Tag Archives: Bruce Levin

Questions from Jeremy Fox about the LTEE, part 1

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

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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.

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  • 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.”

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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.”

Footnotes

*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.

References

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

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Lenski Interview with The Molecular Ecologist

John Stanton-Geddes asked me some great questions for a series on “People Behind the Science” at The Molecular Ecologist blog.  He gave me permission to repost the interview here.

1) Did you always think you’d become an evolutionary biologist?

No!  I always enjoyed being outdoors (sports and hiking), but I didn’t have any particular interest in biology.  However, my mother (who dropped out of college when she married, but then co-authored a sociology textbook with my father) was very interested in biology.  She would give me articles she had read and enjoyed from Natural History and elsewhere.

I went to Oberlin College, where I thought that I might major in government.  But I disliked my first government class.  I also took a team-taught biology class for non-majors.  All of the instructors spoke on topics about which they cared deeply, and I was hooked!  I took more biology courses, and I was especially drawn to ecology because there were so many ideas and questions.  At that time, I wrongly viewed evolutionary biology as a more descriptive, old-fashioned field with fewer questions that one might still address.  (By the way, several other evolutionary biologists were at Oberlin when I was there including Deborah Gordon, Joe Graves, Kurt Schwenk, and Ruth Shaw. Not bad for a small school!)

I went to graduate school at the University of North Carolina, where Nelson Hairston, Sr., was my advisor.  Nelson was interested in the interface of ecology and evolution, and that opened my eyes.  I was also influenced by Janis Antonovics, then at Duke University.  I took his Ecological Genetics course, and he served on my committee.  Janis had written a paper in which he argued that “The distinction between ‘ecological time’ and ‘evolutionary time’ is artificial and misleading.”  That really got me thinking.  I tried to develop a couple of field-based projects that would address evolutionary questions, but I didn’t know what I was doing and they failed.  In the end, my dissertation project was pure ecology.

By then, though, I knew I wanted to pursue evolutionary biology.  While we were finishing our doctoral projects, a fellow grad student Phil Service and I spent a lot of time discussing model systems for studying evolution.  For his postdoc, Phil chose to work with Drosophila.  I recalled an undergrad course in which we read about elegant experiments with microbes that addressed fundamental questions, such as one by Salvador Luria and Max Delbrück showing that mutations happen at random and not in response to selection.  Meanwhile, in a graduate seminar, we read a paper by Lin Chao and Bruce Levin on the coevolution of bacteria and viruses.  I wrote Bruce to ask if he might have an opening for a postdoc.  Lucky for me, Bruce knew Nelson and invited me for a visit.

2) You’ve described the theme of your research as “the tension between chance and necessity”. Can you comment on how chance and necessity have shaped your career?

The ancient Greek philosopher Democritus said, “Everything existing in the universe is the fruit of chance and necessity.”  In my long-term evolution experiment with E. coli, we can explore the tension between chance and necessity because we have replicate populations started with the same ancestor and evolving under identical conditions, and because we can replay evolution from different points along the way.  But it’s difficult, if not impossible, to tease apart the roles of chance and necessity with a sample size of one, which is the life that each of us has experienced, and without the ability to replay our own lives.  (On that last point, let me recommend Replay, a science-fiction novel by Ken Grimwood.)

I would say, though, that most people who have had some success in their adult lives also started out very lucky.  We were fortunate to be born at times and in places where we had food, familial love, education, and opportunity.

3) Reading your blog it’s clear that you are a student of the philosophy and history of science. Do you think we should include more history and philosophy in scientific training? Any advice on something we should all go out and read?

I do think that the history and philosophy of science deserve more emphasis in science and education than they usually receive.  But I didn’t have any formal education in those areas.  Instead, I became interested in these issues through teachers, mentors, colleagues, and my own explorations.

For something to read in this area, I suggest Darwin’s Century by Loren Eiseley.  (Originally published in 1958, it was republished in 2009 by Barnes & Noble.)  The book discusses the fascinating history of evolutionary thought in the decades before and after the publication of The Origin of Species.  I first read Darwin’s Century in a course at Oberlin taught by James Stewart.

