# Mutation supply and the repeatability of selection for antibiotic resistance

## TL;DR

This study investigates how mutation supply and selective pressure influence the repeatability of evolution towards antibiotic resistance in bacteria, revealing that evolution is largely predictable but less so with larger mutation libraries due to multiple mutations increasing resistance.

## Contribution

It demonstrates that mutation supply and population size largely determine evolutionary outcomes, and highlights the role of large-effect mutations and multiple mutations in resistance levels.

## Key findings

- Evolution is predictable based on mutation effects, mutation rate, and population size.
- Large mutation libraries show less repeatability when only large-effect mutations are considered.
- Multiple mutations confer higher resistance, affecting repeatability at large library sizes.

## Abstract

Whether evolution can be predicted is a key question in evolutionary biology. Here we set out to better understand the repeatability of evolution. We explored experimentally the effect of mutation supply and the strength of selective pressure on the repeatability of selection from standing genetic variation. Different sizes of mutant libraries of an antibiotic resistance gene, TEM-1 $\beta$-lactamase in Escherichia coli, were subjected to different antibiotic concentrations. We determined whether populations went extinct or survived, and sequenced the TEM gene of the surviving populations. The distribution of mutations per allele in our mutant libraries- generated by error-prone PCR- followed a Poisson distribution. Extinction patterns could be explained by a simple stochastic model that assumed the sampling of beneficial mutations was key for survival. In most surviving populations, alleles containing at least one known large-effect beneficial mutation were present. These genotype data also support a model which only invokes sampling effects to describe the occurrence of alleles containing large-effect driver mutations. Hence, evolution is largely predictable given cursory knowledge of mutational fitness effects, the mutation rate and population size. There were no clear trends in the repeatability of selected mutants when we considered all mutations present. However, when only known large-effect mutations were considered, the outcome of selection is less repeatable for large libraries, in contrast to expectations. Furthermore, we show experimentally that alleles carrying multiple mutations selected from large libraries confer higher resistance levels relative to alleles with only a known large-effect mutation, suggesting that the scarcity of high-resistance alleles carrying multiple mutations may contribute to the decrease in repeatability at large library sizes.