Ecology and Phylogenetics
The new Cit+ lineage in population #9 arose ~31,000 generations into the LTEE, and became dominant ~33,000 generations. However, the Cit+ lineage did not completely sweep the population. Instead, a small subpopulation of Cit– cells that could not use the citrate coexisted with the Cit+ subpopulation for over 10,000 generations.
Cit- and Cit+ Coexistence
How were Cit– and Cit+ able to coexist? For two or more types of organisms to coexist in an environment as part of a diverse ecology, they have to occupy different niches. For example, different organisms could coexist if they used different resources, or if they used the same resources, but to different degrees. In the case of Cit+ and Cit–, the answer involves a bit of both of these possibilities. The Cit+ cells, of course, had exclusive access to the citrate resource, but both Cit+ and Cit– could use the glucose resource. However, in the course of evolving to be better at growing on citrate, the Cit+ lineage lost some of its ability to compete with Cit– for glucose, giving Cit– a glucose growth advantage.
The difference in ability to compete for glucose is not the whole story, though. As discussed in the Cell Biology section, the CitT antiporter that Cit+ uses to bring citrate into the cell works by pumping citrate into the cell while pumping succinate out of the cell. This means that the Cit+ cells produce a pool of succinate in the broth while it grows on citrate. This succinate, while not as energy-rich as either glucose or citrate, is a third resource that the bacteria can grow on. As with the glucose, both cell types can grow on succinate, but Cit– appears to have had some advantage in competition for it. The evolution of Cit+ therefore turned the broth that previously had one resource, glucose, into one that contains three: glucose, citrate, and succinate. Cit+ can eat all three, while Cit– could eat glucose and succinate, and because it can compete a bit better for them than Cit+, Cit– was able to persist in the population despite being a tiny minority. Did Cit+ evolve to compete better for glucose and succinate, nearly driving Cit– out of the population, or was it more random? Research is currently under way to figure out that question.
Phylogenetics is the study of the evolutionary relationships between groups of organisms. Those relationships are inferred by comparing sets of traits of the organisms under study. Shared traits indicate shared ancestry. Those traits that are not shared might have been lost by one or more lineages after they diverged, or they might have evolved after a lineage diverged, in which case they are referred to as derived. While many traits have been used to infer evolutionary relationships, DNA and protein sequences have come to be the traits most commonly used.
Once evolutionary relationships have been inferred, they are usually depicted visually with phylogenetic trees. In a phylogenetic tree, the organisms from which traits were compared are represented as branch tips, and inferred common ancestors are represented as the nodes from which branches emerge. A clade is a common ancestor and all of its descendants. On a phylogenetic tree, a clade is a node and all of the branches that emerge from it, meaning that it is monophyletic. Because all organisms share a common ancestor at some point, a phylogenetic tree can be thought of as a series of nested clades, with the most inclusive clade being the entire tree.
The phylogeny of LTEE population #9 was determined using data from the sequencing of the whole genomes of clones isolated from various time points in the frozen fossil record. The phylogeny shows that the population was heterogeneous for most of its history even before Cit+ evolved. At least three major clades coexisted in the population from before 20,000 generations until after 33,000 generations. As discussed earlier, such coexistence suggests that the different clades were occupying slightly different niches. What these niches were and what sorts of ecological interactions took place between the three clades is the subject of ongoing research. The phylogeny also shows that all Cit+ clones belong to a single lineage that diverged from Clade 3. The original Cit+ clone and all of the Cit+ cells that descend from it are now a new clade, the Cit+ Clade.