Well, guess what just came in the mail today? Boxes upon boxes of frozen samples, and 12 tiny flasks sealed with parafilm and packed in bubble wrap. And a hand-written note from Jeff, with a picture enclosed. The note reads:
This LTEE of yours is just too much work. Day in and day out for almost a year, we’ve transferred the 12 lines to fresh medium, just like you said we should. But when we look at the cells under the microscope, they’re still just little bacteria – not even a decent yeast cell among them, much less a worm or something more interesting.
And all I have to show for it is a broken arm from doing all that pipetting, after everyone else quit. So, I’m sending back all those boxes and boxes of frozen samples that you foisted on sent us last year, along with the 12 lines as of when I last transferred them, maybe a week or two ago. I’m not sure of the exact number of generations, because we lost count a while back. But I’m pretty sure it started with a 7.
Good luck continuing this fool’s errand the LTEE back in your lab. Maybe you’ll eventually see something interesting, but I doubt it.
My wife, infant son, and I moved to Amherst the first weekend of April 1982. A beautiful snow fell on Sunday. Then, early on Monday morning, my new boss Bruce Levin cross-country skied by the old house we were renting, knocked on the door, and asked me when I’d be coming to the lab!
I had much to learn, of course. I remember learning how to use a pipettor from a technician in Bruce’s lab, and how exciting it was to estimate the number of cells in a flask (typically many millions or even billions). That estimation is done not by counting the cells directly, but instead involves precisely diluting small amounts through a series of test tubes, each tube containing a large, known volume of a sterile solution. At the end of the dilution series, one takes a tiny amount from the final tube and spreads it across an agar plate. The plate is then incubated for a day or so, during which time each of the few hundred cells that survived the dilutions grows into a separate colony. A colony is a clump of millions of cells that can be seen with the naked eye, unlike the individual cells that can be seen only by using a microscope. One counts the colonies on the plate and, using that number and the dilutions that one made, one can then back-calculate the density of cells in the original flask.
In my first effort at this most basic procedure, I did three replicates from the same flask. I was thrilled when I counted the colonies on the first two plates, and the numbers differed by only a few percent. The third plate, however, differed by perhaps a factor of two, which meant I had done something wrong—maybe I’d let an air bubble into the pipettor’s tip, displacing some of the liquid—and I realized the importance of attention to details.
A little later, while I was still learning the ropes, Bruce had me perform a more complicated experiment to measure the rate at which a certain virus, called T6, adsorbs to and infects E. coli cells. The experiment required a lot of repetitive dilutions and plating of samples that I had to process quickly and accurately. The basic idea is that free viruses should decline in number over time as more and more of them enter cells. (This decline continues only until the first viruses to infect cells have had enough time to produce the next generation of viruses, hence the need to process the samples quickly.) Alas, my experiment was a total failure. What was I doing wrong? I think Bruce had me repeat the experiment, with the same lousy outcome. Though he never said it, perhaps he would regret hiring me. After all, given my lack of experience, Bruce had also taken a leap of faith.
Oh, and what about my experiment to look for evolutionary changes that compensated for the cost of bacterial resistance to infection by viruses? That’s what I had proposed in my letter to Bruce asking about a postdoc. I never got to that experiment while I was in Bruce’s lab. However, it provided the seed for a project that I eventually conducted as an early-career faculty member at the University of California, Irvine.
[Bacterial colonies growing on agar plates. Photo credit: Brian Baer, MSU.]
I did my graduate work at the University of North Carolina, Chapel Hill, in what was then called the Department of Zoology. I had several important and formative experiences during those years: clear advice from my advisor, Nelson Hairston (1917-2008), about the value of well-designed experiments in ecology; an eye-opening course on the integration of ecological and evolutionary perspectives, taught by Janis Antonovics (then at Duke University, just a few miles from UNC); an abysmal failure in my own attempt at an experiment with praying mantises; an enlightening collaboration with a fellow grad student, Phil Service; and a dissertation project on the effects of forest clearcutting and competition on beetles in the mountains of southwestern North Carolina.
Although that dissertation project was reasonably successful, I realized it was not a good fit to my skills and interests. Many of my fellow students were excellent naturalists with a love for the organisms they studied. While I enjoy being outdoors, I’m not a naturalist. Instead, I’m intrigued by the conceptual questions that biologists ask about the living world. And as my graduate work moved forward, I realized that questions about evolution, including especially the mechanisms and dynamics of evolution, interested me most. However, the beetles I was studying were not well-suited to those questions. So how could I pursue my interests?
