Diploidy alters the path of fluconazole adaptation in C. glabrata

C. glabrata (syn. Nakaseomyces glabratus) is a major fungal pathogen, typically isolated as a haploid but occasionally found in diploid form. We isolated a spontaneous diploid variant of the type strain CBS138 and performed experimental evolution under fluconazole. At physiologically relevant concentrations of fluconazole, we found that haploids and diploids both acquired PDR1 mutations, as is commonly observed in C. glabrata. Diploids additionally acquired heterozygous ERG11 and ERG25 mutations, and were more likely to acquire aneuploidies. Despite an ancestral fitness advantage for haploids, after [~]200 generations the highest-fitness clone, as measured in competitive assays with and without fluconazole, was a diploid PDR1 V329F/+ ERG11 K152E/+ double heterozygote. Diploid clones also had higher MICs. In a follow-up experiment in which we rapidly increased the fluconazole concentration to [~]1 mg/mL, haploids and diploids adapted via entirely different paths: haploids via co-mutation in ERG3 and CgOSH3, a previously unreported path to fluconazole resistance, and diploids via heterozygous mutation in ERG25 coupled with trisomies of chrF, chrG, and chrI (in all clones) and chrC (in most).