An improved likelihood ratio test for detecting site-specific functional divergence among clades of protein-coding genes.

Maximum likelihood codon substitution models have proven useful for studying when and how protein function evolves, but they have recently been criticized on a number of fronts. The strengths and weaknesses of such methods must therefore be identified and improved upon. Here, using simulations, we show that the Clade model C versus M1a test for functional divergence among clades is prone to false positives under simple evolutionary conditions. We then propose a new null model (M2a_rel) that better accounts for among-site variation in selective constraint. We show that the revised test has an improved false-positive rate and good power. Applying this test to previously analyzed data sets of primate ribonucleases and mammalian rhodopsins reveals that some conclusions may have been misled by the original method. The improved test should prove useful for identifying patterns of divergence in selective constraint among paralogous gene families and among orthologs from ecologically divergent species.