There is a growing need for accurate evolutionary forecasting, but we must first understand how possible evolutionary paths can be constrained by silent genetic features. Here we show that synonymous sequence variation determines extreme parallel evolution during the evolutionary rescue of flagellar motility. An immotile variant of the soil microbe, Pseudomonas fluorescens, swiftly recovers flagellum-dependent motility through parallel de novo mutation. This typically manifests within 96 h under strong selection through repeatable mutation within the nitrogen pathway's histidine kinase gene, ntrB. We found that evolution was parallel to nucleotide resolution in over 95% of cases in minimal medium (M9), with lineages repeatedly fixing an identical mutation (ntrB A289C). There was no evidence that this substitution is context-specific, as repeatable de novo mutation was robust to nutrient condition despite evidence for antagonistic pleiotropy. Competition assays against alternative motile alleles revealed some evidence for selection enforcing repeated fixation of ntrB mutants, but there was no evidence for clonal interference driving parallel evolution to nucleotide resolution. Instead, the introduction of 6 synonymous substitutions surrounding the mutational hotspot reduced parallel evolution from >95% to 0% at the site. In a reciprocal experiment, we introduced 6 synonymous substitutions into a homologous strain that did not ancestrally evolve in parallel and observed that parallel evolution at the site rose from 0% to 80%. We propose that these silent mutations facilitate extremely localised heterogeneity in de novo mutation. Our results reveal that unique quirks in how DNA is structured at specific loci can strongly bias evolutionary outcomes.

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