LAUSR

Significance The “centromere paradox” refers to rapidly evolving and highly diverse centromere DNA sequences even in closely related eukaryotes. However, factors contributing to this rapid divergence are largely unknown. Here, we identified large regional, LTR retrotransposon-rich centromeres in a group of human fungal pathogens belonging to the Cryptococcus species complex. We provide evidence that loss-of-functional RNAi machinery and possibly cytosine DNA methylation trigger instability of the genome by activation of centromeric retrotransposons presumably suppressed by RNAi. We propose that RNAi, together with cytosine DNA methylation, serves as a critical determinant that maintains repetitive transposon-rich centromere structures. This study explores the direct link between RNAi and centromere structure evolution. 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.