Publications

@article{Barrick2023,

title = {Daily Transfers, Archiving Populations, and Measuring Fitness in the Long-Term Evolution Experiment with Escherichia coli},

author = {Jeffrey E. Barrick and Zachary D. Blount and Devin M. Lake and Jack H. Dwenger and Jesus E. Chavarria-Palma and Minako Izutsu and Michael J. Wiser},

doi = {10.3791/65342},

issn = {1940-087X},

year  = {2023},

date = {2023-08-18},

urldate = {2023-08-18},

journal = {Journal of Visualized Experiments},

volume = {198},

pages = {e65342},

abstract = {The Long-Term Evolution Experiment (LTEE) has followed twelve populations of \textit{Escherichia coli} as they have adapted to a simple laboratory environment for more than 35 years and 77,000 bacterial generations. The setup and procedures used in the LTEE epitomize reliable and reproducible methods for studying microbial evolution. In this protocol, we first describe how the LTEE populations are transferred to fresh medium and cultured each day. Then, we describe how the LTEE populations are regularly checked for possible signs of contamination and archived to provide a permanent frozen "fossil record" for later study. Multiple safeguards included in these procedures are designed to prevent contamination, detect various problems when they occur, and recover from disruptions without appreciably setting back the progress of the experiment. One way that the overall tempo and character of evolutionary changes are monitored in the LTEE is by measuring the competitive fitness of populations and strains from the experiment. We describe how co-culture competition assays are conducted and provide both a spreadsheet and an R package (fitnessR) for calculating relative fitness from the results. Over the course of the LTEE, the behaviors of some populations have changed in interesting ways, and new technologies like whole-genome sequencing have provided additional avenues for investigating how the populations have evolved. We end by discussing how the original LTEE procedures have been updated to accommodate or take advantage of these changes. This protocol will be useful for researchers who use the LTEE as a model system for studying connections between evolution and genetics, molecular biology, systems biology, and ecology. More broadly, the LTEE provides a tried-and-true template for those who are beginning their own evolution experiments with new microbes, environments, and questions. },

keywords = {Fitness Trajectories, Methods and Miscellaneous},

pubstate = {published},

tppubtype = {article}

}

@article{Turner2023,

title = { Evolution of a cross-feeding interaction following a key innovation in a long-term evolution experiment with \textit{Escherichia coli} },

author = {Caroline B. Turner and Zachary D. Blount and Daniel H. Mitchell and Richard E. Lenski},

doi = {10.1099/mic.0.001390},

year  = {2023},

date = {2023-08-01},

urldate = {2023-08-01},

journal = { Microbiology (Reading)},

volume = {169},

number = {8},

abstract = {The evolution of a novel trait can profoundly change an organism's effects on its environment, which can in turn affect the further evolution of that organism and any coexisting organisms. We examine these effects and feedbacks following the evolution of a novel function in the Long-Term Evolution Experiment (LTEE) with \textit{Escherichia coli}. A characteristic feature of \textit{E. coli} is its inability to grow aerobically on citrate (Cit^{−}). Nonetheless, a Cit^{+} variant with this capacity evolved in one LTEE population after 31 000 generations. The Cit^{+} clade then coexisted stably with another clade that retained the ancestral Cit^{−} phenotype. This coexistence was shaped by the evolution of a cross-feeding relationship based on C4-dicarboxylic acids, particularly succinate, fumarate, and malate, that the Cit^{+} variants release into the medium. Both the Cit^{−} and Cit^{+} cells evolved to grow on these excreted resources. The evolution of aerobic growth on citrate thus led to a transition from an ecosystem based on a single limiting resource, glucose, to one with at least five resources that were either shared or partitioned between the two coexisting clades. Our findings show that evolutionary novelties can change environmental conditions in ways that facilitate diversity by altering ecosystem structure and the evolutionary trajectories of coexisting lineages. },

keywords = {Citrate Evolution, Demography and Ecology, Historical Contingency},

pubstate = {published},

tppubtype = {article}

}

@article{Blount2020,

title = {Genomic and phenotypic evolution of \textit{Escherichia coli} in a novel citrate-only resource environment},

