Tag Archives: Zachary Blount

A Birthday Sonnet

This past weekend, I celebrated my 60th birthday with friends and family from all over. One of the roasters was Ben “The Bard” Kerr, a professor at the University of Washington and colleague in the BEACON Center for the Study of Evolution in Action.

Borrowing from another bard, Ben waxed poetic about one of the lineages in the long-term evolution experiment and raised a toast with this Shakespearean flask.

 

Ben Kerr's Skakespearean flask

ODE TO AN LTEE LINEAGE

Shall I compare Ara-3 to a summer’s day?

Thou start more humbly, but sure potentiate.

Rough spins do shake the darling bugs of Rich’s gaze,

And latecomer’s “fleece” hath all to port citrate.

One line’s long-shot passed by eleven lines,

And how was its controlled complex “skin” pinned?

Promoter capture, over some time refined.

By chance, with nature’s arranging force, trimmed.

But thy Cit-minus partner shall not fade

Nor gain possession of the flair of most

C4 shall Cit snag, now spawned by carbon trade

Then on it turns ‘til lines will species now boast

     So long these cells can achieve, so wise to see,

     So long lives this work- and awe is rife, Lenski.

 

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Birthday Haiku

This past weekend I had my 60th birthday. I was delighted to celebrate it with wonderful colleagues, students, friends, and family.

At a dinner roast and toast, everyone sang When We’re Sixty Four (Thousand), a tribute from the E. coli in the LTEE to the People of the Lab. And several friends came up with new contributions at the intersection of science and culture.

This beauty is from Andy Ellington, a professor in the Center for Systems and Synthetic Biology at the University of Texas and a member of the BEACON Center. As background, Andy coauthored a recent paper that helps to elucidate how one LTEE population evolved the novel ability to use citrate.

Without further ado, here’s his haiku …

Citrate just beyond.

Acetate potentiates.

Glucose is all gone.

 

Citrate

[Image of citrate molecule from Wikimedia Commons]

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On the Evolution of Citrate Use

Those who follow the long-term evolution experiment (LTEE) with E. coli know that the most dramatic change we have observed to date is the origin of the new ability to grow on citrate. It’s dramatic for several reasons including the fact (external to the LTEE) that E. coli has been historically defined as a species based in part on its inability to grow on citrate in oxic environments and the fact (internal to the LTEE) that it was so difficult for the bacteria to evolve this ability that only one of the populations did so, and that it took over 30,000 generations even though an abundance of citrate has been present in the medium throughout the LTEE. Even after 64,000 generations, only the Ara–3 population has evolved that new ability.

Zachary Blount, formerly a graduate student and now a postdoc in my lab, has spent the last decade studying the evolution of this population and its new ability. His two first-authored papers in PNAS (2008) and Nature (2012) demonstrated, respectively, that (i) the origin of the ability to grow on citrate in the LTEE was contingent on one or more “potentiating” mutations that happened before the “actualizing” mutation that conferred the new function first appeared, and (ii) the actualizing mutation was a physical rearrangement of the DNA that brought together a structural gene, citT, that encodes a transporter and a previously unconnected regulatory region to generate a new module that caused the phenotypic transition to Cit+. These papers presented and discussed much more than these two points, of course, but they are the key findings. More recently, Zack was a coauthor on a paper in eLife (2015) by Erik Quandt, Jeff Barrick, and others that identified two mutations in the gene for citrate synthase—one that potentiated the evolution of citrate utilization, and another that subsequently refined that new function.

So we were keenly interested when we saw a new paper titled “Rapid evolution of citrate utilization by Escherichia coli by direct selection requires citT and dctA” by Dustin Van Hofwegen, Carolyn Hovde, and Scott Minnich. The paper is posted online as an accepted manuscript by the Journal of Bacteriology. What follows here are some overall impressions of their paper that Zack and I put together. We may follow these impressions later with some further analysis and comments.

* * * * *

Let’s begin by saying that it’s great to see other groups working on interesting systems and problems like the evolution of citrate utilization in E. coli.

