Identifying the drivers of computationally detected correlated evolution among sites under antibiotic selection

The ultimate causes of correlated evolution among sites in a genome remain difficult to tease apart. To address this problem directly, we performed a high‐throughput search for correlated evolution among sites associated with resistance to a fluoroquinolone antibiotic using whole‐genome data from clinical strains of Pseudomonas aeruginosa, before validating our computational predictions experimentally. We show that for at least two sites, this correlation is underlain by epistasis. Our analysis also revealed eight additional pairs of synonymous substitutions displaying correlated evolution underlain by physical linkage, rather than selection associated with antibiotic resistance. Our results provide direct evidence that both epistasis and physical linkage among sites can drive the correlated evolution identified by high‐throughput computational tools. In other words, the observation of correlated evolution is not by itself sufficient evidence to guarantee that the sites in question are epistatic; such a claim requires additional evidence, ideally coming from direct estimates of epistasis, based on experimental evidence.