4) If you were starting your career today, what would you study? 

If I were starting today, and at my present age, I might choose to study the history of science, especially evolutionary biology and its antecedents.

But if I were starting out young, as one usually does, I’d like things to unfold as they did.  It might be tempting to skip the rough patches, but dissatisfaction with my early research led me to make the switch to microbial evolution.  Would I have enjoyed this lab-based work as much, if I hadn’t discovered that I was not nearly as good at fieldwork as many of my peers?

5) How close have you come to giving up as a researcher and doing something completely different?

The job market was tough when I was a postdoc, and I had a growing family to support.  So after a slew of applications and rejections, and a period of uncertain funding, I started to think about other possibilities.  Luckily for me, things turned around before I had to make a switch.  (You can read more about it in my blog post, The Good Old Days.)

6) What’s the meaning of life?

I think that some understanding of evolution—at a basic level accessible to anyone with an open mind and a decent education—gives perspective about our place, both as individuals and as a species, in the grand sweep of time and space.  Recognizing the transience of my personal existence fills me with awe and respect for the continuity of life and ideas.  And belonging to a species that is profoundly altering the world that enabled the continuity of life reminds me of our responsibility for ensuring its future.

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Remembering Barbara McClintock’s Laugh

A twitter conversation about women scientists who should be honored with Bobbleheads reminded me of the time I met Barbara McClintock.  My postdoctoral advisor, Bruce Levin, had brought me along to a small discussion meeting on the possible ecological and evolutionary effects of genetically modified organisms being released into the environment.

The meeting was held at the Cold Spring Harbor Laboratory in 1984.  There were two Nobel Prize winners there, at least part of the time, along with seven ecologists and evolutionary biologists.  The Nobelists were James Watson, who was then director of the Laboratory, and Barbara McClintock.  The ecologists and evolutionary biologists were Jim Brown, Rob Colwell, Monte Lloyd, Phil Regal, Dan Simberloff, Bruce Levin, and myself. I think there were also a few participants from the EPA and other agencies with an interest in the issues.  As I recall, Barbara McClintock was the only woman at the meeting.

The meeting was held in an old house.  There were two small restrooms, one for men and one for women; each had just a single toilet and a sink.

Because there were many men at the meeting, there was a line for the men’s room at the coffee breaks.  Bruce Levin, being the smart guy that he is, decided during one break that it would be faster and more efficient to use the women’s room.  The door was open, and so he went in.  Meanwhile, Barbara McClintock also had to use the facilities.  Seeing that the door was closed, she waited her turn.

As Bruce came out, he was already a bit embarrassed to see Barbara waiting.  And then she said, in a serious, almost scolding, tone, “Did you put the seat down?”  Bruce – who usually has a quick comeback – was befuddled and didn’t know what to say.  After a comic pause, Barbara just laughed heartily and went on in.

So there was a warm and funny person, as well as a visionary scientist, behind that Nobel Prize!

McClintock

[This picture is from the Wikipedia entry for Barbara McClintock; it has no known copyright restrictions listed there.  It comes from the Smithsonian Institution.]

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Teaching Competition and Predation from a Microbiological Perspective

Life has been busy, very busy.  And life has been good!  But the busy-ness has made it hard for me to keep up with this blog.  In the next few weeks, I hope to share some of the things that have kept me so occupied this past month.

For starters, I’d like to discuss some recent teaching where I tried to emphasize the interplay between theory and experiments in ecology.

I recently taught part of our graduate-level course called “Integrative Microbial Biology.”  Some years ago this course replaced several other graduate courses (microbial ecology, microbial physiology, microbial diversity, etc.) that each had a low enrollment.  The idea is that we now offer a single, annual, intensive, team-taught course that covers all these topics, albeit more superficially but with the hope that it encourages students and faculty alike to develop a more integrated perspective of microorganisms as organisms.  More specialized courses, with a focus on reading and discussion, are offered as occasional seminar-style courses.