While we were finishing our doctoral projects, Phil and I spent a lot of time discussing potential systems for studying evolution. As he moved on in his career, Phil chose to study evolution using fruit flies, a long-standing model system for studying genetics. I recalled an undergrad course I had taken, where we learned about elegant experiments done with microbes, including one by Salvador Luria and Max Delbrück that showed mutations happen at random, not in response to selection.
Phages are viruses that infect bacteria, and the paper provided an elegant demonstration of the interplay of ecological and evolutionary processes on a time scale of a few weeks. It documented the coevolution of E. coli and a virus, called T7, that can infect and kill the bacteria. The authors showed that the bacteria evolved resistance, then the virus evolved the ability to infect the resistant cells, and finally the bacteria evolved resistance to the viruses with the extended host range. Moreover, they showed that virus-sensitive and virus-resistant host genotypes coexisted because the sensitive types were better competitors for the limiting resources in the environment. That paper and others by Bruce Levin cemented my interest in using microbes to study evolution in action.
In March of 1981, about a year before I defended my dissertation, I wrote Bruce to ask if he would consider me for a postdoctoral position in his lab. I admitted I had no experience working with microbes, but I proposed an experiment. His team’s work showed that bacteria that evolved resistance to phage were outcompeted by their sensitive progenitors when those viruses were not present. I wondered whether the tradeoff was an unavoidable metabolic cost, or whether bacteria could evolve compensatory changes that reduced the cost of resistance. My proposed experiment suggested a way to look for such compensatory changes.
Bruce invited me to visit his lab and give a talk at the University of Massachusetts, Amherst, that spring. I remember him greeting me when I got off a bus at the town square and being surprised by just how young he looked. Although he was 40 years old and a full professor, Bruce could easily have passed for an undergrad. More importantly, I recall our intense discussions over the next two days with Bruce at a chalkboard, writing equations that described the growth of various interacting microbes, and using terms that I barely understood.
Despite my limited experience and knowledge of microbiology, Bruce offered me a postdoctoral position in his lab. I was thrilled, but also worried about doing research in a new field where I lacked experience and knowledge. Nonetheless, I took that leap of faith. And I’m so glad I did.
[Nelson Hairston after his retirement from UNC (left) and Bruce Levin in the mid-1980s (right).]
I wrote about Stephen Jay Gould’s book, Wonderful Life, in a previous post. While that book wasn’t an inspiration for starting the LTEE, I often quote passages from it when I give talks on the LTEE, because those passages frame the big-picture question about the repeatability of evolution.
I first heard Gould speak at a multi-day conference in Irvine, California, in 1994. The conference was on Tempo and Mode in Evolution, with the talks celebrating and building upon the ideas in a landmark 1944 book of that same title written by the paleontologist George Gaylord Simpson (1902-1984).
Gould’s talk began with several pictures of dramatic newspaper headlines that read something like these: Darwin Hammered, Darwin Rejected, and Darwin Trounced Yet Again. I remember nodding in agreement about the lack of respect for Charles Darwin and his ideas in the press, and I’m sure many of the others in attendance did as well.
But then Gould turned the tables to reveal his sly humor. These were all headlines from the sports section of Boston newspapers about ill-fated outings by Danny Darwin, who pitched for the Red Sox. Gould was not only an expert on fossils; he was an aficionado of baseball as well. In fact, he wrote many interesting and scientifically minded essays about baseball including, for example, a memorable piece on the extinction of the .400 hitter in his book Full House. (And see this interview with Gould on that subject.)
I had hoped to meet Gould at this meeting, or at least I hoped he might hear me speak when I gave a talk about the LTEE. (Here’s a link to the paper that I covered in my talk.) Alas, Gould gave his talk and then left the conference before my talk, and before I could meet him.
Luckily, though, I met Gould when he came to MSU, first as a commencement speaker in 1999, and then in 2000 when he gave a public lecture here. On that second visit, I served as one of his hosts. When I picked Gould up at the airport, I brought along two Lansing Lugnuts caps. The Lugnuts are a local minor-league baseball team. I explained to Gould that I’d have liked to take him to a Lugnuts game, but the season had ended before his visit. I gave him one of the caps, and I asked if would autograph the other cap as a souvenir for me.
Gould hesitated for a moment. He explained he had been asked to autograph books by Darwin and others. He would sign books that he had authored, but nothing else. When he looked at the Lugnuts cap, however, he realized this was a different kind of request. And so, he signed it: “To the bacterial allstars, Stephen Jay Gould.” Now that’s a souvenir!
Gould and I also had the chance to have a meal together, just the two of us. We discussed our shared interest in the repeatability of evolution, and how our disparate study systems—fossils and flasks—could shed light on that fascinating question.
