Centromeres in budding yeasts are conserved in chromosomal location but not in structure.
The budding yeast Saccharomyces cerevisiae has point centromeres, which are much smaller and simpler than centromeres of most other eukaryotes and have a defined DNA sequence. Other yeast taxa have different and highly diverse centromere structures, but a clear picture of how yeast centromeres have evolved is lacking. Here, we investigated nine yeast species in two taxonomic orders that are close outgroups to S. cerevisiae. We find that they have a wide diversity of centromere structures, indicating that multiple transitions of structure have occurred within the last 200 Myr. Some species have centromeres with defined sequence motifs (17 - 200 bp), others consist of Inverted Repeats (IRs), and others have Ty5-like retroelement clusters. Strikingly, the chromosomal locations of centromeres have largely been conserved across taxonomic orders, even as their structures have changed, which suggests that structure replacement occurs in situ. In some Barnettozyma species we find that a single genome can contain chromosomes with different centromere structures - some with IRs and some without - which suggests that a structural transition is underway in this genus. We identified only one example of a centromere moving by a long distance: a new centromere formed recently at the MAT locus of Barnettozyma californica, 250 kb from the previous centromere on that chromosome. Author summaryCentromeres are an evolutionary paradox. Their molecular function is highly conserved, but their structures vary tremendously among eukaryotes. The "point" centromeres of the yeast Saccharomyces cerevisiae are among the most unusual: they are tiny (< 200 bp) and non-repetitive, and unlike other centromeres they contain a single copy of a well-defined sequence motif. However, we have little knowledge about where these point centromeres came from, or more generally about how changes of centromere structure occur during evolution. Here, we characterized centromere structure and location in nine species of budding yeasts, spanning an evolutionary depth of approximately 200 million years. We find that the chromosomal locations of centromeres are extraordinarily well conserved, whereas their structures vary greatly. We show that sequence-defined centromeres are older and more widely distributed than previously realised. We identify some species in which the centromeres of different chromosomes have different structures, which suggests that they are in transition from one structure to another. The centromere variation observed makes it difficult to infer the ancestral structure.