bacteria to outcompete ancestral bacteria under the conditions in the long-term evolution experiment. However, although this mutation increased fitness under these conditions, it also increased the bacteria's sensitivity to osmotic stress and decreased their ability to survive long periods in stationary phase cultures. Ecological specialization Over the course of the experiment, the populations have evolved to specialize on the glucose resource on which they grow. This was first described in 2000, when Cooper and Lenski demonstrated that all populations had experienced decay of unused metabolic functions after 20,000 generations, restricting the range of substances on which the bacteria could grow. Their analysis suggested that this decay was due to antagonistic pleiotropy, in which mutations that improved ability to grow on glucose had reduced or eliminated the ability to grow on other substances. A later study by Leiby and Marx that used more advanced techniques showed that much of the decay Cooper and Lenski had identified were experimental artifacts, that loss of unused functions was not as extensive as first thought, and that some unused functions had improved. Moreover, they concluded that the metabolic losses were not due to antagonistic pleiotropy, but the neutral accumulation of mutations in unused portions of the genome, suggesting that adaptation to a simple environment might not necessarily lead to specialization. Evolution of balanced polymorphism and simple ecosystems Two distinct variants, S and L, were identified in the population designated Ara-2 at 18,000 generations based on their formation of small and large colonies, respectively. Clones of the S and L types could co-exist stably in co-culture with each other, indicating they occupied distinct niches in the population. This was verified by the finding that the L type had an advantage during growth on glucose, but that S had an advantage during stationary phase, after glucose had run out. The two types were found to have initially evolved prior to 6,000 generations, and then co-existed thereafter. Phylogenetic analysis of clones of the two types isolated from different generations demonstrated that the S and L types belonged to distinct, co-existing lineages in the population, and might be undergoing incipient speciation. Evolution of aerobic citrate usage in one population BackgroundE. coli is normally unable to grow aerobically on citrate due to the inability to express a citrate transporter when oxygen is present. However, E. coli has a complete citric acid cycle, and therefore metabolizes citrate as an intermediate during