Mutational hotspots can determine evolutionary outcomes and make evolution repeatable. Hotspots are products of multiple evolutionary forces including mutation rate heterogeneity, but this variable is often hard to identify. In this work, we reveal that a near-deterministic genetic hotspot can be built and broken by a handful of silent mutations. We observe this when studying homologous immotile variants of the bacteria Pseudomonas fluorescens, AR2 and Pf0-2x. AR2 resurrects motility through highly repeatable de novo mutation of the same nucleotide in >95% lines in minimal media (ntrB A289C). Pf0-2x, however, evolves via a number of mutations meaning the two strains diverge significantly during adaptation. We determine that this evolutionary disparity is owed to just 6 synonymous variations within the ntrB locus, which we demonstrate by swapping the sites and observing that we are able to both break (>95% to 0%) and build (0% to 80%) a deterministic mutational hotspot. Our work reveals a key role for silent genetic variation in determining adaptive outcomes.

Original languageEnglish
Article number6092
JournalNature Communications
Issue number1
Publication statusPublished - 19 Oct 2021


  • Experimental evolution
  • Mutation bias
  • Molecular microbiology
  • Predicting evolution
  • Pseudomonas fluorescens

ASJC Scopus subject areas

  • Chemistry(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Physics and Astronomy(all)


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