Moreover, the actual science that was done and reported looks fine and interesting, though we have a few quibbles with some details that we will overlook for now. By and large, the work confirms many of the findings that were reported in our papers cited above:

(i) the ability to grow on citrate in the presence of oxygen can and does evolve in E. coli (Blount et al., 2008);

(ii) when aerobic growth on citrate evolves, it does not do so quickly and easily (Blount et al., 2008) but instead takes weeks or longer—more on that below;

(iii) all strains that have evolved this new ability have physical rearrangements that involve the citT gene and appear also to involve a so-called “promoter capture” whereby a copy of this transporter-encoding gene acquires a new upstream regulatory region (Blount et al., 2012); and

(iv) genetic context matters—the strain one uses affects the likelihood of evolving the Cit+ function (Blount et al., 2008) and the resulting ability to grow on citrate (Blount et al., 2012; Quandt et al., 2015).

The problem, then, is not with the experiments and data. Rather, the problem is that the results are wrapped in interpretations that are, in our view, flawed and fallacious.

“No new genetic information”

The authors assert repeatedly (last sentence of their Importance statement, and first and last paragraphs of their Discussion) that “no new genetic information evolved.” However, that statement flatly contradicts the fact that in their experiments, and ours, E. coli gained the new ability to grow on citrate in the presence of oxygen. We would further add (which we have not emphasized before) that these Cit+ strains can grow on citrate as a sole carbon source—when E. coli grows anaerobically on citrate, it requires a second substrate for growth in order to use the citrate (a phenomenon called “co-metabolism”).

The claim that “no new genetic information evolved” is based on the fact that the bacteria gained this new ability by rearranging existing structural and regulatory genetic elements. But that’s like saying a new book—say, Darwin’s Origin of Species when it first appeared in 1859—contains no new information, because the text has the same old letters and words that are found in other books.

In an evolutionary context, a genome encodes not just proteins and patterns of expression, but information about the environments where an organism’s ancestors have lived and how to survive and reproduce in those environments by having useful proteins, expressing them under appropriate conditions (but not others), and so on. So when natural selection—that is, differential survival and reproduction—favors bacteria whose genomes have mutations that enable them to grow on citrate, those mutations most certainly provide new and useful information to the bacteria.

That’s how evolution works—it’s not as though new genes and functions somehow appear out of thin air. As the bacterial geneticist and Nobel laureate François Jacob wrote (Science, 1977): “[N]atural selection does not work as an engineer works. It works like a tinkerer—a tinkerer who does not know exactly what he is going to produce but uses whatever he finds around him, whether it be pieces of string, fragments of wood, or old cardboards; in short, it works like a tinkerer who uses everything at his disposal to produce some kind of workable object.”

To say there’s no new genetic information when a new function has evolved (or even when an existing function has improved) is a red herring that is promulgated by the opponents of evolutionary science. In this regard, it seems relevant to point out that the corresponding author, Scott Minnich, is a fellow of the Discovery Institute and was an expert witness for the losing side that wanted to allow the teaching of “intelligent design” as an alternative to evolution in public schools in the landmark Kitzmiller v. Dover case.

“Rapid evolution of citrate utilization”

In the title of their paper and throughout, Van Hofwegen et al. emphasize that, in their experiments, E. coli evolved the ability to grow aerobically on citrate much faster than the 30,000 generations and ~15 years that it took in the LTEE. That’s true, but it also obscures three points. First, we already demonstrated in replay experiments that, in the right genetic background and by plating on minimal-citrate agar, Cit+ mutants sometimes arose in a matter of weeks (Blount et al. 2008). Second, rapid evolution of citrate utilization—or any evolution of that function—was not a goal of the LTEE. So while it is interesting that Van Hofwegen et al. have identified genetic contexts and ecological conditions that accelerate the emergence of citrate utilization (as did Blount et al., 2008), that in no way undermines the slowness and rarity of the evolution of this function in the context of the LTEE (or, for that matter, the rarity of Cit+ E. coli in nature and in the lab prior to our work). Third, the fastest time that Van Hofwegen et al. saw for the Cit+ function to emerge was 19 days (from their Table 1), and in most cases it took a month or two. While that’s a lot faster than 15 years, it’s still much longer than typical “direct selections” used by microbiologists where a readily accessible mutation might confer, for example, resistance to an antibiotic after a day or two.

So while we commend the authors’ patience, we do not think the fact that their experiments produced Cit+ bacteria faster than did the LTEE is particularly important, especially since that was not a goal of the LTEE (and since we also produced them much faster in replay experiments). However, in a manner that again suggests an ulterior nonscientific motive, they try to undermine the LTEE as an exemplar of evolution. The final sentence of their paper reads: “A more accurate, albeit controversial, interpretation of the LTEE is that E. coli’s capacity to evolve is more limited than currently assumed.” Alas, their conclusion makes no logical sense. If under the right circumstances the evolution of citrate utilization is more rapid than it is in the LTEE, then that means that E. coli’s capacity to evolve is more powerful—not more limited—than assumed.

