Evolutionary switches between two serine codon sets are driven by selection

Significance When a rare evolutionary event is observed, such as substitution of two adjacent nucleotides, the question emerges whether such rare changes are caused by mutational bias or by selection. Here we address this question through genome-wide analysis of double substitutions that lead to switch of the codon sets for the amino acid serine, the only one that is encoded by two disjoint sets of codons. We show that selection is the primary factor behind these changes. These findings suggest that short-term evolution of proteins is subject to stronger purifying selection than previously thought and has significant implications for methods of phylogenetic analysis. Serine is the only amino acid that is encoded by two disjoint codon sets so that a tandem substitution of two nucleotides is required to switch between the two sets. Previously published evidence suggests that, for the most evolutionarily conserved serines, the codon set switch occurs by simultaneous substitution of two nucleotides. Here we report a genome-wide reconstruction of the evolution of serine codons in triplets of closely related species from diverse prokaryotes and eukaryotes. The results indicate that the great majority of codon set switches proceed by two consecutive nucleotide substitutions, via a threonine or cysteine intermediate, and are driven by selection. These findings imply a strong pressure of purifying selection in protein evolution, which in the case of serine codon set switches occurs via an initial deleterious substitution quickly followed by a second, compensatory substitution. The result is frequent reversal of amino acid replacements and, at short evolutionary distances, pervasive homoplasy.