author = {Zachary D. Blount and Rohan Maddamsetti and Nkrumah A. Grant and Sumaya T. Ahmed and Tanush Jagdish and Jessica A. Baxter and Brooke A. Sommerfeld and Alice Tillman and Jeremy Moore and Joan L. Slonczewski and Jeffrey E. Barrick and Richard E. Lenski},

url = {https://elifesciences.org/articles/55414},

doi = {10.7554/eLife.55414},

issn = {2050-084X},

year  = {2020},

date = {2020-05-01},

urldate = {2020-05-01},

journal = {eLife},

volume = {9},

pages = {1--64},

abstract = {Evolutionary innovations allow populations to colonize new ecological niches. We previously reported that aerobic growth on citrate (Cit^{+}) evolved in an \textit{Escherichia coli} population during adaptation to a minimal glucose medium containing citrate (DM25). Cit^{+} variants can also grow in citrate-only medium (DM0), a novel environment for \textit{E. coli}. To study adaptation to this niche, we founded two sets of Cit^{+} populations and evolved them for 2500 generations in DM0 or DM25. The evolved lineages acquired numerous parallel mutations, many mediated by transposable elements. Several also evolved amplifications of regions containing the \textit{maeA} gene. Unexpectedly, some evolved populations and clones show apparent declines in fitness. We also found evidence of substantial cell death in Cit^{+} clones. Our results thus demonstrate rapid trait refinement and adaptation to the new citrate niche, while also suggesting a recalcitrant mismatch between \textit{E. coli} physiology and growth on citrate.},

keywords = {Citrate Evolution, Demography and Ecology, Descendant Experiments, Genotypes and Phenotypes},

pubstate = {published},

tppubtype = {article}

}

@article{Blount2018,

title = {Contingency and determinism in evolution: Replaying life's tape},

author = {Zachary D. Blount and Richard E. Lenski and Jonathan B. Losos},

url = {https://www.sciencemag.org/lookup/doi/10.1126/science.aam5979},

doi = {10.1126/science.aam5979},

issn = {0036-8075},

year  = {2018},

date = {2018-11-01},

urldate = {2018-11-01},

journal = {Science},

volume = {362},

number = {6415},

pages = {eaam5979},

abstract = {Historical processes display some degree of "contingency," meaning their outcomes are sensitive to seemingly inconsequential events that can fundamentally change the future. Contingency is what makes historical outcomes unpredictable. Unlike many other natural phenomena, evolution is a historical process. Evolutionary change is often driven by the deterministic force of natural selection, but natural selection works upon variation that arises unpredictably through time by random mutation, and even beneficial mutations can be lost by chance through genetic drift. Moreover, evolution has taken place within a planetary environment with a particular history of its own. This tension between determinism and contingency makes evolutionary biology a kind of hybrid between science and history. While philosophers of science examine the nuances of contingency, biologists have performed many empirical studies of evolutionary repeatability and contingency. Here, we review the experimental and comparative evidence from these studies. Replicate populations in evolutionary "replay" experiments often show parallel changes, especially in overall performance, although idiosyncratic outcomes show that the particulars of a lineage's history can affect which of several evolutionary paths is taken. Comparative biologists have found many notable examples of convergent adaptation to similar conditions, but quantification of how frequently such convergence occurs is difficult. On balance, the evidence indicates that evolution tends to be surprisingly repeatable among closely related lineages, but disparate outcomes become more likely as the footprint of history grows deeper. Ongoing research on the structure of adaptive landscapes is providing additional insight into the interplay of fate and chance in the evolutionary process.},

keywords = {Historical Contingency, Parallelism and Divergence, Review Articles},

pubstate = {published},

tppubtype = {article}

}

@article{nokey,

title = {On the deformability of an empirical fitness landscape by microbial evolution},

author = {Djordje Bajić and Jean C. C. Vila and Zachary D. Blount and Alvaro Sánchez},

url = {http://www.pnas.org/lookup/doi/10.1073/pnas.1808485115},

doi = {10.1073/pnas.1808485115},

issn = {0027-8424},

year  = {2018},

date = {2018-10-30},

urldate = {2018-10-30},

journal = {Proceedings of the National Academy of Sciences of the United States of America},