I teach two parts of the course – one on aspects of microbial ecology, the other on microbial evolution.   Many of the students have not had an undergraduate course in general ecology or evolutionary biology, and so I try to bring them up to speed, albeit with examples that focus on microorganisms.

So, for the ecology portion I begin with population growth and competition.  I’m a fan of resource-based competition theory, as opposed to the more familiar logistic growth and Lotka-Volterra competition models.  The key strength of resource-based competition theory is that one can predict the outcome of competition based on parameters that can be measured separately for each species or strain, without requiring that one compete them in order to understand their competition.  Of course, there are many reasons the predictions might fail, but the resource-based model (and extensions to it) provide a mechanistic framework for understanding competition.

I then present predator-prey interactions, surveying the extraordinary diversity of microbe-on-microbe predation and parasitism, and then providing again a dynamical framework for understanding those interactions.  Here, Lotka-Volterra predator-prey models do provide a reasonable starting point because one can measure key parameters that have mechanistic interpretations (e.g., attack rates, conversion efficiencies) and use them to make new predictions about the dynamics of the system as a whole.

Besides presenting the general theory, I also present empirical studies from the primary literature.  In some cases, I summarize the papers in my lectures, while in other cases the students read the papers and we then discuss them.  Here are four of the papers with summaries; I hope to blog someday in greater depth on at least the Hansen & Hubbell and Rainey & Travisano papers, which I view as “must-read” papers in the field of ecology.

Hansen, S. R., and S. P. Hubbell.  1980.  Single-nutrient microbial competition: qualitative agreement between experimental and theoretically forecast outcomes.  Science 207:1491-1493.

This paper presented an early, concise, and compelling demonstration of the utility of resource-based competition theory.  By choosing three pairs of competitors that differed in various parameters, and then competing them in chemostats, the authors showed that the outcome depended on the two competitors’ relative “break-even” (equilibrium) concentrations of the growth-limiting resource.  For any student who wants more information on this approach – and every year at least some students ask for more – I recommend they read David Tilman’s outstanding book, Resource Competition and Community Structure (1982, Princeton University Press).

Rainey, P. B., and M. Travisano.  1998.  Adaptive radiation in a heterogeneous environment.  Nature 394:69-72.

This paper is a beauty.  The authors showed that the evolutionary emergence of diversity can sometimes depend on something as simple as whether a flask is shaken or not.  In the absence of shaking, an initially monotypic population of Pseudomonas fluorescens evolved into a community of three distinct ecotypes that differentially exploit the environmental gradients that arise without constant mixing; that diversity is stably maintained, as was shown by analyzing pairwise interactions.  By contrast, simply shaking the flask, with all else being equal, homogenizes the environment and the ecotypic diversity does not evolve; and if the diversity had already evolved, then it was eliminated as a single type came to dominate the well-mixed system.

Lenski, R. E., and B. R. Levin.  1985.  Constraints on the coevolution of bacteria and virulent phage: a model, some experiments, and predictions for natural communities.  American Naturalist 125:585-602.

Virulent phage infect bacteria, and they have life-cycles like those of insect parasitoids; that is, a successful infection is lethal to the host, and many phage are produced from a single infection.  In this paper, we examined the ecological and evolutionary dynamics of the interactions between E. coli and four different virulent phages.  First, the Lotka-Volterra predator-prey model – modified to include resource-based growth for the prey (bacteria) and a time-lag associated with predator reproduction (phage replicating inside bacteria) – predicted reasonably well the short-term dynamics of the interaction between E. coli and one of the phages, called T4.  Second, the model was extended to include the evolution of bacteria that are resistant to phage attack.  Resistance mutations changed the equilibrium density of the bacteria by several orders of magnitude, as the bacterial population went from top-down predator limitation to bottom-up resource limitation.  Yet despite complete resistance, the phage population persisted because there was a “cost of resistance” – in the absence of phage, the sensitive bacteria out-competed the resistant mutants.  In essence, the system becomes one of predator-mediated coexistence of sensitive and resistant prey populations.  Third, the interactions between E. coli and three other phages were examined.  Each interaction had somewhat different dynamics depending on whether resistance was costly or not, whether resistance was partial or complete, and whether the phage population produced host-range mutants that could infect the mutant bacteria that had become resistant to the progenitor phage.  [This paper built on related work that Lin Chao had done a few years earlier with Bruce Levin, and which inspired me to contact Bruce about joining his lab.]

