Mutations with large fitness benefits and mutations occurring at high rates may both cause parallel evolution, but their contribution is predicted to depend on population size. Moreover, high-rate and large-benefit mutations may have different long-term adaptive consequences. We show that small and 100-fold larger bacterial populations evolve resistance to a β-lactam antibiotic by using similar numbers, but different types of mutations. Small populations frequently substitute similar high-rate structural variants and loss-of-function point mutations, including the deletion of a low-activity β-lactamase, and evolve modest resistance levels. Large populations more often use low-rate, large-benefit point mutations affecting the same targets, including mutations activating the β-lactamase and other gain-of-function mutations, leading to much higher resistance levels. Our results demonstrate the separation by clonal interference of mutation classes with divergent adaptive consequences, causing a shift from high-rate to large-benefit mutations with increases in population size.
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There are no restrictions on data availability. Accession codes for reference sequences used are provided in Supplementary Information (REL606 genome: Genbank NC_012967.1; pACTEM1 plasmid: Genbank MN386081). Raw sequencing reads have been submitted to the NCBI Sequence Read Archive (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA790633). Other data and code have been made available in Supplementary Information and at Dryad (https://doi.org/10.5061/dryad.b2rbnzsh2), and are organized per figure.
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We thank B. Koopmanschap (deceased) for practical help, and D. Aanen, S.-C. Park, S. Das, M. Lässig and A. Stoltzfus for comments and discussion. This work was supported by DFG grant SFB680 to M.F.S., M.P.Z., S.H., J.K. and J.A.G.M.d.V.; DFG grant CRC1310 to J.K. and J.A.G.M.d.V.; HFSP Research Grant RGP0010/2015 to P.R. and J.A.G.M.d.V.; and an EMBO fellowship (ALTF 273-2017) to P.R. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.
The authors declare no competing interests.
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CTX concentrations during the evolution experiment based on daily 20.25-fold increases when the OD600 was higher than 75% of the ancestral value without CTX (see text). Red lines represent small populations (S), blue lines large populations (L). Shades and line types have been varied randomly to better distinguish replicate populations. Note that for one large population the CTX concentration was increased during every round of passaging, as the cultures always reached a high density. For two other large populations, this was the case on all but one round of passaging.
Histogram of the number of mutations per clone, for all 112 populations.
Mutation frequency per mutation class and per treatment for the 107 non-mutator populations. Error bars indicate the standard error of the mean. SNPs are green, Indels are blue, and SVs are purple. Error bars indicate the standard error of the mean.
(a) Gene-level H-index is given for all mutational events in clones from non-mutator populations. (b) Gene-level H-index is given for three classes of mutational events, as indicated by the legend.
Regression analysis of the fraction of SVs among all mutations per clone against CTX concentration.
Distribution of fixed mutations at the evolutionary endpoint obtained from the optimized WF model in comparison to the experimental data. Column heights represent the mean number of mutations and error bars show the corresponding standard deviation.
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Schenk, M.F., Zwart, M.P., Hwang, S. et al. Population size mediates the contribution of high-rate and large-benefit mutations to parallel evolution. Nat Ecol Evol 6, 439–447 (2022). https://doi.org/10.1038/s41559-022-01669-3
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