volume = {115},

number = {44},

pages = {11286-11291},

abstract = {A fitness landscape is a map between the genotype and its reproductive success in a given environment. The topography of fitness landscapes largely governs adaptive dynamics, constraining evolutionary trajectories and the predictability of evolution. Theory suggests that this topography can be deformed by mutations that produce substantial changes to the environment. Despite its importance, the deformability of fitness landscapes has not been systematically studied beyond abstract models, and little is known about its reach and consequences in empirical systems. Here we have systematically characterized the deformability of the genome-wide metabolic fitness landscape of the bacterium \textit{Escherichia coli}. Deformability is quantified by the noncommutativity of epistatic interactions, which we experimentally demonstrate in mutant strains on the path to an evolutionary innovation. Our analysis shows that the deformation of fitness landscapes by metabolic mutations rarely affects evolutionary trajectories in the short range. However, mutations with large environmental effects produce long-range landscape deformations in distant regions of the genotype space that affect the fitness of later descendants. Our results therefore suggest that, even in situations in which mutations have strong environmental effects, fitness landscapes may retain their power to forecast evolution over small mutational distances despite the potential attenuation of that power over longer evolutionary trajectories. Our methods and results provide an avenue for integrating adaptive and eco-evolutionary dynamics with complex genetics and genomics. },

keywords = {Citrate Evolution, Demography and Ecology, Theory and Simulations},

pubstate = {published},

tppubtype = {article}

}

@article{Blount2016,

title = {A case study in evolutionary contingency},

author = {Zachary D. Blount},

url = {https://linkinghub.elsevier.com/retrieve/pii/S1369848615001806},

doi = {10.1016/j.shpsc.2015.12.007},

issn = {13698486},

year  = {2016},

date = {2016-08-01},

urldate = {2016-08-01},

journal = {Studies in History and Philosophy of Science Part C: Studies in History and Philosophy of Biological and Biomedical Sciences},

volume = {58},

pages = {82--92},

publisher = {Elsevier Ltd},

abstract = {Biological evolution is a fundamentally historical phenomenon in which intertwined stochastic and deterministic processes shape lineages with long, continuous histories that exist in a changing world that has a history of its own. The degree to which these characteristics render evolution historically contingent, and evolutionary outcomes thereby unpredictably sensitive to history has been the subject of considerable debate in recent decades. Microbial evolution experiments have proven among the most fruitful means of empirically investigating the issue of historical contingency in evolution. One such experiment is the \textit{Escherichia coli} Long-Term Evolution Experiment (LTEE), in which twelve populations founded from the same clone of \textit{E. coli} have evolved in parallel under identical conditions. Aerobic growth on citrate (Cit+), a novel trait for \textit{E. coli}, evolved in one of these populations after more than 30,000 generations. Experimental replays of this population's evolution from various points in its history showed that the Cit+ trait was historically contingent upon earlier mutations that potentiated the trait by rendering it mutationally accessible. Here I review this case of evolutionary contingency and discuss what it implies about the importance of historical contingency arising from the core processes of evolution.},

keywords = {Review Articles},

pubstate = {published},

tppubtype = {article}

}

@article{nokey,

title = {Sustained fitness gains and variability in fitness trajectories in the long-term evolution experiment with \textit{Escherichia coli}},

author = {Richard E. Lenski and Michael J. Wiser and Noah Ribeck and Zachary D. Blount and Joshua R. Nahum and J. Jeffrey Morris and Luis Zaman and Caroline B. Turner and Brian D. Wade and Rohan Maddamsetti and Alita R. Burmeister and Elizabeth J. Baird and Jay Bundy and Nkrumah A. Grant and Kyle J. Card and Maia Rowles and Kiyana Weatherspoon and Spiridon E. Papoulis and Rachel Sullivan and Colleen Clark and Joseph S. Mulka and Neerja Hajela},

