Swapping five most likely adaptive residues in the CENP-O C-terminal RWD domain is insufficient to reduce centromere binding. Mouse oocytes expressing the indicated CENP-O–GFP constructs were fixed in meiosis I and stained for centromeres (CREST) and DNA (DAPI). One construct is rat CENP-O with the mouse C-terminal RWD domain, and the other is identical except for five mutations in the RWD domain swapping mouse-specific residues with the highest probability of having evolved under positive selection (probability ≥ 0.9; R225G, C226T, T228A, N247G, P261H) to the corresponding rat-specific residues. (A–C) Images (A) show maximum projections; scale bars, 5 μm. Graphs show CENP-O-GFP intensity at centromeres (B) or in the cytoplasm (C); for each construct, n ≥ 950 centromeres from ≥25 cells from three independent experiments. Each spot represents one cell; bars, mean intensities with standard deviation; ns: not significant, two-tailed Student's T test.