On February 24, 1988, Richard Lenski seeded 12 flasks with E. coli and set them up to shake overnight at 37ºC. But he seeded them with only enough nutrients to grow until early the next morning. Every single afternoon since then, he (or someone in his lab) has taken 100 microliters of each bacterial solution, put them into a new flask with fresh growth media, and put the new flask in the shaker overnight. Every 75 days—about 500 bacterial generations—some of the culture goes into the freezer.

The starvation conditions are a strong pressure for evolution. And the experiment includes its own time machine to track that evolution.

The pivotal piece of technology enabling this experiment is the -80ºC freezer. It acts essentially, Lenski says, as a time machine. The freezer holds the bacterial cultures in a state of suspended animation; when they are thawed, they are completely viable and their fitness can be compared to that of their more highly evolved descendants shaking in their flasks. As an analogy, imagine if we could challenge a hominin from 50,000 years ago to a hackathon. (Which she would probably win, because the paleo diet.)

So cool, right? The MacArthur Foundation thought so, too—it gave Lenski a grant in 1996, all the way back at around generation 17,000 or so. The experiment is now at generation 68,113 (approximately).

The bacteria have been maintained in the same medium—the same environment—over the course of the experiment. Their food source is glucose, which is calibrated to wane over the 24 hours before the passage to the next flask. This diminishing food supply is the only selective pressure the bacteria experience.

The competitive fitness of all 12 cultures has improved over time; the cells are bigger than they were at the start of the experiment, they utilize glucose more efficiently, and they grow faster. The rate of improvement has declined over the course of the experiment, but the rate at which genetic mutations accrue does not.

The 12 populations tend to get mutations in the same set of genes, but they don’t get the same mutations within those genes; they are each finding their own path toward the same goal of optimal fitness, much like climbers each find their own paths to the same peak. And although the rate of mutation has slowed, it has not ceased. So Lenski concludes that—even in their simple, relatively static environment, and even after 68,113 generations—there are still molecular tweaks the bacteria can make to become fitter.

At about 20,000 generations, one of the 12 cultures evolved the ability to survive by eating citrate in addition to glucose. It has remained the only one of the 12 to have developed this ability and, over time, it became less able to deal with glucose as an energy source. Since the inability to metabolize citrate is kind of a hallmark of E.coli, are these guys even E. coli anymore? Or a new species?

It is not only the fitness of current bacteria that can be compared to their ancient unfrozen forbears. Lenski sequenced the genomes of each frozen culture so he can disentangle the dynamics of evolution at the molecular level.

Six of the 12 initial populations have become hypermutators. They picked up early mutations in genes controlling DNA repair, which then enabled them to accrue more mutations in the rest of their genomes. These bacteria undergo bouts of molecular evolution that yield jumps in their degree of genetic diversity. The other six populations are nonmutators; these guys accumulate mutations at a much more stately pace. The strain that eats citrate started as a nonmutator, but once it gained the ability to exploit a new food source, it began to mutate more rapidly to refine its new ability.

The length of time that each mutation sticks around sheds light on the selective forces at play. It does not seem to be the case that one beneficial mutation arises at a time and sweeps through a population. Rather, a few occur in rapid succession, and these compete for dominance. But one doesn’t always win; in most populations, the mutations segregate into groups, creating different subcultures within each flask. These subcultures have a tenuous coexistence, with their relative abundance shifting over time.

Random, stochastic mutations allow species to diversify. But selective pressures push them toward sameness, by forcing them to thrive under limiting conditions. The 60,000 generations of E. coli already in Richard Lenski’s freezer have started to show how these opposing forces shape evolution; who knows what the next 60,000 will reveal?

Nature, 2017. DOI: 10.1038/nature24287 (About DOIs).