“Speciation Event”

To us, one of the most interesting facets of the evolution of the citrate-using E. coli in the LTEE is its implications for our understanding of the evolutionary processes by which new species arise. Part of the reason for this interest—and the one that’s most easily stated in a popular context—is that the inability to grow on citrate is part of the historical definition for E. coli as a species, going back almost a century. But the deeper interest to us lies not in labeling a new species or debating where to draw the line between species—various criteria are used by different scientists, and inevitably there are many cases that lie in grey areas. Rather, as evolutionary biologists, we are most interested in the process of speciation—the ecological and genetic dynamics that lead to changing biological forms that, over time, are more and more like a new species until, eventually, perhaps far in the future, there is no doubt that a new species has evolved.

In short, speciation is not an event. As Ptacek and Hankison (2009, in Evolution: The First Four Billion Years) put it, “[S]peciation is a series of processes, with a beginning stage of initial divergence, a middle stage wherein species-specific characteristics are refined by various forces of evolution, and an end point at which a new species becomes a completely separate evolutionary lineage on its own trajectory of evolutionary change with the potential for extinction or further diversification into new lineages.” We realize that scientists (ourselves included) often use shorthand and jargon instead of writing more carefully and precisely. We have no doubt that one can find solid scientific papers that talk about speciation events; but except for cases that involve hybridization leading to polyploids that are reproductively isolated in a single generation (as sometimes occurs in plants), this is simply an imprecise shorthand.

In our first paper on the citrate-using E. coli that arose in the LTEE, we clearly emphasized that becoming Cit+ was only a first step on the road to possible speciation (Blount et al., 2008). One criterion that many biologists would apply to investigate speciation is whether a later form merely replaced an earlier form (evolution without speciation) or, alternatively, one lineage split into two lineages that then coexisted (incipient speciation). In fact, we showed that, after the new function evolved, the Cit+ and Cit lineages coexisted (and their coexistence was confirmed using genomic data in Blount et al., 2012). We concluded the 2008 paper by asking explicitly: “Will the Cit+ and Cit– lineages eventually become distinct species?” (emphasis added) and discussing how we might assess their ongoing divergence.

By contrast, Van Hofwegen et al. dismiss the idea of speciation out of hand, not only by calling it an event but by treating the issue as though it hinges, literally, on the individual mutations that produced a Cit+ cell. For example, they write: “[B]ecause this adaptation did not generate any new genetic information … generation of E. coli Cit+ phenotypes in our estimation do not warrant consideration as a speciation event.” And in the penultimate sentence of their paper, they say: “[W]e argue that this is not speciation any more than any other regulatory mutant of E. coli.” (We also note that this is a rather bizarre generalization, as though the gain of function that gave access to a new resource is equal in regards to its speciation potential to, say, the loss of regulation of a function that is no longer used by a lineage in its current environment. Both might well be adaptations, but one seems much more likely to begin the process of speciation.)

In conclusion, Van Hofwegen, Hovde, and Minnich have done some interesting experiments that shed further light on the nature of the mutations and ecological conditions that allow E. coli cells to evolve the ability to grow aerobically on citrate, a function that this species cannot ordinarily perform. However, they misunderstand and/or misrepresent the relevance of this system for evolutionary biology in several important respects. 

And the meaning of historical contingency

The paper by Hofwegen et al. is accompanied by a commentary by John Roth and Sophie Maisnier-Patin. Their abstract begins: “Van Hofwegen et al. demonstrate that E. coli rapidly evolves ability to use citrate when long selective periods are provided. This contrasts with the extreme delay (15 years of daily transfers) seen in the long-term evolution experiments of Lenski and coworkers. Their idea of ‘historical contingency’ may require reinterpretation.”

Historical contingency is a complicated notion, but it essentially means that history matters. In Blount et al. (2008), we made it clear what we mean by historical contingency in the context of the evolution of the Cit+ lineage in one of the LTEE populations. Was this an extremely rare event that could have happened at any time? Or did it instead depend on the occurrence of a sequence of events, a particular history, whereby an altered genetic context evolved—a potentiated background—in which this new function could now evolve?

