Synonymous mutations in representative yeast genes are mostly strongly nonneutral

Synonymous mutations in protein-coding genes do not alter protein sequences so are generally presumed neutral or nearly so 1 – 5 . To experimentally verify this presumption, we constructed 8,341 yeast mutants each carrying a synonymous, nonsynonymous, or nonsense mutation in one of 21 endogenous genes with diverse functions and expression levels, and measured their fitness relative to the wild-type in a rich medium. Surprisingly, three-quarters of synonymous mutations reduce the fitness significantly, and the distribution of fitness effects is overall similar albeit nonidentical between synonymous and nonsynonymous mutations. We find that both synonymous and nonsynonymous mutations frequently disturb the mutated gene’s mRNA level and that the extent of the disturbance partially predicts the fitness effect. Investigations in additional environments reveal greater across-environment fitness variations for nonsynonymous than synonymous mutants despite their similar fitness distributions in each environment, suggesting a smaller proportion of nonsynonymous than synonymous mutants that are always non-deleterious in a changing environment to permit fixation, potentially explaining substantially lower nonsynonymous than synonymous substitution rates commonly observed. The strong non-neutrality of most synonymous mutations, if also true for other genes and in other organisms, would require reexamining numerous biological conclusions about mutation, selection, effective population size, divergence time, and disease mechanism that rely on the neutral assumption of synonymous mutations.