Tag Archives: history of science

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.

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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


Filed under Education, Science

Erdös with a non-kosher side of Bacon

Erdös number

Paul Erdös was a prolific and important mathematician. He also had hundreds of collaborators from around the world who coauthored papers with him.

Years ago, Casper Goffman explained an idea, called the Erdös number, that describes the “collaborative distance” between Erdös and someone else, where that distance is defined by the smallest number of steps based on coauthored papers. Erdös himself has an Erdös number of 0, while the 511 mathematicians who wrote papers with Erdös have an Erdös number of 1. One of these people is Persi Diaconis, a professional magician and Stanford mathematician specializing in probability theory.

Over 9,000 people have Erdös numbers of 2, meaning they wrote a paper with one or more of Erdös’s coauthors but never wrote a paper with Erdös himself.  Two of these people are Berkeley professors Bernd Sturmfels, in the field of algebraic geometry, and Lior Pachter, a computational biologist.  (Sturmfels coauthored three papers with Diaconis, and he wrote other papers with two more people with Erdös numbers of 1.  Pachter wrote several papers with mathematician Daniel Kleitman, an Erdös coauthor.)

In 2007, I coauthored a paper with Pachter and Sturmfels in which we analyzed epistatic interactions to describe the geometric structure of a fitness landscape:

Beerenwinkel, N., L. Pachter, B. Sturmfels, S. F. Elena, and R. E. Lenski. 2007. Analysis of epistatic interactions and fitness landscapes using a new geometric approach. BMC Evolutionary Biology 7:60.

So that paper gives me an Erdös number of 3.

Bacon number

A group of students later came up with the idea of a Bacon number, a Hollywood version of the Erdös number that equals the smallest number of film links separating any other actor from Kevin Bacon. (Bacon had been previously described as the “center of the Hollywood universe” after a 1994 interview in which he said he worked with everybody in Hollywood or someone who’s worked with them, according to Wikipedia.)

So Kevin Bacon has a Bacon number of 0, while actors who have appeared in a film with him have Bacon numbers of 1. An actor who appeared in a film with any actors who appeared with Bacon, but not in a film with Bacon himself, have a Bacon number of 2.

Morgan Freeman has a Bacon number of 1 based on a 2013 documentary film called “Eastwood Directs: The Untold Story.” (You missed that one? Me, too.) Well, a couple of weeks ago, I appeared in an episode of the show “Through the Wormhole with Morgan Freeman.”

Erdös-Bacon number

Now there’s a really special number called the Erdös-Bacon number, which is the sum of a person’s Erdös and Bacon numbers. Not many people have an Erdös number, and not many have a Bacon number. And very few people have an Erdös-Bacon number because you have to have written a math or science paper and appeared in a film, and of course with known connections to Erdös and Bacon along both paths.

Cornell mathematics professor Steven Strogatz has an Erdös-Bacon number of just 4, having appeared in a TV documentary film with Kevin Bacon called “Connected: The Power of Six Degrees.” Of course, that film is about the very sort of mathematical links we’re talking about here!

So someone just suggested to me that I now have an Erdös-Bacon number of 5. If so, that would put me ahead of such luminaries as Carl Sagan and Richard Feynman! Awesome!!

The fine print

As I was looking into this exciting possibility, I discovered a website called “The Oracle of Bacon.” It seems to be the semi-official arbiter of Bacon numbers, and it says: “We do not consider links through television shows, made-for-tv movies, writers, producers, directors, etc.”

That documentary about Cliff Eastwood, with both Morgan Freeman and Kevin Bacon in it, apparently doesn’t qualify.  So Morgan Freeman’s Bacon number rises to 2 (via many different paths through his many major films).

Even worse, though, my Bacon number evaporates entirely, since my link to Kevin Bacon goes through my appearance on a television show with Morgan Freeman.

So there you have it. I have an officially non-kosher Erdös-Bacon number of 5.

I guess I can live with that.

But if Kevin Bacon, Morgan Freeman, or any of their Hollywood friends invites me to appear in a real film, I’ll probably accept!


Note:  It looks like Steven Strogatz’s Erdös-Bacon number of 4 is also compromised because his Bacon number is through a TV movie.  You need to use non-default settings for it to show up on The Oracle of Bacon website.  But maybe it’s less non-kosher, since it was a TV movie, not just a TV show.


Filed under Humor

Tiny Things that Live in Little Bottles

As I mentioned in my previous post, it can be a fun challenge to explain your scientific research to people who aren’t scientists.

A week or so ago I came across a website that challenges you to explain something complicated using only the thousand most commonly used words.

