Parallel evolutionary dynamics of adaptive diversification in Escherichia coli

Abstract

The causes and mechanisms of evolutionary diversification are central issues in biology. Geographic isolation is the traditional explanation for diversification, but recent theoretical and empirical studies have shown that frequency-dependent selection can drive diversification without isolation and that adaptive diversification occurring in sympatry may be an important source of biological diversity. However, there are no empirical examples in which sympatric lineage splits have been understood at the genetic level, and it is unknown how predictable this process is-that is, whether similar ecological settings lead to parallel evolutionary dynamics of diversification. We documented the genetic basis and the evolutionary dynamics of adaptive diversification in three replicate evolution experiments, in which competition for two carbon sources caused initially isogenic populations of the bacterium Escherichia coli to diversify into two coexisting ecotypes representing different physiological adaptations in the central carbohydrate metabolism. Whole-genome sequencing of clones of each ecotype from different populations revealed many parallel and some unique genetic changes underlying the derived phenotypes, including changes to the same genes and sometimes to the same nucleotide. Timelines of allele frequencies extracted from the frozen "fossil" record of the three evolving populations suggest parallel evolutionary dynamics driven at least in part by a co-evolutionary process in which mutations causing one type of physiology changed the ecological environment, allowing the invasion of mutations causing an alternate physiology. This process closely corresponds to the evolutionary dynamics seen in mathematical models of adaptive diversification due to frequency-dependent ecological interactions. The parallel genetic changes underlying similar phenotypes in independently evolved lineages provide empirical evidence of adaptive diversification as a predictable evolutionary process.

Figure 1. Mutations found in sequenced clones from generation 1045 and inferred genealogical relationships among the clones.

(a) population 18. (b) population 19. (c) population 20. Black circles indicate the time point samples from the frozen “fossil” record. Δ, deletion; +, insertion. Numbers following gene names indicate the affected codon within the gene. Two gene names separated by a forward slash (e.g., yfbV/ackA) indicate that the mutation is in the intergenic region between the indicated genes. Underlined gene names indicate nonsynonymous changes (including indels in coding regions). Colored gene names (other than black) indicate changes in or upstream of the same gene. Timing of mutations and divergences were inferred from the fossil record: a mutation found in a clone was assumed to have arisen at the midpoint between the first time step at which the mutation was detected in the fossil record and the previous time step; divergences between clones are assumed midway between the last mutation they share and the first mutation they do not share. Because of limitations on time resolution and minimum detectable frequency, timing of all such events should be viewed as approximations. Mutations found in clones but not in time point samples are assumed to have occurred near the end of the experiment and are marked with asterisks (*).

Figure 2. Dynamics of the frequencies of mutations detected in the fossil record of three evolving populations.

Shades of blue (above) indicate the mutations associated with FS clones as identified in Figure 1, and shades of green (below) indicate the mutations associated with SS clones as identified in Figure 1 (mutations with a * in Figure 1 are not shown, because their frequency was not high enough to be detected in the time point samples). Gold indicates ancestral strains (which may include mutations not associated with any sequenced clone). The white region in (b) indicates an independent origin of the SS phenotype. Mutations within a lineage are cumulative—that is, mutations corresponding to lighter regions appear in the genetic background corresponding to the darker regions in which the lighter regions are nested. For example, in population 20 the first mutations to appear in the SS lineage were an 1,160 bp deletion in the rbs operon and a substitution in codon 454 of spoT. An IS150 insertion in the intergenic region between mokB and trg appeared on this background around generation 300 and remained at low frequency for the rest of the experiment. Around generation 650, a single bp deletion in codon 394 of nadR appeared on the rbs Δ1160 bp+spoT-454+mokB/trg IS150 background. Grouping of mutations into lineages was based on their presence together in sequenced clones (in this case 20-SS2), and their order of appearance was inferred from the time point sample in which each was first detected. In addition, three mutations not found in any of the sequenced clones but whose association with SS and FS can be inferred (explained in SI) are shown in gray [FS-associated spoT-414 in (a), and SS-associated nadR-235 in (a) and nadR-290 in (c)]. For visual clarity, mutations of similar frequency within a lineage have been lumped together and their frequencies averaged. See Figures 3–5, S1, and S2 for the frequencies of individual mutations. The * on the time axis indicates the time when the sequenced clones (Figure 1) were extracted.

Figure 3. Dynamics of frequencies of mutations found in the SS clones.

(a) population 18. (b) population 19. (c) population 20. Δ, deletion; +, insertion. Numbers following gene names indicate the affected codon within the gene. Two gene names separated by a forward slash (e.g., mokB/trg) indicate that the mutation is in the intergenic region between the indicated genes. Mutations shown are in both SS clones from the population except where indicated otherwise.

Figure 4. Dynamics of frequencies of mutations found in the FS clones.

(a) population 18. (b) population 19. (c) population 20. Δ, deletion; +, insertion. Numbers following gene names indicate the affected codon within the gene. Two gene names separated by a forward slash (e.g., yfbV/ackA) indicate that the mutation is in the intergenic region between the indicated genes. Mutations shown are in both FS clones from the population except where indicated otherwise. The +T insertion between yfbV and ackA is identical in populations 18 and 19.

Figure 5. Frequencies of mutations in the nadR gene.

(a) population 18. (b) population 19. (c) population 20. Δ, deletion; +, insertion. Numbers following gene names indicate the affected codon within the gene. FS, SS1, etc. in parentheses indicate which clones (if any) have the mutation. The nadR-290 mutation is identical in populations 19 and 20; nadR-290 in population 18 is a different mutation in the same codon. The nadR-294 mutation is identical in populations 18 and 20; nadR-294 in population 19 is a different mutation in the same codon. Note that the highest frequency on the y-axis is 0.35.