Unconventional centromere architectures in Tapirus indicus reveal hotspots for satellite-free centromere formation in Perissodactyla.

Centromeres, the chromosomal loci responsible for segregation during cell division, play a key role in genome evolution and speciation. While centromere function is highly conserved and epigenetically defined by CENP-A, the underlying DNA sequences are among the most rapidly evolving. Although mammalian centromeres are typically associated with satellite DNA, we previously showed that equids carry numerous satellite-free centromeres. Here, we investigate centromere and karyotype evolution in Tapirus indicus, a non-equid perissodactyl with exceptional karyotypic plasticity. Through CENP-A ChIP-seq analysis on the same individual for which a near-gapless diploid genome assembly generated by the Vertebrate Genome Project was available, we identify 23 canonical satellite-based centromeres, two completely satellite-free centromeres, and one centromere with a very low content of satellite tracts. The unconventional centromeres arose through centromere repositioning, thereby redefining the evolutionary prevalence of satellite-free centromeres across mammals. Comparative genomic analysis uncovers evolutionary hotspots for satellite-free centromere formation across Perissodactyla. Finally, analysis of CENP-B binding shows that T. indicus displays uncoupling between CENP-A and CENP-B, a feature previously observed only in equids. These findings reveal that high centromere plasticity is not unique to equids and support a broader model in which centromere plasticity and CENP-B uncoupling contribute to karyotype evolution in mammals.