Bohannan, B. J. M., and R. E. Lenski.  2000.  Linking genetic change to community evolution: insights from studies of bacteria and bacteriophage.  Ecology Letters 3:362-377.

This paper reviews the research that Brendan Bohannan did for his dissertation in my lab.  His work examined the same four bacteria-phage interactions studied in the Lenski and Levin paper above, but the work was extended to include some elegant new manipulations and analyses.  In particular, by changing the levels of resource available to the bacteria, the classic “paradox of enrichment” predicted by Lotka-Volterra predator-prey models was confirmed, with respect to the effects of enrichment on both equilibrium densities and the temporal fluctuations in population densities.  These experiments also provided compelling evidence for predator-prey cycles and the effects of bacterial resistance on the dynamics of the interaction between the remaining sensitive bacteria and phage populations.

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The Good Old Days

I started applying for faculty jobs in 1983, after about a year and a half as a postdoc.  The job market was tight then, as the US was coming out of a deep recession.  (Sound familiar?)  And faculty jobs in microbial evolution simply didn’t exist in those days.  So I applied for any and all positions that had anything to do with ecology or evolution, whether at big universities, small colleges, or anywhere else.  The first year I sent out around 75 applications, as I recall.  And I do mean sent out, because in those days applicants had to copy things and mail them.  I had one interview, but no offer.  Meanwhile, Bruce Levin’s grant that was going to support me going forward got rejected.

My wife and I had one child, and our second was on the way.  I wasn’t panicked, although maybe I should have been!  However, I did start vaguely thinking about back-up plans.  I was good with numbers, and I’d written a couple of papers on the analysis of life-table data with Phil Service when we were grad students.  So when I saw an announcement for some talk on actuarial analysis, it caught my eye.  I thought that might be a possible alternative career.  I went to the talk, and I might even have gotten a business card from the speaker.

In the months ahead, Bruce revised and resubmitted the grant and, thankfully, it was funded, so I was secure for a while longer.  The next year, I again sent out applications far and wide.  I had a couple more papers, so my CV was stronger and the economy was improving, too.  I got three interviews and, with my postdoc secure, I actually declined another one.  However, the interviews were near the due-date for our second child, so I worried that I might have to cancel (and reschedule, if they’d allow it) the interviews.  Luckily, #2 arrived in time.  So I left my wife at home with a 10-day old baby and a toddler … and took off for back-to-back-to-back interviews.  Soon after I got home, I got two job offers on the same day.  (When it rains, it pours.)  One offer was from UC-Irvine, and that’s where we went after deferring the start for a year so I could get more research done as a postdoc.

I see there’s a lot of angst out there about the job market in academics.  And rightfully so.  Positions are scarce, and the competition is extraordinary.  I feel fortunate that I got a very good faculty position to start my career.  Things were tough back in the day, but they are much tougher now.  I admire all of you who are pursuing your dreams, but it never hurts to consider a backup plan – whether you need to use it or not.  There are, after all, many roads to happiness and success.

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Chao and Levin, 1981, PNAS

This is the third in my series of must-read papers.  It’s an elegant paper that sits right at the interface of ecology, evolution, and behavior.  And like the last paper that I wrote about, this one is superb for teaching and capturing the interest of students.

Chao, L., and Levin, B. R.  1981. Structured habitats and the evolution of anticompetitor toxins in bacteria.  Proc. Natl. Acad. Sci. USA 78, 6324-6328.

