The emergence of a long term stable polymorphism has been shown to require three mutations for one the two partners. Complex epistatic interactions between these mutations and allele specificity appear to be key components in the establishment of the frequency dependency sustaining the stability of

Ecological opportunities promote population divergence into coexisting lineages. However, elucidation of the genetic mechanisms that enable new lineages to exploit these opportunities has been limited to cases of single mutations. We examined how two E. coli lineages diverged from their common ancestor at the outset of a long-term coexistence. By sequencing genomes and reconstructing the genetic history of one lineage, we showed that three mutations together were sufficient to produce the frequency-dependent fitness effects that allowed this lineage to invade and stably coexist with the other. These mutations all affected regulatory genes and collectively caused substantial metabolic changes. Moreover, the particular derived alleles were critical for the initial divergence and invasion, indicating that the establishment of this polymorphism depended on specific epistatic interactions.