Extreme parallel evolution of flagellar motility facilitated by silent mutations

12 There is a growing need for accurate evolutionary forecasting, but we must first understand how 13 possible evolutionary paths can be constrained by silent genetic features. Here we show that 14 synonymous sequence variation determines extreme parallel evolution during the evolutionary rescue 15 of flagellar motility. An immotile variant of the soil microbe, Pseudomonas fluorescens, swiftly 16 recovers flagellum-dependent motility through parallel de novo mutation. This typically manifests 17 within 96 h under strong selection through repeatable mutation within the nitrogen pathway’s histidine 18 kinase gene, ntrB. We found that evolution was parallel to nucleotide resolution in over 95% of cases 19 in minimal medium (M9), with lineages repeatedly fixing an identical mutation (ntrB A289C). There 20 was no evidence that this substitution is context-specific, as repeatable de novo mutation was robust to 21 nutrient condition despite evidence for antagonistic pleiotropy. Competition assays against alternative 22 motile alleles revealed some evidence for selection enforcing repeated fixation of ntrB mutants, but 23 there was no evidence for clonal interference driving parallel evolution to nucleotide resolution. Instead, 24 the introduction of 6 synonymous substitutions surrounding the mutational hotspot reduced parallel 25 evolution from >95% to 0% at the site. In a reciprocal experiment, we introduced 6 synonymous 26 substitutions into a homologous strain that did not ancestrally evolve in parallel and observed that 27 parallel evolution at the site rose from 0% to 80%. We propose that these silent mutations facilitate 28 extremely localised heterogeneity in de novo mutation. Our results reveal that unique quirks in how 29 DNA is structured at specific loci can strongly bias evolutionary outcomes. 30