Short summary:  Some bacterial strains produce and release toxins that kill members of their own species – except, that is, close kin that possess a linked immunity function.  The production of the toxins is also lethal to the small fraction of cells that actually do so in any given generation.  Lin Chao and Bruce Levin sought to understand when and how this trait would be beneficial.  When killer and sensitive strains competed in liquid, the killer strain prevailed, but only if it started out above a threshold frequency.  That raised the question of how the killer strain could reach that frequency, because it was at a disadvantage when it was below that threshold.  When the same strains competed in a structured environment (a gel-like matrix), this conundrum was resolved—the killer strain could invade a population of sensitive cells even if the killers started at an arbitrarily low frequency.  The difference arises because, in the structured environment, the resources made available by the killers accrue disproportionately to the killers’ kin.  This paper was ahead of its time, but it set the conceptual stage for the now-blossoming field that uses microbes to study the evolution of social traits and interactions.

Some additional background and explanation:  Many bacteria can produce and release toxins that kill other members of the same species.  These toxins are called bacteriocins in general; those studied by Chao and Levin are also called colicins because they are produced by, and used against, E. coli.  The toxin production and immunity functions are tightly linked in a genetic module, and such modules are often located on extra-chromosomal elements called plasmids.  Interestingly, the production of the toxin is lethal to the individual cell that does so, because the cell must lyse to release the toxin.  However, only a small proportion (maybe 1%) of the potential killers that carry the toxin/immunity module actually produce toxin in a given generation, while the others constitutively express the immunity function.

How can a function evolve that is lethal to the individual organism that expresses it?  Chao and Levin began by competing two otherwise identical E. coli strains—one that carries the toxin/immunity module, the other sensitive to the toxin—in a well-mixed liquid medium.  Let’s call the strains K and S for killer and sensitive, respectively.  If there were enough K cells (above ~2% in their experimental conditions), then K rose in frequency and drove the S type extinct.  Although the K population experienced some deaths from the production of the toxin, the resulting toxin concentration was so high that the death rate of S exceeded its growth rate.

But if the initial frequency of the K type was below that ~2% threshold, then the outcome was reversed—the S population rose in frequency, and the K population declined, although the exclusion played out more slowly than when K started out above the threshold.  What’s happening here?  The K cells still had the extra cell deaths caused by the release of toxin, but the concentration of toxin was not sufficient to wipe out the S population.  Some S cells were killed, and their resources—those released upon their death plus those they could no longer consume—became available to other cells.  Because the competition environment was well mixed, any cell—whether K or S—had equal access to the freed-up resources.  If the death rate of the K type (the proportion that produces toxin and then lyses) were greater than the kill rate of the S type, then K would decline in frequency because the resulting benefit—the extra resource that became available—was equally available to all survivors, regardless of whether they had the K or S genotype.

From an ecological standpoint, it’s a nice example of a dynamically unstable equilibrium between two competitors.  However, it raises a problem from an evolutionary perspective.  If possession of the toxin/immunity module is beneficial when it is common in a population, but disadvantageous when it is rare, then how can it go from being rare to common?

Chao and Levin recognized that a physically structured environment might be important, because it would change the distribution of the freed-up resources to the two cell types.  So they repeated the competitions between K and S strains, again varying the initial frequency of the K type, except now in a semi-solid medium called “soft agar.”   (The procedures get more complicated here; to propagate the competing cell types, each day they had to free the cells from the soft-agar matrix and transfer them into a new matrix.)  When the two types competed in this structured environment, the unstable equilibrium disappeared, and the K strain could invade and take over from an arbitrarily low initial frequency.  That is, the K genotype could now go from being rare to common.

Why this difference between the liquid and semi-solid environments?  In the structured environment, the bacteria grew as colonies, not as individuals floating about at random.  As a consequence, the extra resources made available by the killers flowed disproportionately to their own kin.  Here a picture is worth a thousand words; I show a figure from Chao and Levin below that makes this point graphically.  In a sea of crowded S colonies, there’s one K colony.  The K colony is larger than most of the S colonies.  Each colony began from a single cell; the fact that the K colony is larger than most means that it got more than its share of resources.  Even more strikingly, the K colony is surrounded by a large zone that is entirely devoid of colonies—the toxins released by the small proportion of K cells that lysed have diffused into this zone and prevented growth of S cells.  The resources diffused randomly, but the K colony sat alone in the middle of this zone of inhibition that it generated, and so indeed it got more than its share of resources.

