On the unpredictability of evolution and potentiation in Lenski's long-term evolution experiment

Richard Lenski's long-term evolution experiment (LTEE) has yielded a number of interesting results over the past two decades. Back in 1988, he set up twelve flasks of E. coli B growing in minimal medium. The cultures were diluted 1/100 every day. There were 6.64 generations per day or almost 2,500 per year. By now, the cultures have evolved for 60,000 generations.

All twelve cultures are under strong selection for rapid growth and all twelve cultures have evolved. One, and only one, of the cultures evolved to utilize citrate as a carbon source (normal E. coli cultures cannot use citrate but it's in the medium as a chelating agent). You can read about the mutations that gave rise to this phenotype in: Lenski's long-term evolution experiment: the evolution of bacteria that can use citrate as a carbon source.

It's important to understand that only one culture evolved this ability. That's because a complex series of mutations had to arise and become fixed in order for the bacteria to be able to use citrate. Because there have been so many generations, every single nucleotide in the genome has been mutated several times in each culture but the probability of just the right mutations happening in just the right order is very low.

The original analyses were done by Blount et al. (2008) and Blount et al. (2012). They discovered that three different steps were required for efficient citrate utilization. Today, I want to discuss the latest contribution to this study from Quandt et al. (2013).¹ It's worth quoting the introduction to their paper since it explains the three stages.

Key innovations in the history of life are often caused by the acquisition of a qualitatively new trait that is "an evolutionary novelty which allows the exploitation of new resources or habitats and thus triggers an adaptive radiation." Such innovations are typically rare and difficult to predict because they result from complex nonadditive (i.e., epistatic) genetic interactions or ecological interactions, within or between species, that develop only over the course of long evolutionary trajectories. Evolution of a new trait can be conceptually divided into three steps: potentiation, actualization, and refinement. First, one or more potentiating events may be necessary to generate a genetic background or environmental conditions that make a new trait accessible to evolution. Genetic potentiation, for example, may involve a period of nonadaptive genetic drift wherein a phenotype stays constant or the accumulation of mutations that are immediately advantageous for reasons unrelated to the new trait. Then, a keystone actualizing mutation or environmental shift may lead to expression of the new trait, possibly by coopting latent changes in a cellular network or physical structure for a new use. Finally, there may be many subsequent opportunities for further refinement mutations that improve an emergent trait so that a newly colonized niche can be fully exploited.

The key mutation was a mutation that altered expression of the citT gene allowing uptake of citrate from the medium. This gave rise to a weak Cit⁺ phenotype that persisted in the population for many generations but did not become fixed. This original mutation occurred in a background of pre-existing mutations that promoted rapid growth.

The weak Cit⁺ phenotype arose in a single culture after about 31,500 generations. The mutant gene was introduced into earlier ancestors of this strain and into the other eleven strains but it had no effect. This tells you that there was something special about the culture at 35,000 generations—something that "potentiated" the development of the Cit⁺ phenotype. The citT mutation is the "actualization" step.

The main result of the Quandt et al paper is characterization of a second mutation that enhanced the weak Cit⁺ phenotype to a strong Cit⁺⁺ phenotype that took over the entire population. This is the "refinement" step. There are actually two different mutations that lead to enhanced utilization of citrate. The first was duplication of the citT gene leading to production of even more enzymes. This event was characterized by Blount et al. in their 2012 paper.

The second was a mutation in the promoter of the dctA gene leading to an increase in the synthesis of a C₄-dicarboxylate transporter. C₄-dicarboxylates are important metabolites related to citrate. (Succinate is an example. Recall that succinate and citrate are both part of the citric acid cycle.) Quandt et al. show that this mutation (in dctA) arose after the original citT gene mutation and that the combination of the two mutations is responsible for converting the weak Cit⁺ to the strong Cit⁺⁺ phenotype. Carl Zimmer on his blog The Loom describes the result in more detail: Evolution Hidden in Plain Sight.

Although this is an important finding, the most interesting part of the Quandt et al. paper—as far as I'm concerned—is the negative results in the search for potentiating mutations. The authors designed an experiment to analyze large sections of the E. coil genome from the ancestors of the Cit⁺ strain. Recall that these ancestors carried mutations than potentiated the development of the phenotype while other cultures did not have these mutations so they couldn't give rise to the utilization of citrate.

There were more than 70 mutations that could have been responsible for potentiation but none of them were uniquely identified with potentiation. I like how the authors describe their conclusions since they are clearly aware of modern evolutionary theory and the possibility that mutations can be neutral or even deleterious. This is a characteristic of the people trained in Lenski's lab and it's a refreshing change.

The question remains: how did the potentiating mutations make the highly advantageous Cit⁺⁺ phenotype more accessible to evolution in this particular LTEE population? In light of our results that argue against the all-or-none epistasis hypothesis, we propose that the potentiating mutations have a quantitative epistatic effect that makes the two-step mutational path required for evolution of the complex Cit⁺⁺ phenotype from Cit^– cells more likely to be realized in the context of an evolving population. Both the evolved citT and dctA alleles may have deleterious or nearly neutral effects on fitness when they are added alone to the ancestral strain or most genetic backgrounds that existed in the LTEE. In this case, weakly Cit⁺ variants with the citT mutation would rapidly be lost from the population after they arose, due to competition with other alleles that are beneficial for glucose utilization. In fact, however, genotypes with the citT mutation persisted in the population for >1,500 generations, and a small, but significant, fitness benefit was found when the citT allele was added to a Cit^– isolate believed to have the potentiating mutations.

Therefore, on the basis of our results with dctA*, we hypothesize that potentiation may have altered central metabolism in some way that was directly beneficial for growth on glucose but fortuitously increased the supply of C₄-dicarboxylates available to power citrate import by the CitT transporter, such that the activating citT mutation became slightly beneficial, rather than neutral or deleterious. This change could have enabled weakly Cit⁺ lineages to persist in the population long enough to pick up dctA* or other refinement mutations that yielded the highly beneficial, self-sufficient Cit⁺⁺ phenotype. Assessment of the fitness effect of the citT mutation at various points along the lineage that achieved Cit⁺⁺ in the future may provide insight into the identity of the potentiating mutations. Finally, it is also possible that the initially very subtle benefit of the Cit⁺ phenotype depended to some extent on the ecological interactions involving differential uptake and secretion of nutrients by cells in this genetically diverse population.

The important lesson here is that evolution can be highly contingent and largely accidental. The mutations required to "potentiate" the novel pathway may have been neutral or even deleterious. We need to stop thinking that novel traits can be explained solely by gradualistic natural selection operating sequentially on single alleles.

Not only is evolution more complex than that but it can also explore a much greater range of possibilities than the simple model would suggest.

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1. Many Sandwalk readers will recognize one of the authors. It's "deaddog" from talk.origins.

Blount, Z.D., Borland, C.Z. and Lenski, R.E. (2008) Historical contingency and the evolution of a key innovation in an experimental population of Escherichia coli. Proceedings of the National Academy of Sciences 105:7899-7906. [doi: 10.1073/pnas.0803151105]

Blount, Z.D., Barrick, J.E., Davidson, C.J. and Lenski, R.E. (2012) Genomic analysis of a key innovation in an experimental Escherichia coli population. Nature 489:513-518. [doi: 10.1038/nature11514]

Quandt, E.M., Deatherage, D.E., Ellington, A.D., Georgiou, G., and Barrick, J.E. (2013) Recursive genomewide recombination and sequencing reveals a key refinement step in the evolution of a metabolic innovation in Escherichia coli. Proc. Natl. Acad. Sci. published online December 2013 [doi: 10.1073/pnas.1314561111]