Roth and Maisnier-Patin’s suggestion that our idea of “historical contingency” may require reinterpretation reflects a false dichotomy between historical contingency, on the one hand, and the effects of different selection schemes, on the other. The fact that evolution might be fast and not contingent on genetic background (though the evidence of Van Hofwegen et al. is, at best, ambiguous in this regard) in one set of circumstances has no bearing on whether it is contingent in another set of circumstances. The historical contingency of Cit+ evolution is not mere conjecture. We showed that the evolution of this new function in the LTEE was contingent. In replay experiments, Blount et al. (2008) showed that that the Cit+ trait arises more often in later-generation genetic backgrounds than in the ancestor or early-generation backgrounds. Moreover, Blount et al. (2012) performed genetic manipulations and showed that a high-copy-number plasmid carrying the evolved module that confers the Cit+ function had very different phenotypic effects when put in a Cit clone from the lineage within which Cit+ evolved than when placed in the ancestor or even other late-generation lineages not on the line of descent leading to the emergence of the Cit+ bacteria. In the clone on the line of descent, this module conferred strong, immediate, and consistent growth on citrate. In the other genetic backgrounds, growth on citrate was weak, delayed, and/or inconsistent.

The hypothesis of historical contingency is not mutually exclusive with respect to causal factors of an ecological or genetic nature—it simply says that factors that changed over time were important for the eventual emergence of Cit+. Moreover, historical contingency was invoked and demonstrated in a specific context, namely that of the emergence of Cit+ in the LTEE—it does not mean that the emergence of Cit+ is historically contingent in other experimental contexts, nor for that matter that other changes in the LTEE are historically contingent—in fact, some other evolved changes in the LTEE have been highly predictable and not (or at least not obviously) contingent on prior mutations in the populations (e.g., Woods et al., PNAS, 2006). [For more on historical contingency and the LTEE, you can download a preprint of Zack’s latest paper from his website: Blount, Z. D. A Case Study in Evolutionary Contingency. Studies in the History and Philosophy of Biology and Biomedical Sciences.]

Erik Quandt offers this analogy to illustrate our point that contingency depends on context: “It’s kind of like the difference between being an average person attempting to dunk a basketball when all by yourself, with unlimited time, and maybe even with a trampoline versus having to get to the rim in a game with LeBron James and the Cavs playing defense. Just because you can do it by yourself under optimal conditions, does this negate the difficulty of doing it in an NBA game or say anything about the kind of history (training and/or genetics) that you would need for that situation?”

* * * * *

LTEE lines centered on citrate #11

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63,000 Strong

Ever wonder about those big numbers posted in a window in that tall building on the east side of Farm Lane, across from the entrance to the MSU Dairy Store?

Right now, the digits read 63000. That’s the number of generations in an experiment that’s been running in my lab for over a quarter century.

We call it the LTEE, which stands for the Long-Term Evolution Experiment. There are 12 populations of E. coli bacteria in the experiment, and they all started from the same strain.

Every day—weekends and holidays included—a member of my team takes 1% of the cells in each population and puts them in a flask with fresh food. Over the next 24 hours, the population grows 100-fold and then runs out of food. These dilutions and renewals go on day after day, week after week, month after month, year after year, decade after decade. I hope the experiment will continue long after I’m gone, so that someday someone can write “and century after century.”

Bacteria grow by binary fission: 1 cell makes 2 cells, 2 cells make 4, 4 make 8, etc. So the 100-fold growth in the fresh medium represents about 6.6 doublings, or generations, every day. (There’ve been some interruptions since the LTEE began in 1988, but not many.)

Now consider a bacterial cell that gets a mutation in its DNA that lets it acquire more food and grow a little faster. That cell will leave more descendants than its competitors—that’s adaptation by natural selection. Over time, the bacteria are becoming stronger and fitter in their flask-worlds.

By watching the 12 populations evolve, we can answer questions about the dynamics and repeatability of evolution in a group of organisms—bacteria—that are essential for life on Earth as well as important players in health and disease. We measure the growth rates of the bacteria, we sequence their DNA, and we see just how much evolution can achieve even in short order.

Oh, about the sign. Zachary Blount is a talented postdoc who works on this project, and he likes to have fun with science. He put up the window display which, if you look closely, has a picture of Charles Darwin on the left, “The E. coli Long-Term Evolution Experiment” over the number, and “Generations and Counting” to the right. Every 1,000 generations or so, Zack updates the sign.

