Numerical data underlying all graphs.

Antimicrobial resistance (AMR) is a global health threat emerging through microbe adaptation, driven by genetic variation, genome plasticity or epigenetic processes. In this study, we investigated how the Mucor circinelloides species complex adapts to the antifungal natural product FK506, which binds to FKBP12 and inhibits calcineurin-dependent hyphal growth. In Mucor bainieri, most FK506-resistant isolates (90%) were found to be unstable and transient, readily reverting to being drug sensitive when passaged without drug, and with no associated DNA mutations. In half of the isolates (50%), FK506-resistance was conferred by RNAi-dependent epimutation in which small interfering RNAs (siRNAs) silenced the fkbA encoding FKBP12 post-transcriptionally. In contrast, most of the remaining FK506-resistant isolates (40%) were found to have undergone heterochromatin-mediated silencing via H3K9 dimethylation, transcriptionally repressing fkbA and neighboring genes. In these heterochromatic epimutants, only minimal enrichment of siRNA to the fkbA locus was observed, but in three of the four examples, siRNA was significantly enriched at a locus distant from fkbA. A similar mechanism operates in Mucor atramentarius, where FK506 resistance was mediated by ectopic heterochromatin silencing of fkbA and associated genes with siRNA spreading across the region. Heterochromatin-mediated fkbA epimutants exhibited stability during in vivo infection, suggesting epimutation could impact pathogenesis. These findings reveal that antifungal resistance arising through distinct, transient epimutation pathways involving RNAi or heterochromatin, highlighting adaptive AMR strategies employed by ubiquitous eukaryotic microbes.