url = {https://royalsocietypublishing.org/doi/10.1098/rspb.2015.2292},

doi = {10.1098/rspb.2015.2292},

issn = {0962-8452},

year  = {2015},

date = {2015-12-22},

urldate = {2015-12-22},

journal = {Proceedings of the Royal Society B: Biological Sciences},

volume = {282},

number = {1821},

pages = {20152292},

abstract = {Many populations live in environments subject to frequent biotic and abiotic changes. Nonetheless, it is interesting to ask whether an evolving population's mean fitness can increase indefinitely, and potentially without any limit, even in a constant environment. A recent study showed that fitness trajectories of \textit{Escherichia coli} populations over 50 000 generations were better described by a power-law model than by a hyperbolic model. According to the power-law model, the rate of fitness gain declines over time but fitness has no upper limit, whereas the hyperbolic model implies a hard limit. Here, we examine whether the previously estimated power-law model predicts the fitness trajectory for an additional 10 000 generations. To that end, we conducted more than 1100 new competitive fitness assays. Consistent with the previous study, the power-law model fits the new data better than the hyperbolic model. We also analysed the variability in fitness among populations, finding subtle, but significant, heterogeneity in mean fitness. Some, but not all, of this variation reflects differences in mutation rate that evolved over time. Taken together, our results imply that both adaptation and divergence can continue indefinitely—or at least for a long time—even in a constant environment.},

keywords = {Fitness Trajectories, Mutation Rates, Parallelism and Divergence},

pubstate = {published},

tppubtype = {article}

}

@article{Turner2015,

title = {Replaying Evolution to Test the Cause of Extinction of One Ecotype in an Experimentally Evolved Population},

author = {Caroline B. Turner and Zachary D. Blount and Richard E. Lenski},

editor = {Frederick M. Cohan},

url = {https://dx.plos.org/10.1371/journal.pone.0142050},

doi = {10.1371/journal.pone.0142050},

issn = {1932-6203},

year  = {2015},

date = {2015-11-01},

urldate = {2015-11-01},

journal = {PLOS ONE},

volume = {10},

number = {11},

pages = {e0142050},

abstract = {In a long-term evolution experiment with \textit{Escherichia coli}, bacteria in one of twelve populations evolved the ability to consume citrate, a previously unexploited resource in a glucoselimited medium. This innovation led to the frequency-dependent coexistence of citrate-consuming (Cit+) and non-consuming (Cit-) ecotypes, with Cit-bacteria persisting on the exogenously supplied glucose as well as other carbon molecules released by the Cit+ bacteria. After more than 10,000 generations of coexistence, however, the Cit-lineage went extinct; cells with the Cit-phenotype dropped to levels below detection, and the Cit-clade could not be detected by molecular assays based on its unique genotype. We hypothesized that this extinction was a deterministic outcome of evolutionary change within the population, specifically the appearance of a more-fit Cit+ ecotype that competitively excluded the Cit-ecotype. We tested this hypothesis by re-evolving the population from a frozen population sample taken within 500 generations of the extinction and from another sample taken several thousand generations earlier, in each case for 500 generations and with 20-fold replication. To our surprise, the Cit-type did not go extinct in any of these replays, and Cit-cells also persisted in a single replicate that was propagated for 2,500 generations. Even more unexpectedly, we showed that the Cit-ecotype could reinvade the Cit+ population after its extinction. Taken together, these results indicate that the extinction of the Cit-ecotype was not a deterministic outcome driven by competitive exclusion by the Cit+ ecotype. The extinction also cannot be explained by demographic stochasticity alone, as the population size of the Cit-ecotype should have been many thousands of cells even during the daily transfer events. Instead, we infer that the extinction must have been caused by a rare chance event in which some aspect of the experimental conditions was inadvertently perturbed.},

keywords = {Citrate Evolution, Demography and Ecology, Historical Contingency},

pubstate = {published},

tppubtype = {article}

}

@article{Quandt2015,

title = {Fine-tuning citrate synthase flux potentiates and refines metabolic innovation in the Lenski evolution experiment.},

author = {Erik M. Quandt and Jimmy Gollihar and Zachary D. Blount and Andrew D. Ellington and George Georgiou and Jeffrey E. Barrick},

url = {http://www.ncbi.nlm.nih.gov/pubmed/26465114 

http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=PMC4718724},

doi = {10.7554/eLife.09696},

issn = {2050-084X},

year  = {2015},

date = {2015-10-01},

urldate = {2015-10-01},

journal = {eLife},

volume = {4},

number = {October},

pages = {e09696},

abstract = {Evolutionary innovations that enable organisms to colonize new ecological niches are rare compared to gradual evolutionary changes in existing traits. We discovered that key mutations in the \textit{gltA} gene, which encodes citrate synthase (CS), occurred both before and after \textit{Escherichia coli} gained the ability to grow aerobically on citrate (Cit(+) phenotype) during the Lenski long-term evolution experiment. The first \textit{gltA} mutation, which increases CS activity by disrupting NADH-inhibition of this enzyme, is beneficial for growth on the acetate and contributed to preserving the rudimentary Cit(+) trait from extinction when it first evolved. However, after Cit(+) was refined by further mutations, this potentiating \textit{gltA} mutation became deleterious to fitness. A second wave of beneficial \textit{gltA} mutations then evolved that reduced CS activity to below the ancestral level. Thus, dynamic reorganization of central metabolism made colonizing this new nutrient niche contingent on both co-opting and overcoming a history of prior adaptation.},

