Replaying Evolution to Test the Cause of Extinction of One Ecotype in an Experimentally Evolved Population

In a long-term evolution experiment with Escherichia coli, bacteria in one of twelve populations evolved the ability to consume citrate, a previously unexploited resource in a glucose-limited medium. This innovation led to the frequency-dependent coexistence of citrate-consuming (Cit+) and non-consuming (Cit-) ecotypes, with Cit- bacteria persisting on the exogenously supplied glucose as well as other carbon molecules released by the Cit+ bacteria. After more than 10,000 generations of coexistence, however, the Cit- lineage went extinct; cells with the Cit- phenotype dropped to levels below detection, and the Cit- clade could not be detected by molecular assays based on its unique genotype. We hypothesized that this extinction event was a deterministic outcome of evolutionary change within the population, specifically the appearance of a more-fit Cit+ ecotype that competitively excluded the Cit- ecotype. We tested this hypothesis by re-evolving the population from one frozen sample taken just prior to the extinction and from another sample taken several thousand generations earlier, in each case for 500 generations and with 20-fold replication. To our surprise, the Cit- type did not go extinct in any of these replays, and Cit- cells also persisted in a single replicate that was propagated for 3,000 generations. Even more unexpectedly, we showed that the Cit- ecotype could reinvade the Cit+ population after its extinction. Taken together, these results indicate that the extinction of the Cit- ecotype was not a deterministic outcome driven by competitive exclusion by the Cit+ ecotype. The extinction also cannot be explained by demographic stochasticity, as the population size of the Cit- ecotype should have been many thousands of cells even during the daily transfer events. Instead, we infer that the extinction must have been caused by a rare chance event in which some aspect of the experimental conditions was inadvertently perturbed.