The roles of chance, contingency, and necessity in evolution is unresolved, because they have never been assessed in a single system or on timescales relevant to historical evolution. We combined ancestral protein reconstruction and a new continuous evolution technology to mutate and select B-cell-lymphoma-2-family proteins to acquire protein-protein-interaction specificities that occurred during animal evolution. By replicating evolutionary trajectories from multiple ancestral proteins, we found that contingency generated over long historical timescales steadily erased necessity and overwhelmed chance as the primary cause of acquired sequence variation; trajectories launched from phylogenetically distant proteins yielded virtually no common mutations, even under strong and identical selection pressures. Chance arose because many sets of mutations could alter specificity at any timepoint; contingency arose because historical substitutions changed these sets. Our results suggest that patterns of variation in BCL-2 sequences – and likely other proteins, too – are idiosyncratic products of a particular, unpredictable course of historical events.

The extent to which evolutionary outcomes reflect the unpredictable influences of chance and contingency is a central but unanswered question in evolutionary biology 1–4. A precise characterization requires evolutionary trajectories to be repeated multiple times under identical environmental conditions from multiple starting points across history, a scenario that rarely, if ever, occurs in nature. Here we combine continuous experimental evolution 5 with ancestral protein reconstruction 6 and manipulative genetic experiments to identify the causes and consequences of chance and contingency in the genetic outcomes of molecular evolution. By repeatedly evolving ancestral proteins in the B-cell lymphoma-2 (BCL-2) family of apoptosis regulators 7 to acquire the same protein-protein interaction specificities that evolved during history, we found that contingency and chance interact to make sequence evolution increasingly unpredictable over phylogenetic timescales. Although replicates from the same starting genotype sometimes share mutations – indicating partial predictability – there are multiple alternative sets of changes that can alter specificity, and chance decides which of these paths is taken. Contingency has a stronger effect: when trajectories are initiated from different starting points, outcomes are even more divergent, because substitutions that occurred during phylogenetic history repeatedly changed the potential of other mutations to confer new binding specificities. The impact of contingency increased steadily with phylogenetic distance and magnified the effects of chance, resulting in a >3-fold increase in genetic variance among evolutionary trajectories initiated from different starting points across the timescale of metazoan evolution. Our findings show how a particular cascade of chance evolutionary steps throughout history makes the outcomes of molecular evolution increasingly idiosyncratic and unpredictable, even under strong selection.

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