Evolutionary history of R-spondins. Homologs of R-spondin (each contains two Fu domains followed by a thrombospondin protein 1 (TSP1) domain) are shown. Two non-chordate R-spondin sequences with its characteristic domain architecture were detected in the hemichordate acorn worm and the echinodermate sea urchin. These two were used to root the gene phylogeny. The tree clearly shows the two successive whole genome duplications that generated present day R-spondin diversity in vertebrate species such as fish and mammals. Sequences were aligned using MAFFT . The tree was constructed using neighbor-joining as implemented in the clustalx package and visualized using iToL [81, 82]. SkRspo is D1LXC5_SACKO from the hemichordate acorn worm Saccoglossus kowalevskii, BfRspo is C3Y1K8_BRAFL from the primitive chordate amphioxus Branchiostoma floridae, and SpRspo is XP_796266.2 from the echinodermate sea urchin Strongylocentrotus purpuratus. Vertebrate protein identifiers used for this tree are as follows: HsRspo4 ENSP00000217260, MmRspo4 ENSMUSP00000041578, DrRspo4 ENSDARP00000123862, DrRspo2 ENSDARP00000100941, HsRspo2 ENSP00000276659, MmRspo2 ENSMUSP00000067325, HsRspo1 ENSP00000348944, MmRspo1 ENSMUSP00000030687, DrRspo1 ENSDARP00000058458, HsRspo3 ENSP00000349131, MmRspo3 ENSMUSP00000090287 and DrRspo3 ENSDARP00000058577.