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 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 a frozen population sample taken ...