The structure of an ancient genotype-phenotype map shaped the functional evolution of a protein family.

Mutations are more likely to produce some phenotypes than others, but the causal role of these production propensities in the evolution of phenotypic diversity remains unclear. There are two major challenges: it is difficult to separate the effect of the genotype-phenotype (GP) map from that of natural selection when analysing natural diversity, and most extant phenotypes evolved long ago in species whose GP maps cannot be recovered. Here, using two reconstructed ancestral transcription factors that are closely related but differ in function, we created libraries containing all possible amino acid combinations at historically variable sites in the proteins' DNA binding interface (the genotypes) and measured their capacity to specifically bind DNA elements containing all possible combinations of nucleotides at historically variable sites (the phenotypes). The ancestral GP maps were strongly anisotropic (the distribution of phenotypes encoded by genotypes is highly non-uniform) and heterogeneous (the phenotypes accessible around each genotype vary greatly among genotypes), but the extent and direction of these properties differed substantially between the maps. In both cases, these properties steered evolution towards the lineage-specific phenotypes that evolved during history. Our findings establish that ancient properties of the GP relationship were causal factors in the evolutionary process that produced present-day patterns of functional conservation and diversity.