keywords = {Citrate Evolution, Genotypes and Phenotypes, Historical Contingency},

pubstate = {published},

tppubtype = {article}

}

@article{Blount2012,

title = {Genomic analysis of a key innovation in an experimental \emph{Escherichia coli} population},

author = {Zachary D. Blount and Jeffrey E. Barrick and Carla J. Davidson and Richard E. Lenski},

url = {http://www.nature.com/articles/nature11514},

doi = {10.1038/nature11514},

issn = {0028-0836},

year  = {2012},

date = {2012-09-01},

urldate = {2012-09-01},

journal = {Nature},

volume = {489},

number = {7417},

pages = {513--518},

abstract = {Evolutionary novelties have been important in the history of life, but their origins are usually difficult to examine in detail. We previously described the evolution of a novel trait, aerobic citrate utilization (Cit +), in an experimental population of \textit{Escherichia coli}. Here we analyse genome sequences to investigate the history and genetic basis of this trait. At least three distinct clades coexisted for more than 10,000 generations before its emergence. The Cit + trait originated in one clade by a tandem duplication that captured an aerobically expressed promoter for the expression of a previously silent citrate transporter. The clades varied in their propensity to evolve this novel trait, although genotypes able to do so existed in all three clades, implying that multiple potentiating mutations arose during the population's history. Our findings illustrate the importance of promoter capture and altered gene regulation in mediating the exaptation events that often underlie evolutionary innovations.},

keywords = {Citrate Evolution, Genome Evolution, Genotypes and Phenotypes, Historical Contingency, Mutation Rates},

pubstate = {published},

tppubtype = {article}

}

@article{Blount2008,

title = {Historical contingency and the evolution of a key innovation in an experimental population of \emph{Escherichia coli}},

author = {Zachary D. Blount and Christina Z. Borland and Richard E. Lenski},

url = {http://www.pnas.org/cgi/doi/10.1073/pnas.0803151105},

doi = {10.1073/pnas.0803151105},

issn = {0027-8424},

year  = {2008},

date = {2008-06-01},

urldate = {2008-06-01},

journal = {Proceedings of the National Academy of Sciences of the United States of America},

volume = {105},

number = {23},

pages = {7899--7906},

abstract = {The role of historical contingency in evolution has been much debated, but rarely tested. Twelve initially identical populations of \textit{Escherichia coli} were founded in 1988 to investigate this issue. They have since evolved in a glucose-limited medium that also contains citrate, which \textit{E. coli} cannot use as a carbon source under oxic conditions. No population evolved the capacity to exploit citrate for >30,000 generations, although each population tested billions of mutations. A citrate-using (Cit^{+}) variant finally evolved in one population by 31,500 generations, causing an increase in population size and diversity. The long-delayed and unique evolution of this function might indicate the involvement of some extremely rare mutation. Alternately, it may involve an ordinary mutation, but one whose physical occurrence or phenotypic expression is contingent on prior mutations in that population. We tested these hypotheses in experiments that “replayed” evolution from different points in that population's history. We observed no Cit^{+} mutants among 8.4 × 10^{12} ancestral cells, nor among 9 × 10^{12} cells from 60 clones sampled in the first 15,000 generations. However, we observed a significantly greater tendency for later clones to evolve Cit^{+}, indicating that some potentiating mutation arose by 20,000 generations. This potentiating change increased the mutation rate to Cit^{+} but did not cause generalized hypermutability. Thus, the evolution of this phenotype was contingent on the particular history of that population. More generally, we suggest that historical contingency is especially important when it facilitates the evolution of key innovations that are not easily evolved by gradual, cumulative selection.},

keywords = {Citrate Evolution, Demography and Ecology, Historical Contingency},

pubstate = {published},

tppubtype = {article}

}