Alterations in chromosome number and size are hallmarks of cancer, and yet they are also crucial for species evolution. Recent insight on chromosome structure and segregation, together with significant advances in genomic sequencing, established previously underappreciated parallels between the mechanisms of karyotypic evolution during speciation and cancer. Importantly, alterations in chromosome number and size pose significant challenges for the cell division machinery. However, how dividing cells adapt to cope with karyotypic alterations remains an outstanding fundamental question with strong clinical implications. To address this, we propose an innovative approach that integrates super-resolution microscopy, large-scale 3D cryo-electron tomography reconstructions, computational modelling and cell fusion experiments in Indian and Chinese muntjacs, two closely related deer species with identical genomes, but extremely divergent chromosome number (2n=6/7 vs. 2n=46, respectively) and size. We will focus on dissecting mechanistic aspects underlying mitotic spindle organization, chromosome dynamics, checkpoint control of mitotic progression and segregation fidelity in both muntjac species, with the goal of identifying genes differentially required for efficient mitosis in cells with karyotypic alterations. In parallel, we will exploit functional genomics in Cervidae, including telomere-to-telomere sequencing of Indian and Chinese muntjac genomes, to determine and manipulate the exact chromosome fusion sites between both species, while testing the novel hypothesis of a viral mechanism in the evolution of their karyotypes. Lastly, to test the limits of karyotypic evolution for chromosome segregation, we will engineer mammalian cells with only 1 or 2 chromosomes and investigate whether and how cell division tolerates this extreme genome reorganization. This pioneer work will unveil vital cell adaptive mechanisms to karyotypic evolution relevant for speciation and cancer.