it, the module was strongly detrimental. The gltA mutation therefore seems to have permitted early, weak Cit+ variants to persist in the population until later refining mutations could occur, consistent with their earlier conclusions. After a strong Cit++ phenotype evolved, the increased citrate synthase activity became detrimental. The researchers found that later mutations in gltA countered the first mutation, reducing citrate synthase activity, and further improving growth on citrate. They concluded that the series of mutation in gltA first potentiated, and then refined growth on citrate. They also suggested that the lineage in which Cit+ arose might have occupied a niche in Ara-3 based on growth on acetate, and that the potentiating mutations that led to evolution of Cit+ in Ara-3 were originally adaptive for acetate use. Investigation of post-Cit+ ecology and persistent diversity A small subpopulation of Cit− cells unable to grow on citrate, and belonging to a separate clade persisted in the population after the Cit+ cells became dominant. Early findings showed that this diversity was partly due to the Cit− cells being better at growing on the glucose in the medium. Turner et al. later found that another factor behind the coexistence was that the Cit− cells evolved the ability to cross feed on the Cit+ majority. They showed that the Cit+ cells release succinate, malate, and fumarate during growth on citrate, as the CitT transporter pumps these substances out of the cell while pumping citrate into the cell. The Cit− cells had rapidly evolved the ability to grow on these substances due to a mutation that restored expression of an appropriate transporter protein that was silent in the ancestor. The Cit− subpopulation eventually went extinct in the population between 43,500 and 44,000 generations. This extinction was shown to not be due to the Cit+ majority evolving to be able to invade the niche occupied by the Cit− minority. Indeed, Cit− clones could invade Cit+ populations from after the extinction event. Moreover, in an experiment in which they restarted twenty replicates of the Ara-3 population from the sample frozen 500 generations before the extinction, Turner et al. found that the Cit− subpopulation had not gone extinct in any of the replicates after 500 generations of evolution. One of these replicates was continued for 2,500 generations, over which Cit− continued to coexist. The researchers concluded that the extinction of Cit− had been due to some unknown "rare environmental