Overall bile salt variation across vertebrates. Variation of bile salts is overlaid on a vertebrate phylogeny, with turtles placed as sister group to birds/crocodiles, frogs and salamanders as sister groups, and placental mammals and marsupials as sister groups . Lizards are paraphyletic but are indicated here as a single group for comparison purposes. Unresolved relationships are depicted as polyotomies. The figure shows two "shifts to the right" from 5α-C27 bile alcohols to C24 bile acids as the bile salt synthetic pathway presumably grew in length: (1) from Agnatha to ray-finned fish and (2) from lobe-finned fish to tetrapods. Note that C27 bile acids are common in reptiles and amphibians but uncommon in fish. Within amphibians, there is only preliminary data on caecilians based on analysis of two specimens, both of which showed only bile alcohols but with the orientation of the 5-hydroxyl group as yet undetermined pending additional analyses (LR Hagey, unpublished data).