>, the last common ancestor of Deuterostomes. Particularly, in some RSPO3 subtypes of Actinopterygii (ray-finned fishes), an FU repeated motif with three conserved cysteines was identified. Further conservative analysis of amino acids and alignment of tertiary protein structure revealed the potential functional sites for each subgroup. The results provide insight into the phylogenetic relationships and evolutionary patterns of conserved motifs of <i>RSPO</i> family genes in animal kingdoms, which will guide further studies on the biological functions of RSPO in other non-model species.