Effects of Synonymous Mutations beyond Codon Bias: The Evidence for Adaptive Synonymous Substitutions from Microbial Evolution Experiments

Abstract

Synonymous mutations are often assumed to be neutral with respect to fitness because they do not alter the encoded amino acid and so cannot be "seen" by natural selection. Yet a growing body of evidence suggests that synonymous mutations can have fitness effects that drive adaptive evolution through their impacts on gene expression and protein folding. Here, we review what microbial experiments have taught us about the contribution of synonymous mutations to adaptation. A survey of site-directed mutagenesis experiments reveals the distributions of fitness effects for nonsynonymous and synonymous mutations are more similar, especially for beneficial mutations, than expected if all synonymous mutations were neutral, suggesting they should drive adaptive evolution more often than is typically observed. A review of experimental evolution studies where synonymous mutations have contributed to adaptation shows they can impact fitness through a range of mechanisms including the creation of illicit RNA polymerase binding sites impacting transcription and changes to mRNA folding stability that modulate translation. We suggest that clonal interference in evolving microbial populations may be the reason synonymous mutations play a smaller role in adaptive evolution than expected based on their observed fitness effects. We finish by discussing the impacts of falsely assuming synonymous mutations are neutral and discuss directions for future work exploring the role of synonymous mutations in adaptive evolution.

Keywords: distribution of fitness effects; experimental evolution; positive selection; synonymous mutations.

Fig. 1.

Distribution of fitness effects of all mutations, with fitness relative to the ancestor (ω) along the x axis and counts of mutations along the y axis. Blue represents nonsynonymous mutations, red represents synonymous mutations. Bars indicate mutation count data. The vertical black solid lines at ω = 1 indicate the fitness of the ancestor and the dashed vertical lines on either side indicate an estimate of 95% CI around that estimate based on mean measurement error reported. Study K did not report measurement error for its fitness estimates and so no dashed line is plotted. Blue and red curves indicate smoothed density fits of the nonsynonymous and synonymous mutations, respectively, using the “density” function in R. Letter labels correspond to study letter labels in table 1. The shapes of the DFEs of synonymous and nonsynonymous mutations are significantly different in all panels except I and J (K–S test, P < 0.05). Note.—The x axis in panel K is not strictly a fitness measure, but instead fold-increase in minimum inhibitory concentration (MIC) relative to the ancestor.

Fig. 2.

Distribution of fitness effects of mutations with relative fitness greater than 1 (ω > 1). Relative fitness is shown along the x axis and counts of mutations along the y axis. Blue represents nonsynonymous mutations, red represents synonymous mutations. Bars indicate mutation count data. The vertical black solid lines at ω = 1 indicate the fitness of the ancestor and the dashed vertical lines indicate the estimated 95% CI based on mean measurement error reported. Study K did not report measurement error for its fitness estimates and so no dashed line is plotted. Blue and red curves indicate smoothed density fits of the nonsynonymous and synonymous mutations, respectively, using the “density” function in R. Letters correspond to studies summarized in table 1. Panel J is blank because this study did not observe beneficial mutations. The shapes of the DFEs of synonymous and nonsynonymous mutations are significantly different in panels A, B, F, K, and L (K–S test, P < 0.05). Note.—The x axis in panel K is not strictly a fitness measure, but instead fold-increase in minimum inhibitory concentration (MIC) relative to the ancestor.

Fig. 3.

Probability that the next mutation fixed during an adaptive is synonymous over the number of unique clones competing for fixation. Points along each line represent the outcomes of random draws of a range of different number of beneficial clones with fitness drawn from experimentally quantified distributions of fitness effects from the studies summarized in table 1 (legend letter labels correspond to letter labels in table 1).