A high aneuploidy supply rate underlies parallel evolution of fluconazole resistance in Saccharomyces cerevisiae

Abstract

Copy number variants (CNVs) of ERG11 play a major role in acquisition of resistance to the commonly used azole family of antifungal drugs in many pathogenic yeast species. However, the conditions in which these CNVs arise, and the factors that underlie their emergence and selection in evolving populations, remain poorly understood. Here, we studied the dynamics of de novo CNVs at the ERG11 locus in Saccharomyces cerevisiae to determine the effect of different drug concentrations and temperatures on CNV formation and selection. We found that ERG11 CNVs emerge reproducibly at fluconazole concentrations around the IC50. With increases in temperature, CNVs emerged more rapidly and had a higher tendency towards fixation. Evolved ERG11 CNV strains provided a significant growth advantage across a narrow range of fluconazole concentrations near the IC50 of the wildtype strain. Whole genome sequencing of 35 independent evolved lineages revealed that all ERG11 CNVs are aneuploidies of chromosome VIII. Relocation of ERG11 to chromosome XI also results exclusively in selection for chromosome XI aneuploids. We show that fluconazole does not increase the frequency at which aneuploids are generated. Therefore, we conclude that a high spontaneous rate of aneuploidy formation underlies recurrent acquisition of resistance to fluconazole in a temperature and drug concentration dependent manner. Author SummaryCopy number variations (CNV), defined as duplications or deletions of genomic regions, are pervasive throughout all forms of life. Although CNVs can provide a route toward rapid adaptation, they can also be associated with high fitness costs. In pathogenic yeast species, CNVs with diverse genetic structures are known to play a role in antifungal drug resistance. However, the conditions that favor CNV formation and selection are unclear. We applied a fluorescent CNV reporter system to track newly formed CNVs and found that they arise reproducibly at a narrow range of drug concentrations, and that increases in temperature generally lead to more rapid emergence of CNVs. DNA sequencing revealed that the evolved CNVs contained duplications of the entire chromosome, rather than just the region surrounding the gene under selection. Thus, aneuploidy - gain or loss of an entire chromosome - is a mechanism for rapid evolution of drug resistance, potentially without the fitness costs thought to be associated with this mechanism.