Epistasis among clustered lineage-specific 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 population genetic screen for spatially-clustered and lineage-specific adaptive amino acid substitutions in Drosophila proteins, we systematically identify experimentally tractable candidates for functional analysis. As proof of concept, we focus on the Trio protein, a Rho guanine nucleotide exchange factor that exhibits three spatially-clustered putatively adaptive amino acid substitutions in the D. melanogaster lineage. By systematically reconstructing evolutionary intermediates in vivo using genome editing, we find that all possible intermediate states exhibit reduced viability and/or locomotor defects, providing strong evidence for epistatic constraints on evolutionary trajectories. Notably, these deleterious effects are recessive, suggesting that intermediate combinations of epistatically interacting amino acid substitutions can accumulate in heterozygotes prior to fixation, thereby circumventing apparent constraints imposed by maladaptive intermediate states. Together, these findings provide a rare empirical view of the fitness landscape shaped by intramolecular epistasis and establish a framework for investigating the constraints on adaptive protein evolution in diploid multicellular organisms.