The role of plasmid copy number and mutation rate in evolutionary outcomes.

Multicopy plasmids are widespread in nature and compose a common strategy for spreading beneficial traits across microbes. However, the role of plasmids in supporting the evolution of encoded genes remains underexplored due to challenges in experimentally manipulating key parameters such as plasmid copy number and mutation rate. Here we developed a strategy for controlling copy number in the plasmid-based Saccharomyces cerevisiae continuous evolution system, OrthoRep, and used our resulting capabilities to investigate the evolution of a conditionally essential gene under varying copy number and mutation rate conditions. Our results show that low copy number facilitated the faster enrichment of beneficial alleles whereas high copy number promoted robustness through the maintenance of allelic diversity. High copy number also slowed the removal of deleterious mutations and increased the fraction of non-functional alleles that could hitchhike during evolution. This study highlights the nuanced relationships between plasmid copy number, mutation rate and evolutionary outcomes, providing insights into the adaptive dynamics of genes encoded on multicopy plasmids and nominating OrthoRep as a versatile tool for studying plasmid evolution.