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Comparative genomics has proven a fruitful approach to acquire many functional and evolutionary insights into core cellular processes. Here it is argued that in order to perform accurate and interesting comparative genomics, one first and foremost has to be able to recognize, postulate, and revise different evolutionary scenarios. After all, these studies lack a simple protocol, due to different proteins having different evolutionary dynamics and demanding different approaches. The authors here discuss this challenge from a practical (what are the observations?) and conceptual (how do these indicate a specific evolutionary scenario?) viewpoint, with the aim to guide investigators who want to analyze the evolution of their protein(s) of interest. By sharing how the authors draft, test, and update such a scenario and how it directs their investigations, the authors hope to illuminate how to execute molecular evolution studies and how to interpret them. Also see the video abstract here https://youtu.be/VCt3l2pbdbQ.

Kinetochores are highly specialized macromolecular machines built on top of centromeric chromatin to orchestrate the synchronous and equal distribution of chromosomes during mitosis and meiosis. Each kinetochore consists of up to hundreds of copies of a core complex of ~75 proteins that together constitute and regulate the chromosomal attachment site for microtubules that emanate from the bipolar spindle. Although microtubule-based chromosome segregation is evolutionary conserved in eukaryotes, kinetochore compositions seem to evolve rapidly. To systematically inventory kinetochore diversity and to reconstruct its evolution, we determined the orthologs of 75 kinetochore proteins in 100 phylogenetically diverse eukaryotes. The resulting ortholog sets imply that the last eukaryotic common ancestor (LECA) possessed a complex kinetochore and highlight that many present-day kinetochores have diverged through rapid sequence evolution, extensive gene loss, duplication, invention and displacement. Striking examples include the mutually exclusive presence absence patterns of the microtubule plus-end tracking Dam1 and SKA complex, the recurrent loss of the largest part of a 16-subunit inner kinetochore complex (CCAN) that is essential in vertebrate and fungal cells and the degenerate composition of kinetochores in various alveolate and excavate lineages. To harness the wealth of sequence data of our manually determined ortholog sets and to capture patterns of highly divergent sequence evolution that are characteristic of many kinetochore subunits, we developed a de novo sequence discovery workflow (ConFeaX) to trace the eukaryote-wide (co-)evolution of short linear motifs, domains and proteins of the kinetochore network. This versatile approach proved useful as the advanced detection of conserved elements and their (co-)evolutionary reconstructions provided testable hypotheses on various aspects of eukaryotic kinetochore biology. For example, ConFeaX guided the discovery of functional short linear motifs for the kinetochore proteins Knl1, BubR1 and Spindly in human cells. The extensive diversity of kinetochore compositions raises many questions on the evolutionary dynamics of a core eukaryotic cellular process such as chromosome segregation.