Bount et al (2012) describes a very interesting study of the appearance (evolution) of the ability of the common gut bacteria, E. coli, to utilize citrate in the presence of O2, something that E. coli normally cannot do. This work was based on the original observation that described the evolutionary origin of citrate utilizing E. coli (Bount et al., 2008). The Lenski lab has been growing (and freezing) samples of this population for over 40,000 generations (here is an interesting paper on how these populations have been used to study competition within a population (Le Gac et al., 2012).
The evolution of the ability to utilize citrate in the presence of O2 in E. coli involved "potentiating mutations", which occurred in one specific lineage somewhere around the 20000th generation of the experiment. These mutations have no (as yet discovered) overt phenotype on their own.
Later on in this lineage, around the 31,000th generation, there was a mutation that involved the duplication and generation of a novel fusion protein derived from the citrate-succinate antiporter (a membrane-transport protein of a type we discuss generically here in biofundamentals). This mutation allows the cell's carrying it to import and grow (albeit) poorly on citrate. Subsequent mutations improve the efficiency of citrate metabolism.
What is interesting is that because they had "frozen ancestors", Blout et al could "replay" their evolution, and ask, when new citrate utilizing lines arose (which they did), whether they had similar (although not identical) mutations to that found in the originally identified lines. Interestingly, they did.
Now it becomes an interesting question whether, given that E. coli is usually defined in part by its inability to grow on citrate under aerobic conditions, whether the appearance of cells, derived from E. coli, but able to grow on citrate in the presence of O2 represents a new species or not.