So here’s my effort about our long-term evolution experiment with E. coli:

My team works with really tiny things that live in little bottles. We watch the tiny things change over time – over a really long time. The tiny things that do the best have learned to eat their food faster and faster, before the other guys can eat their lunch, so to say.  Well, the tiny things don’t really learn, but it’s kind of like learning – and even better, the best ones pass along what they learned to their kids.  A really cool guy came up with the idea of how this works more than a hundred years ago. My team’s work shows he got it pretty much right. But there’s a lot of stuff he didn’t know, and we’re figuring that out, too.

Several other biologists followed up including Nicole King, Graham Coop, and Josie Chandler (the links are to the simple-words-only descriptions of their own research).

Give it a try, and add your contributions in the comments below!


Filed under Education, Humor, Science

Thirty Years

No, the LTEE did not suddenly jump forward by almost 3 years. That milestone will be reached on February 24, 2018.

Next Friday is the end of the semester at MSU and, for me, it will mark 30 years that I’ve been on the faculty: six at UC-Irvine, and 24 here at MSU. (I also taught for one semester at Dartmouth as a sabbatical replacement, while I was doing a postdoc at UMass.)

Holy cow: 30 years. Where did all that time go?

Well, a lot of it was spent advising, supervising, and mentoring graduate students. And those have been some of the most interesting, enjoyable, and rewarding professional experiences that I can imagine.

In fact, this afternoon Caroline Turner defended her dissertation – congratulations Dr. Turner! Her dissertation is titled “Experimental evolution and ecological consequences: new niches and changing stoichiometry.” It contains four fascinating and meaty chapters, two on the interplay between evolutionary and ecological processes in the LTEE population that evolved the ability to grow on citrate, and two on evolved changes in the elemental stoichiometry of bacterial cells over experimental time scales.

Caroline is the 20th student to complete her Ph.D. with me serving as the advisor or co-advisor. Here they all are, with links to their professional pages or related sites.

  1. Felisa Smith, Ph.D. in 1991 from UC-Irvine.
  2. John Mittler, Ph.D. in 1992 from UC-Irvine.
  3. Mike Travisano, Ph.D. in 1993 from MSU.
  4. Paul Turner, Ph.D. in 1995 from MSU.
  5. Greg Velicer, Ph.D. in 1997 from MSU.
  6. Brendan Bohannan, Ph.D. in 1997 from MSU.
  7. Phil Gerrish, Ph.D. in 1998 from MSU.
  8. Farida Vasi, Ph.D. in 2000 from MSU.
  9. Vaughn Cooper, Ph.D. in 2000 from MSU.
  10. Danny Rozen, Ph.D. in 2000 from MSU.
  11. Kristina Hillesland, Ph.D. in 2004 from MSU.
  12. Elizabeth Ostrowski, Ph.D. in 2005 from MSU.
  13. Bob Woods, Ph.D. in 2005 from MSU.
  14. Dule Misevic, Ph.D. in 2006 from MSU.
  15. Gabe Yedid, Ph.D. in 2007 from MSU.
  16. Sean Sleight, Ph.D. in 2007 from MSU.
  17. Zack Blount, Ph.D. in 2011 from MSU.
  18. Justin Meyer, Ph.D. in 2012 from MSU.
  19. Luis Zaman, Ph.D. in 2014 from MSU. (Charles Ofria was the primary advisor.)
  20. Caroline Turner, Ph.D. in 2015 from MSU.

There are also 8 doctoral students at various stages currently in my group at MSU including Brian Wade (Ph.D. candidate), Mike Wiser (Ph.D. candidate), Rohan Maddamsetti (Ph.D. candidate), Alita Burmeister (Ph.D. candidate), Elizabeth Baird, Jay Bundy, Nkrumah Grant, and Kyle Card.

My own advisor – the late, great Nelson Hairston, Sr. – said that he expected his graduate students to shed sweat and maybe even occasional tears, but not blood. I would imagine the same has been true for my students.

Thirty years, holy cow. Time flies when you’re working hard and having fun!

Added November 4, 2015:  And now #21 in my 31st year, as  Mike Wiser successfully defended his dissertation today!


Filed under Education, Science

Window Dressing

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

62K window dressing

Comments Off on Window Dressing

Filed under Humor, Science

Questions from Jeremy Fox about the LTEE, part 1

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


Over at the Dynamic Ecology blog, Jeremy Fox asked me some interesting questions about the history, philosophy, and science of the E. coli long-term evolution experiment. Perhaps mistakenly—in terms of time management, not my interest!—I agreed to try to answer them … though over what time frame, I’m not sure. Anyhow, here is Jeremy’s first question followed by my (very) short and (too) long answers.


  • When you first started the LTEE, did you consider it to be a low risk or high risk experiment? Because I could see arguing both ways. In some ways, it’s low risk, because one can imagine lots of different possible outcomes, all of which would be interesting if they occurred. But in other ways, it’s high risk–I imagine that many of the interesting outcomes (including those that actually occurred!) would’ve seemed unlikely, if indeed they’d even occurred to you at all. Or did you not worry much about the range of possible outcomes because the experiment was basically a lottery ticket? “This’ll be cheap and not much work, let’s just do it and see what happens. Something really cool might happen, but if it turns out boring that’s ok because it wasn’t a big investment.”


