Whether or not evolutionary change is inherently irreversible remains a controversial topic. Some examples of evolutionary irreversibility are known; however, this question has not been comprehensively addressed at the molecular level. Here, we use data from 221 human genes with known pathogenic mutations to estimate the rate of irreversibility in protein evolution. For these genes, we reconstruct ancestral amino acid sequences along the mammalian phylogeny and identify ancestral amino acid states that match known pathogenic mutations. Such cases represent inherent evolutionary irreversibility because, at the present moment, reversals to these ancestral amino acid states are impossible for the human lineage. We estimate that approximately 10% of all amino acid substitutions along the mammalian phylogeny are irreversible, such that a return to the ancestral amino acid state would lead to a pathogenic phenotype. For a subset of 51 genes with high rates of irreversibility, as much as 40% of all amino acid evolution was estimated to be irreversible. Because pathogenic phenotypes do not resemble ancestral phenotypes, the molecular nature of the high rate of irreversibility in proteins is best explained by evolution with a high prevalence of compensatory, epistatic interactions between amino acid sites. Under such mode of protein evolution, once an amino acid substitution is fixed, the probability of its reversal declines as the protein sequence accumulates changes that affect the phenotypic manifestation of the ancestral state. The prevalence of epistasis in evolution indicates that the observed high rate of irreversibility in protein evolution is an inherent property of protein structure and function.

This work was supported by Plan Nacional grant BFU2009-09271 from the Spanish Ministry of Science and Innovation and by FPU (Formación del Profesorado Universitario) program grant AP2008-01888 from the Spanish Ministry of Education to O.S.