Sadly, Gould died just two years later. However, he managed to complete a massive volume, The Structure of Evolutionary Theory, shortly before his death. That 1400-page tome included a recounting of the history of evolutionary thought—informed by Gould’s collection of rare old books—as well as a synthesis of modern research in evolutionary biology from his perspective. I was pleased and honored that he discussed the LTEE at several places in that book.
I’ve sometimes been asked whether the idea of the LTEE was inspired by Stephen Jay Gould’s book, Wonderful Life. In this bestseller, Gould put forward the idea of “replaying” evolution to explore the idea of whether evolution is repeatable. He wrote (page 48): “I call this experiment ‘replaying life’s tape.’ You press the rewind button and, making sure you thoroughly erase everything that actually happened, go back to any time and place in the past—say, to the seas of the Burgess Shale. Then let the tape run again and see if the repetition looks at all like the original.” However, Gould then went on to say: “The bad news is we can’t possibly perform the experiment.”
Gould (1941-2002) was a paleontologist as well as an historian of science and prolific author, and he had in mind replaying life’s tape on a planetary scale over millions of years. The Burgess Shale is a geological formation in western Canada that contains fossils from about 500 million years ago. The fossils include exceptionally well-preserved early animals, many of which have body plans that are unlike any modern animals. Building on his thought experiment of replaying life’s tape, Gould pondered the potential outcomes: “If each replay strongly resembles life’s actual pathway, then we must conclude that what really happened pretty much had to occur. But suppose that the experimental versions all yield sensible results strikingly different from the actual history of life? What could we then say about the predictability of self-conscious intelligence? or of mammals?”
Of course, Gould’s experiment is impossible at a paleo-planetary scale. But at a more modest scale, one of the main goals of the LTEE is to study the repeatability of evolution. And so, I often quote from Wonderful Life when I’m giving talks about the experiment. Thus, it’s only natural that someone might wonder if Gould’s book had inspired me to start the LTEE.
So, while Gould and I were thinking about similar issues, we were imagining them at vastly different scales. It’s one of the fascinating aspects of evolution that these broad categories of causality—adaptation by natural selection, chance events from mutations to asteroid impacts, and the effects of past history on future opportunities—play out at these different scales.
I was lucky to meet Gould and discuss these issues with him several years later, as I’ll describe in a future post.
Back in February, I wrote about our plan to move the LTEE from here at MSU to Jeff Barrick’s lab at UT-Austin later this year.
Well, Jeff told me he’s had a change of heart. When he explained to his lab team that the lines required daily transfers that included weekends and holidays, everyone went totally berserk and threatened to resign then and there. And if that happened, Jeff would have to do all the transfers himself, 365 days a year.
Jeff is a hard worker … or at least he used to be. But he’s a professor now. Like me, I’ll bet he doesn’t even know where the pipette tips and clean flasks are stored in his lab, much less how to make the culture medium from all those jars of chemicals with strange names.
With that painful possibility in mind, Jeff called me up, and he said we’d have to keep the LTEE going here. I was kind of annoyed because I was in the middle of doing Wordle, and for some reason it wouldn’t accept “ecoli” as a guess. But despite all that, I said ok to Jeff.
So yesterday, when I told the people in my lab that we’d have to keep doing the transfers until I found another sucker lab to take over, everyone here went totally berserk and threatened to resign on the spot. To calm everyone down, I had to promise that today we’d stop the LTEE, empty out the freezers, and autoclave all of the samples.
Over the decades, several lines fell behind others due to cross-contamination (or concerns about the possibility), which we detected by examining the alternating Arabinose marker and seeing the resulting colony colors on TA plates. Those lines were then restarted from whole-population samples, but they would be 500 generations behind the others (or a multiple of 500 generations behind in some cases).
The picture above shows red and white colonies growing on TA agar in a Petri dish. The red colonies cannot grow on the sugar arabinose that is part of the TA medium, while the white ones can use arabinose. Half of the LTEE lines started from red colonies (Ara–1 to Ara–6), and half started from white colonies (Ara+1 to Ara+6). We alternate the red and white lines each day during their propagation. That way, if cross-contamination occurs, we can detect it by the presence of bacteria that make colonies that are the wrong color. We check colonies before every periodic freeze of the LTEE. These days, with DNA sequencing, we can also use derived mutations that are unique to each lineage to check whether a putative contamination event is real or not. (Indeed, in some populations, especially those that evolved hypermutability, the colony markers don’t work like they did when the LTEE started.) If we confirm that a cross-contamination event has occurred, we restart the affected population from the last frozen sample of that population.
So today, Devin Lake will propagate the last two lagging populations. Our lab will continue to propagate them until they, too, reach 75,000 generations. The last one should reach that goal in late May.