63K window

[Photo credit: Zachary D. Blount]

Note:   This piece first appeared at eastlansing.org after an invitation from Alice Dreger to explain the numbers in the window to our community.

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Window Dressing

The window to the lab has been updated, courtesy of Zack Blount.

62K window dressing

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A Day in the Life of …

Today was a great day – busy and wonderful. Pretty typical, I’m happy to say, though a bit busier than usual but all of it great.

Woke up to beautiful Spring day in East Lansing and walked 1.7 miles to work at MSU.

Did the usual email stuff.

Worked on getting ready for teaching for a class on evolutionary medicine taught by my colleague Jim Smith. Today’s focus will be the paper by Tami Lieberman et al. on the evolution of Burkholderia dolosa in cystic fibrosis patients during an outbreak in Boston. Last night I re-read the paper for the umpteenth time, and I still enjoyed it. Today I organized a series of questions for the students – a very interactive and smart group – around three parts.

Part I: Some background about CF, the inheritance of this disease, the frequency of the disease, how that frequency allows one to estimate the frequency of carriers, why the allele might be so common (not understood), side questions about sickle-cell anemia and why it’s so prevalent, and why, if it’s inherited, the paper we read is all about infections.

Part II: Preparing slides so we could work our way, figure by figure and panel by panel, through all of the main points in Lieberman et al.  (Reminder: Explain to students how scientific papers are often written around figures.  Once the figures and tables are there, then start on the results, etc.)

Part III: Follow up questions about the paper, the system, the interface of epidemiology and evolutionary biology, prospects for the future of this field and the students’ careers (most in this class are premed, many with a research bent), etc. And whatever questions they might want to ask of me.

Sometime in the middle of doing all that: Chatted with second-year grad student Jay Bundy, who is reading some of Mike Travisano’s terrific earlier papers on the LTEE. Specifically, why do we sometimes express fitness as a ratio of growth rates (measured in head-to-head competitions) and sometimes as a difference in growth rates?

Also in the middle of doing all that: Had phone conversation with former Ph.D. student Bob Woods, now also an M.D. specializing in infectious disease, about a faculty job offer he has (congrats, Bob!), some of the issues he needs to clarify or negotiate, and some of the amazing work he’s now doing on the population dynamics and evolution of nasty infections.

Email from grad student Mike Wiser that our paper, submitted to PLOS ONE, has been officially accepted. We had posted a pre-submission version at bioRxiv – now it’s gone through peer-review and revisions and is accepted for publication. Congrats, Mike!

Got a draft of the fourth and final chapter of Caroline Turner’s dissertation. The first three chapters are in great shape. Congrats, Caroline! With teaching looming, I had only time to review the figures, tables, and legends on this one, and made some small suggestions. On to the text tomorrow … It’s a beautiful body of work on two fascinating aspects of the interplay between ecology and evolution that have emerged in the LTEE and another evolution experiment that Caroline performed. Stay tuned for these papers!

Took a phone call from an MSU colleague who has friend with a child in high school who is interested in microbiology, who is visiting MSU, and who wanted to see the lab. Yikes, I gotta run teach! But postdoc Zack Blount kindly agreed to give a guided tour as I headed off to teach.  Thanks, Zack!

Beautiful day continues as I walk to teach in another building. Touch base with Jim Smith about what I plan to cover.

Two straight hours of teaching (one 5-minute break) in an overly hot room. Almost all of it interactive, with me asking questions and the students conferring in small groups and then responding. Very interactive, very bright students! The two hours were nearly up, with little time for my third, post-paper set of questions. But all of the students stayed (despite the beautiful weather, hot room, and the dinner hour at hand) an extra 15-20 minutes for a couple of my questions and some great ones from them about the LTEE and the future prospects for microbial evolution in relation to medicine.

It’s 6:20 pm: I’m mentally exhausted but equally invigorated. Beautiful Spring day continues as I walk home. I’m greeted by our lovely hound, Cleopatra. Exercise and feed her. Then an even more lovely creature, Madeleine, returns home and I greet her.

Check email before dinner. Find that paper with grad student Rohan Maddamsetti and former postdoc Jeff Barrick has been provisionally accepted, pending minor revisions, at Genetics. We posted a pre-submission version of that paper, too, at bioRxiv. Though we still need to do some revisions, I think it’s fair to offer congrats to Rohan and Jeff, too!

