Calibration choice, rate smoothing, and the pattern of tetrapod diversification according to the long nuclear gene...

A phylogeny of tetrapods is inferred from nearly complete sequences of the nuclear RAG-1 gene sampled across 88 taxa encompassing all major clades, analyzed via parsimony and Bayesian methods. The phylogeny provides support for Lissamphibia, Theria, Lepidosauria, a turtle-archosaur clade, as well as most traditionally accepted groupings. This tree allows simultaneous molecular clock dating for all tetrapod groups using a set of well-corroborated calibrations. Relaxed clock (PLRS) methods, using the amniote = 315 Mya (million years ago) calibration or a set of consistent calibrations, recovers reasonable divergence dates for most groups. However, the analysis systematically underestimates divergence dates within archosaurs. The bird-crocodile split, robustly documented in the fossil record as being around approximately 245 Mya, is estimated at only approximately 190 Mya, and dates for other divergences within archosaurs are similarly underestimated. Archosaurs, and particulary turtles have slow apparent rates possibly confounding rate modeling, and inclusion of calibrations within archosaurs (despite their high deviances) not only improves divergence estimates within archosaurs, but also across other groups. Notably, the monotreme-therian split ( approximately 210 Mya) matches the fossil record; the squamate radiation ( approximately 190 Mya) is younger than suggested by some recent molecular studies and inconsistent with identification of approximately 220 and approximately 165 Myo (million-year-old) fossils as acrodont iguanians and approximately 95 Myo fossils colubroid snakes; the bird-lizard (reptile) split is considerably older than fossil estimates (< or = 285 Mya); and Sphenodon is a remarkable phylogenetic relic, being the sole survivor of a lineage more than a quarter of a billion years old. Comparison with other molecular clock studies of tetrapod divergences suggests that the common practice of enforcing most calibrations as minima, with a single liberal maximal constraint, will systematically overestimate divergence dates. Similarly, saturation of mitochondrial DNA sequences, and the resultant greater compression of basal branches means that using only external deep calibrations will also lead to inflated age estimates within the focal ingroup.