The short answer: Life was good, and I wasn’t thinking about risk. Or as they say about investing: it’s better to be lucky than smart!

The long, non-linear* answer: I’d already had success with some shorter duration, more traditionally designed experiments (e.g., Lenski, 1988), and so it wasn’t a total shot in the dark—that is, I knew the LTEE would yield data. I also knew, though, that it was an unusually abstract, open-ended, and non-traditional experiment, and that it might not appeal to some people for those reasons. But I loved (and still do) the seemingly simple (but in reality complex) questions, issues, and hypotheses that motivated the LTEE.

I never thought of the LTEE project as a “lottery ticket”, but some follow-up work that grew out of it had that feel.** And, oddly enough, there was one lottery-ticket aspect of the research early on, although that reflected a lack of preparation rather than a well-conceived feature.***

Maybe I was overly confident, but I’d also say that I was pretty sure the outcomes—whatever they might be—would be “cool.” The questions were intriguing, and there hadn’t been many, if any, previous attempts to answer them quite so directly. Data would be forthcoming, and even if the results weren’t definitive, I felt there would be some interest in trying to interpret whatever data emerged.**** Plus, I knew enough about what would happen—based on the experiments I had already done—that I was confident that the data and analyses would be informative with respect to at least some of my questions. Also, the use of microbes to study evolution in action was still uncommon, so the novelty of the approach would ensure some interest among my colleagues—although let me emphasize that Lin Chao, Dan Dykhuizen, Barry Hall, and Bruce Levin, among others, had already demonstrated the power of using microbes for experimental studies of evolutionary questions.*****

I should also say, in case it’s not obvious, that I had no idea or intention that the experiment would continue for anywhere near as long as it has lasted—nor that it might, I now hope, be running long after I’m gone. I had previously performed some experiments that lasted several hundred generations, and as I saw the dynamics and thought about the math behind the dynamics, I realized that over those time scales I might be seeing the effects of only one or two beneficial mutations as they swept to fixation. That hardly seemed satisfactory for experiments to explore the structure of the fitness landscape. So I decided the experiment should run for 2,000 generations, over which time I expected there would be at least several fixations of beneficial mutations in each population (and I was right), and that would deserve calling it long-term. That would take a little less than a year, given the 100-fold dilution and 6.6 generations of re-growth each day.

Of course, propagating the lines for 2,000 generations was one thing—running the competitions to measure fitness, analyzing the data, writing the paper, responding to reviews, all that took longer. So while the experiment began in February 1988, the first paper (Lenski et al., 1991) was not submitted until August 1989, resubmitted September 1990, accepted that November, and finally published in December 1991. Meanwhile, the LTEE itself continued and the generations ticked by. The baseline work of keeping the populations going is not that onerous—yes, somebody has to attend to the transfers every day, but once a lab team reaches a moderate size, it’s not too hard to arrange. And I lived next to the campus in Irvine, so it wasn’t hard for me to come in on the weekends and holidays … and my wife still loves me, and my kids recognized my face ;>)

You also wondered whether some of the interesting possible and actual outcomes had occurred to me when I started. Definitely not! I had made a strategic decision to make the environment of the LTEE very simple in order to eliminate, or at least reduce, certain complications (especially frequency-dependent interactions and clonal interference). And while I think my planning kept these complications from getting out of hand, the tension between the simplicity of the experimental design and all the complications has definitely been part of its interest. That tension, along with time, the evolutionary potential of the bacteria, and the smart, talented, creative** and hard-working students and colleagues have made the LTEE what I call “the experiment that keeps on giving.”


*Hey, that’s what footnotes are for, right?

**I’ve thought that way about some follow-on work that uses the LTEE lines, but not about the project as a whole. Here are a couple of examples of “lottery tickets” that people suggested to me, and that won big. A former postdoc Paul Sniegowski, now on the faculty at Penn, wanted to know whether the actual mutation rate itself might be evolving in the LTEE populations. Bingo! Several lines evolved hypermutability and so, curiously enough, the first mutations we ever mapped affected the mutation rate itself (Sniegowski et al., 1997). Another example: Dominique Schneider is a molecular microbiologist in Grenoble, and we’ve collaborated for over 15 years. He thought we should look at whether DNA topology—the physical supercoiling inside the cell—might have changed in the LTEE lines. Well, I thought to myself, why would it change? But Dom’s lab will do all the work, so sure, why not look? And it turns out, sure enough, that DNA supercoiling changed repeatedly in the LTEE lines (Crozat et al., 2005), and it even led us to discover a gene not previously known to affect supercoiling (Crozat et al., 2010). There’s a lesson here, by the way—work with people who are smarter, who have different interests, and who have different skills than oneself.