Time to crack open a bottle of wine and have some dinner. Fortunately, some of the pre-packaged dinners are pretty tasty and healthy, too, these days ;>)

Refill wine glass. Sit down and start to write a blog on a day in the life of …

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The LTEE as meta-experiment: Questions from Jeremy Fox about the LTEE, part 3

EDIT (23 June 2015): PLOS Biology has published a condensed version of this blog-conversation.

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This is the 3rd installment in my responses to Jeremy Fox’s questions about the LTEE (my lab’s long-term evolution experiment with E. coli), which he asked at the Dynamic Ecology blog. This response addresses his 2nd and 7th questions, which I’ve copied below. I like all of Jeremy’s questions, but I especially like his 2nd one because it forced me—and many readers, I hope—to think carefully about what experiments are and why we do them.

~~~~~

  • Is the LTEE actually an experiment, and wouldn’t it have been even better if it was? It’s just one “treatment”–12 replicates of a single set of conditions. Wouldn’t it have been even more interesting to have, say, two treatments? Two different culture conditions, two different founding genotypes, two different founding species…?
  • Is the LTEE itself now a “model system”? Model systems in biology–systems in which it’s tractable to ask a given question–often are systems that we know a lot about. We can leverage that background knowledge to ask questions that otherwise wouldn’t be tractable. coli of course is a model organism for many purposes, because we know so much about it. But is the LTEE itself now a model system?

 ~~~~~

You’re certainly right, Jeremy, that experiments in the fields of ecology and evolutionary biology typically have two or more treatments. But it’s not an essential part of the definition of an experiment that it has that sort of structure. It would have been nice, perhaps, if the LTEE did have two or more environments and/or two or more ancestors, as you suggest—in fact, we’ve run several of those types of experiments over the years in my lab, and I’ll mention a few of them below.

The reason I didn’t do that with the LTEE, though, was because one of my core motivating questions (see part 2 of my response) concerned the repeatability of evolutionary dynamics across replicate populations. That’s a question about the trajectory of variances over time, which is challenging statistically because estimates of variances have large uncertainties. So if the LTEE had two treatments, I might have been able to say something meaningful about the differences between them, but I would have had less power to say anything about the among-replicate variances for either treatment. In other words, with respect to that motivating question, going from 12 replicate populations down to 6 replicates would have been risky.

It certainly would be nice to have more total populations, say, 24 or even more; and nowadays many labs use 96-well plates for evolution experiments, with each well a replicate population and liquid-handling robots to automate the transfers. When I started the LTEE, though, we worked with flasks (albeit small ones); 12 may not seem like too many, but when we run the competition assays to measure fitness, we then have replicate assays for each population and we analyze multiple generations simultaneously, so the students and postdocs running these assays are handling many dozens or even hundreds of flasks.

The LTEE as a meta-experiment

Stepping back a bit, I’d like to suggest that the LTEE is a sort of meta-experiment, to coin a term. (This idea echoes the question where you suggested that the LTEE has itself become a model system.) By “meta” I mean the LTEE transcends—goes above and beyond—what one usually considers an experiment because the LTEE enables experimentation at several levels.

Level 1: The LTEE as an experiment

First, it is an experiment in the sense that it set out to measure, under defined conditions and with replication, certain specific quantities, such as fitness trajectories. It may not be typical in having a single treatment, but the temporal dimension coupled with being able to analyze multiple time points simultaneously—that is, the “time travel” enabled by the frozen samples across the generations, including the use of the ancestral strain as an internal control in fitness assays—functions in much the same way from an analysis standpoint.

Level 2: The LTEE as a generator of new questions and experiments to answer them

Second, the LTEE has generated a number of new questions and hypotheses that are themselves amenable to structurally independent follow-on experiments. Let me give two examples. We observed fairly early on that several populations had evolved changes in their DNA metabolism and repair that caused their mutation rates to increase by roughly 100-fold (Sniegowski et al. 1997). Such “mutator” mutations can arise by hitchhiking, albeit only occasionally and stochastically, with beneficial mutations that they cause (Lenski 2004, see pp. 246-251). It wasn’t clear, though, whether they would necessarily increase the rate of fitness improvement, given the large populations and correspondingly large potential supply of beneficial mutations in the LTEE. So we designed a separate, shorter-duration experiment with some 48 populations where we varied the mutation rate, population size, and initial fitness of the founding ancestor, and assessed the resulting fitness gains over 1,000 generations (de Visser et al. 1999).

