Possible Evolutionary Paths, Epistatic Effects, and Evolutionary Reversibility in the ancB-ancAP3 Lineage. Possible Evolutionary Paths, Epistatic Effects, and Evolutionary Reversibility in the ancB-ancAP3 Lineage. (A) Overlap of gain-of-interaction sites identified in ancAP3 by Y2H screen and sites that evolved along the ancB-ancAP3 branch, showing a strong association between sites identified (rows) and sites evolved (columns). (B) Total number of sites identified by the reverse Y2H screen in the subdomains of ancAP3. (C) Quantification of Y2H interaction with ancCD of selected ancAP3 double mutants identified by the screen and the corresponding single mutants generated by site-directed mutagenesis, exemplifying the frequent occurrence of positive epistasis. Error bars indicate the se of the mean of three replicates. (D) Mutational trajectory of ancB toward the loss of its interaction with ancCD. Several gradual mutational trajectories are possible (indicated by black arrowheads) that circumvent epistatic constraints (red lines). K96E to L54F/K96E is a nearly neutral substitution that may or may not be accessible depending on the population genetic context (black line without arrowhead). The pie charts represent the interaction between ancB or its variants (AD) and ancCD (BD) as a percentage of the ancB-AD+ancCD-BD amount of Miller units. (E) The observed male and female quartets AP3-PI-AG-SEP3 and AG-AG-SEP3-SEP3 can both be formed along a single axis given a C gradient along this axis. Shown at the bottom is a schematic representation of the parameter space for which a male-female shift can be established along the C gradient (see also Supplemental Figures 5 to 12). Philip Ruelens et al. Plant Cell 2017;29:229-242 ©2017 by American Society of Plant Biologists