RNAi is a critical determinant of centromere evolution in closely related fungi.

The centromere DNA locus on a eukaryotic chromosome facilitates faithful chromosome segregation. Despite performing such a conserved function, centromere DNA sequence as well as the organization of sequence elements is rapidly evolving in all forms of eukaryotes. The driving force that facilitates centromere evolution remains an enigma. Here, we studied the evolution of centromeres in closely related species in the fungal phylum of Basidiomycota. Using ChIP-seq analysis of conserved inner kinetochore proteins, we identified centromeres in three closely related Cryptococcus species: two of which are RNAi-proficient, while the other lost functional RNAi. We find that the centromeres in the RNAi-deficient species are significantly shorter than those of the two RNAi-proficient species. While centromeres are LTR retrotransposon-rich in all cases, the RNAi-deficient species lost all full-length retroelements from its centromeres. In addition, centromeres in RNAi-proficient species are associated with a significantly higher level of cytosine DNA modifications compared with those of RNAi-deficient species. Furthermore, when an RNAi-proficient Cryptococcus species and its RNAi-deficient mutants were passaged under similar conditions, the centromere length was found to be occasionally shortened in RNAi mutants. In silico analysis of predicted centromeres in a group of closely related Ustilago species, also belonging to the Basidiomycota, were found to have undergone a similar transition in the centromere length in an RNAi-dependent fashion. Based on the correlation found in two independent basidiomycetous species complexes, we present evidence suggesting that the loss of RNAi and cytosine DNA methylation triggered transposon attrition, which resulted in shortening of centromere length during evolution.