Overall bile salt variation in reptiles, birds, and mammals. The phylogeny depicted has Testudines as sister-group to Aves/Crocodylia. Lizards are paraphyletic but are indicated here as a single group for comparison purposes. Based on the patterns of bile salt variation across vertebrates, the ancestral bile salt profiles at nodes A, B, and C can be inferred by parsimony. The ancestral phenotype at node A would likely be C27 bile alcohols, the phenotype found in all living lobe-finned, jawless, and cartilaginous fish surveyed to date. The ancestral phenotype at node B would likely be C27 bile alcohols and trihydroxy-C27 bile acids, the major bile salts of Crocodylia and paleognath birds. The most recent common ancestor to tuatara, lizards, and snakes (node C) would additionally have the ability to produce 24R-hydroxylated C27 bile acids, the main bile salts of tuatara and lizards within Anguimorpha and Scinciformata. Based on the phylogeny depicted, there appear to be several 'innovations' unique to certain groups: (1) 24R-hydroxylation of C27 bile acids by lepidosaurs as mentioned above, (2) 22-hydroxylation of C27 bile acids by Testudines, and (3) production of 7-deoxy and C23 bile acids by snakes. The figure does not show all the known bile salt modifications, but only some of the more common ones within in each group.