Tests of parallel molecular evolution in a long-term experiment with Escherichia coli

Abstract

The repeatability of evolutionary change is difficult to quantify because only a single outcome can usually be observed for any precise set of circumstances. In this study, however, we have quantified the frequency of parallel and divergent genetic changes in 12 initially identical populations of Escherichia coli that evolved in identical environments for 20,000 cell generations. Unlike previous analyses in which candidate genes were identified based on parallel phenotypic changes, here we sequenced four loci (pykF, nadR, pbpA-rodA, and hokB/sokB) in which mutations of unknown effect had been discovered in one population, and then we compared the substitution pattern in these "blind" candidate genes with the pattern found in 36 randomly chosen genes. Two candidate genes, pykF and nadR, had substitutions in all 11 other populations, and the other 2 in several populations. There were very few cases, however, in which the exact same mutations were substituted, in contrast to the findings from conceptually related work performed with evolving virus populations. No random genes had any substitutions except in four populations that evolved defects in DNA repair. Tests of four different statistical aspects of the pattern of molecular evolution all indicate that adaptation by natural selection drove the parallel changes in these candidate genes.

Fig. 1.

Mutations substituted by 20,000 generations in four candidate genes in 12 experimental populations of E. coli. Lighter regions indicate protein-coding sequences for and near pykF (A), nadR (B), pbpA-rodA (C), and hokB/sokB (D). Long bars below indicate the range sequenced; short bars show scale (200 bp). Each arrow marks a mutation; the number shows the affected population. The mutations in and near ydcA are of unknown relevance. ∗, An IS150 insertion, and, for populations −1 and +1, these were the original mutations used to identify the candidate genes. ▵, A 1-bp deletion. §, A synonymous mutation. All others in the coding regions are nonsynonymous, except for a 1-bp insertion in ydcA in population −2.

Fig. 2.

Distribution of numbers of substitutions in the 12 populations for the four candidate genes. Observed distributions are shaded. Poisson distributions with the same mean as the observed distribution are shown in outline.