Epistasis among clustered lineage-specific adaptive amino acid substitutions in the Drosophila Trio protein.

Intramolecular epistasis is increasingly recognized as a key factor shaping patterns of evolutionary rate variation among protein sites and constraining adaptive evolution. While genome-wide analyses have revealed that intramolecular epistatic interactions can drive the spatial clustering of amino acid substitutions, direct empirical evidence for such interactions and their evolutionary consequences remains limited. Using a statistical screen for lineage-specific clustering of adaptive substitutions in Drosophila, we systematically identify experimentally tractable candidate proteins with evidence for spatially-clustered adaptive amino acid substitutions for functional genetic analysis. As proof of concept, we focus on the Trio protein, which exhibits three spatially-clustered adaptive amino acid substitutions in along the D. melanogaster lineage. Through systematic reconstruction of evolutionary intermediates in vivo, we find that intermediate states exhibit reduced fitness, providing strong evidence for epistatic constraints on evolutionary trajectories. Notably, we find that the deleterious effects of intermediates are recessive, suggesting that intermediate combinations of epistatically interacting amino acid substitutions can arise and be maintained in heterozygotes before fixation. Our findings provide a rare empirical window into the fitness landscape shaped by intramolecular epistasis and establish a framework for broader investigation of constraints adaptive protein evolution in diploid multicellular organisms.