Another case is the “replay” experiments that Zachary Blount ran after one lineage evolved the ability to grow on citrate in the presence of oxygen, which E. coli generally cannot do (Blount et al. 2008). Zack ran thousands of populations that started from genotypes isolated at different times from the population that eventually evolved this new function, in order to test whether it could have arisen at any time by an appropriate mutation or, alternatively, whether it required first evolving a “potentiated” genetic background, or context, in which the “actualizing” mutation would then confer the citrate-using phenotype.

In both of these examples, the subsequent experiments, though separate and distinct from the LTEE, nonetheless emerged from the LTEE. That is, the questions and hypotheses tested in these later experiments were motivated by observations we had made in the LTEE itself.

Level 3: The LTEE-derived strains as useful ancestors for a variety of experiments meant to address existing questions

The third level of the meta-experiment involves questions that arise outside of the LTEE, but for which the LTEE generates a set of materials—specifically, strains—that are especially useful for experiments to address those questions. Again, I’ll give a couple of examples.

Many ecologists, physiologists, and others are interested in studying adaptation to specific environmental factors—such as resource availability, temperature, etc.—as well as examining possible tradeoffs associated with adaptation to those factors. One difficulty, though, is that by moving organisms from nature into the lab and allowing them to evolve under, say, different temperature regimes, adaptation to the shared features of the lab environments may well outweigh adaptation to the specific variable of interest. If so, that would interfere with one’s ability to identify the mutations and adaptations most relevant to the factor of interest, and it could also obscure tradeoffs that might be important if populations were already well adapted to the other aspects of the environment. With these considerations in mind, Albert Bennett and I took a strain from the LTEE that had evolved in and adapted to those conditions—the resources, pH, absence of predators, etc.—and we used it as the ancestor for a new evolution experiment where 6 replicate populations evolved under each of 4 different thermal regimes: 32C, 37C (the same as in the LTEE), 42C, and daily alternations between 32C and 42C (Bennett et al. 1992, Bennett and Lenski 1993). In that way, we could focus attention on temperature-specific adaptations, which were Al’s main interest, rather than having such changes overwhelmed by adaptation to the lab environment.

My second example where LTEE-derived strains were ancestors for an experiment meant to address an extrinsic question is one of an abstract nature. In this study, we quantitatively partitioned the effects of adaptation, history, and chance on phenotypic evolution by founding 3 replicate populations from 12 different ancestors—each one a genotype sampled from a different one of the LTEE populations—and we then let these 36 populations evolve in a new environment, where we changed the identity of the limiting nutrient (Travisano et al. 1995). By measuring the fitness of the 12 ancestors and 36 derived lines in the changed environment, we were able to disentangle and quantify the relative contributions of adaptation, history, and chance to the observed outcomes (see figure below). That is, adaptation measured the mean tendency for fitness to increase, history reflected the effect of the different starting genotypes on the fitness achieved, and chance the variation in the resulting fitness among the replicates that started from the same ancestor.

Sniegowski, P. D., P. J. Gerrish, and R. E. Lenski. 1997. Evolution of high mutation rates in experimental populations of Escherichia coli. Nature 387:703-705.

Lenski, R. E. 2004. Phenotypic and genomic evolution during a 20,000-generation experiment with the bacterium Escherichia coli. Plant Breeding Reviews 24:225-265.

De Visser, J. A. G. M., C. W. Zeyl, P. J. Gerrish, J. L. Blanchard, and R. E. Lenski. 1999. Diminishing returns from mutation supply rate in asexual populations. Science 283:404-406.

Blount, Z. D., C. Z. Borland, and R. E. Lenski. 2008. Historical contingency and the evolution of a key innovation in an experimental population of Escherichia coli. Proc. Natl. Acad. Sci. USA 105:7899-7906.

Bennett, A. F., R. E. Lenski, and J. E. Mittler. 1992. Evolutionary adaptation to temperature. I. Fitness responses of Escherichia coli to changes in its thermal environment. Evolution 46:16-30.

Bennett, A. F., and R. E. Lenski. 1993. Evolutionary adaptation to temperature. II. Thermal niches of experimental lines of Escherichia coli. Evolution 47:1-12.

Travisano, M., J. A. Mongold, A. F. Bennett, and R. E. Lenski. 1995. Experimental tests of the roles of adaptation, chance, and history in evolution. Science 267:87-90.

[The figure below appeared in Science (Travisano et al. 1995), and it is reproduced here under the doctrine of fair use.]

Adaptation, chance, history image

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