***I actually started two versions of the LTEE—not one experiment with two proper treatments, but two separate experiments that differed in terms of both the starting strain and the environment. Unlike the successful LTEE, I hadn’t done any previous evolution experiments with the other ancestral strain and environment. Anyhow, I soon stopped the other version when the populations evolved a phenotype that made it very difficult to work with them. In brief, the populations evolved to make pinprick-sized colonies that were next-to-impossible to count in the assays we use to measure fitness. Who needed that headache! So, in a way, I guess I had two lottery tickets: I hadn’t done the relevant prior work for one of them, whereas the one that paid off was actually a pretty strategic gamble.

****I was at UC Irvine when I started the LTEE, and Michael Rose was one of my colleagues there. His work on the evolution of aging—postponed senescence—in fruit flies (e.g., Rose 1984) was an inspiration in terms of the importance and power of long experiments. We also spent a lot of time discussing fitness landscapes, the alternative perspectives of Sewall Wight and R. A. Fisher about the dynamics on those landscapes, and what experiments might tell us. Michael didn’t design, direct, or do the lab work for the first LTEE paper, but he helped me clarify my thinking and write the first paper on the LTEE (Lenski et al., 1991). Perhaps more importantly, his interest in the questions and issues made me realize that other smart people would also be interested.

*****I used to complain, mostly in jest, that “Evolutionary biologists say I’m asking the right questions, but studying the wrong organism, and microbiologists tell me I’m studying the right organism but asking the wrong questions.” I got that sort of response occasionally, but many people from both fields were very interested and encouraging. For example, I remember David Wake telling me, after one of my first talks about the LTEE, how much he liked both the questions and the approach.


Lenski, R. E. 1988. Experimental studies of pleiotropy and epistasis in Escherichia coli. II. Compensation for maladaptive pleiotropic effects associated with resistance to virus T4. Evolution 42: 433-440.

Lenski, R. E., M. R. Rose, S. C. Simpson, and S. C. Tadler. 1991. Long-term experimental evolution in Escherichia coli. I. Adaptation and divergence during 2,000 generations. American Naturalist 138:  1315-1341.

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.

Crozat, E., N. Philippe, R. E. Lenski, J. Geiselmann, and D. Schneider. 2005. Long-term experimental evolution in Escherichia coli. XII. DNA topology as a key target of selection. Genetics 169: 523-532.

Crozat, E., C. Winkworth, J. Gaffé, P. F. Hallin, M. A. Riley, R. E. Lenski, and D. Schneider. 2010. Parallel genetic and phenotypic evolution of DNA superhelicity in experimental populations of Escherichia coli. Molecular Biology and Evolution 27:2113-2128.

Rose, M. R. 1984. Laboratory evolution of postponed senescence in Drosophila melanogaster. Evolution 38: 1004-1010.


LTEE flasks repeating


Filed under Science

Wonderful Life Times Two

No, I’m not talking about the movie It’s a Wonderful Life, starring James Stewart, and the eponymous book Wonderful Life by Stephen Jay Gould that presented the case for the role of contingency in the evolution of life.

Rather, I’m celebrating a wonderful end to the week and a wonderful weekend, too.

Last week, we submitted the renewal proposal to the National Science Foundation for phase two of our BEACON Center for the Study of Evolution in Action. We were led, as usual, by our amazingly wonderful director Erik Goodman, and our wonderfully superb managing director Danielle Whitaker, with major work by all of us co-PIs and input from many others. BEACON’s mission is to illuminate and harness the (wonderful) power of evolution in action to advance science and technology and benefit society. And as we move toward phase two, we’re looking forward to even more wonderful research, collaborations, diversity initiatives, training, education, and outreach.

And this weekend, one of my wonderful daughters and her wonderful husband organized a wonderful weekend in Chicago for my wonderful wife and me. We stayed with my son-in-law’s wonderful parents, and we got to spend the weekend with them and our wonderful three-year-old granddaughter. And on Saturday evening, while the in-laws babysat, we went to the wonderful Looking Glass Theater and saw a truly wonderful play, In The Garden: A Darwinian Love Story.

The play is about Charles and Emma Darwin: their childhood—they were cousins—their romance, their marriage, their trials and tribulations as Charles grappled with his science and they struggled to reconcile Emma’s religion with his science, and they both struggled to reconcile their beliefs with the death of their beloved daughter Annie, all the while remaining deeply in love with one another.

The Looking Glass Theater is a tiny, intimate setting—wonderful for an intimate play like In the Garden.

It’s a wonderful life indeed.

[Emma Darwin, in 1840, painted by George Richmond, image via Wikipedia.]

Emma Darwin


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