Diploidy alters the path of fluconazole adaptation in Candida glabrata

Candida glabrata (syn. Nakaseomyces glabratus) is a major fungal pathogen, typically isolated as a haploid. 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 clones, as measured in competitive assays with and without fluconazole, were diploid double heterozygotes having nonsynonymous mutations to PDR1 and ERG11 or PDR1 and ERG25. Diploid clones also had higher minimum inhibitory concentrations (MICs) to fluconazole. These results suggest, in this particular context, an evolvability advantage for diploids. In a follow-up experiment in which we rapidly increased the fluconazole concentration to a very high concentration, haploids and diploids adapted via entirely distinct routes: 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 sampled clones, and chrC in most sampled clones.