Chao and Levin Fig 3

The figure above is from Chao and Levin, 1981, Proc. Natl. Acad. Sci. USA; it is shown here under the doctrine of fair use.  The image is centered on a single colony of toxin-producing bacteria surrounded by an inhibition zone and, further out, by colonies of sensitive bacteria.  The scale bar is 0.5 mm.

A Later, Related Paper:  There’s another nice paper by Ben Kerr, Peg Riley, Marc Feldman and Brendan Bohannan (2002, Nature) that builds on the work by Chao and Levin.  Kerr et al. added a third “player”—a third strain—into these experiments, one that was resistant to the toxin but did not produce it.  In a physically structured environment, the toxin-producing killer strain could invade and displace the sensitive strain, just as Levin and Chao saw.  However, the resistant strain could invade and displace the toxin-producer, because the physiological cost of resistance was less than the combined costs of toxin-production and immunity.  And the sensitive strain could invade and displace the resistant strain, because the sensitive strain did not pay the cost of resistance.  In other words, the pairwise interactions were non-transitive, just like the game of rock-paper-scissors.  But although each pairwise interaction had a winner and a loser, the three types could coexist indefinitely in a spatially structured environment provided different spatial regions were out of phase—in effect, the three populations chased one another around in space and time.

Why I like this paper so much:  First, the paper by Chao and Levin beautifully illustrates how population biologists frame, dissect and analyze a complex problem—one that involves frequency-dependent effects, tradeoffs, spatial structure, and genetic relatedness along with both scramble and interference competition.  Out of all these complications, there comes that “Aha!” moment when it all makes sense—just like the feeling one gets from the Luria and Delbrück experiment.

Second, there’s been a boom in the study of the evolution of social behaviors using microbes over the last 15 years or so.  The current phase began with papers by Paul Tuner and Lin Chao on interactions among viruses infecting the same cell leading to a Prisoner’s Dilemma (Nature, 1999); by Greg Velicer, Lee Kroos, and myself on cheating during multicellular fruiting-body development in the bacterium Myxococcus xanthus (Nature, 2000); and by Joan Strassmann, Yong Zhu, and David Queller on cooperation and cheating in aggregations of the social amoeba Dictyostelium discoideum (Nature, 2000).  Today, there are many groups around the world who study quorum sensing, fruiting-body formation, biofilms, toxin degradation, and other microbial behaviors from an evolutionary perspective.  The 1981 paper by Chao and Levin showed that microbial systems could serve as model systems for studying social evolution while being fascinating in their own right.  (It’s also fitting to note that John Bonner, who pioneered the study of D. discoideum, served as the editor for Chao and Levin’s paper.)

Third, Bruce Levin was my postdoctoral mentor, and Lin Chao did his graduate work with Bruce.  Lin had moved on to a postdoc position before I joined the lab, but this paper was one of my formative exposures to the conceptual elegance and experimental power of using microbes to study population dynamics.  Lin and Bruce had also written two papers on the dynamics of interactions between bacteria and phage (Levin et al., 1977, Am. Nat.; Chao et al., 1977, Ecology), and those papers were the ones that first led me to write Bruce about the possibility of joining his group as a postdoc.

Finally, this paper provides a sobering reminder that we humans are not as special as we often imagine, even in warfare.  Mindless bacteria were killing each other billions of years before we came on the scene.  Perhaps we can use our minds to suppress the worst of our primal urges.

[ADDED 13 Sept. 2013] Lin Chao emailed me that “The inspiration of that work was a lecture that Bruce gave in his Pop Biology class at UMass where he discussed the limitations of Lotka Volterra equations for interference competition.  That sat in my mind for a couple of years until it became a real project.” So this must-read paper also provides a nice example of the productive interplay between teaching and research.

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