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EAST LANSING, MICHIGAN—When most biologists want to understand how evolu- tion unfolds, they look for clues in the fossil The Man Who record or the natural world. simply walks across his Michigan State Uni- versity lab to his freezers. There, stored in Bottled Evolution 4000 vials, are dating back to 1988. That was the year Lenski started a simple but radical experiment. He put samples of Richard Lenski’s 25-year experiment in bacterial evolution into a sugar solution, stop- shows no signs of running out of surprises about how pered the flasks, and waited to see what mutation and selection shape living things would happen. It was a study with no defi ned endpoint, so risky that he didn’t try very hard to get outside funding for it. After 25 years and 58,000 bacterial generations, Lenski’s bacteria are still growing, mutating, and evolving. They are proving as critical to understanding the workings of evolution as classic paleontology studies such as Stephen Jay Gould’s research on the pace of change in mollusks. Lenski’s humble E. coli have shown, among other things, how multiple small mutations can prepare the ground for a major change; how new species can arise and diverge; and that Gould was mistaken on November 24, 2013 when he claimed that, given a second chance, evolution would likely take a completely different course. Most recently, the colonies have demonstrated that, contrary to what many biologists thought, evolution never comes to a stop, even in an unchanging environment. The work is “an

absolutely magnifi cent achievement,” says www.sciencemag.org Douglas Futuyma, an evolutionary biologist at Stony Brook University in New York. Other researchers have done experimental evolution, setting up populations of insects, yeast, and even fish in the lab and in controlled fi eld conditions, and subjecting the organisms to a particular environmental stress for relatively short periods. But Downloaded from Lenski’s long-term experiment “is just orders of magnitude beyond what anyone else has done,” Futuyma says. The project’s quarter-century has witnessed the rise of bioinformatics and the birth of whole-genome sequencing, and Lenski has taken advantage of both technologies to glean new insights. Generations of students have tended and analyzed the microbes, and the project sparked a memorable conflict between Lenski and creationists. Fifteen years ago, he almost abandoned it for digital models of evolution, then reconsidered—and was vindicated when his bacteria took one of their most dramatic evolutionary leaps. As

Time traveler. To turn back evolution’s clock,

Richard Lenski dips into his freezer. (2009) UNIVERSITY STATE G. L. KOHUTH/MICHIGAN CREDIT:

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Scott Edwards, an evolutionary biologist at to describe,” explains Lenski postdoc Noah opportunity to look at before,” says Graham Harvard University, said at a June evolution Ribeck, a physicist. Bell, an evolutionary biologist at McGill meeting,* “the principles of evolution In 1988, Lenski placed identical E. coli University in Montreal, Canada. that Rich has uncovered have touched all populations into 12 fl asks fi lled with a liquid By thawing out frozen vials from of us and caused us to look at evolution in containing nutrients and 25 milligrams generations past, Lenski can go back in a new way.” of glucose per liter. Every 24 hours, with time to look at intermediate stages of constant stirring and a comfortable 37°C evolutionary change. “It’s like the perfect Hard data environment, the bacteria multiplied fossil record,” says Douglas Emlen, an Lenski, 57, has been a MacArthur Fellow, explosively, depleting the sugar. Each day, evolutionary biologist at the University of a member of several editorial boards, and a 1% of the bacteria were transferred to a new Montana in Missoula. “They can pinpoint society president; he belongs to the National glucose-laden fl ask. Every 75 days—about exactly when [a trait] arose.” And Lenski can Academy of Sciences and served on commit- 500 generations for E. coli—Lenski’s team restart the experiment with those ancestral tees that evaluated genetically froze some of the bacteria for bacteria to see if history repeats itself. “A modified organisms and the Online future studies and as backups investigation of the 2001 anthrax sciencemag.org should the experiment become attacks. In August, he became Podcast interview contaminated. This protocol has an avid tweeter and started his with Richard Lenski continued day in and day out, own blog, Telliamed Revisited, (http://scim.ag/pod_6160a). weekends and holidays, virtually named after an 18th century uninterrupted for 25 years. (The book that stressed the importance of under- cultures were temporarily frozen during standing the world through observations and one winter break when the campus was not religious dogma. He has collected old sci- deserted and when Lenski moved his lab entifi c texts and baseball statistics, although from California to Michigan.) these days, he says, “my granddaughter is my Setting up the protocol and current hobby.” sticking with it “took a certain spirit But one thing will always capture his and vision,” Levin says. Early on, attention. Show up at his door with new data, skeptics argued that evolution in and he’s “like a 5-year-old at Christmas,” bacteria, particularly bacteria growing says graduate student Michael Wiser. That hunger for hard data about evolution was what drew him to bacteria in the fi rst place. Lenski never had a course in . As a graduate student at the University of North Carolina, Chapel Hill, he studied the ecology of ground beetles—work he found interesting but limited. “[I]t was diffi cult to imagine feasible experiments that would really test the scientific ideas that most excited me,” he wrote in an August blog. For his postdoctoral training in 1982, he switched gears and joined the lab of Bruce Levin, one of the few researchers doing experimental evolution in microbes. They studied the Evolution on display. A mutation led to a population boom in one of 12 cultures, turning the medium interactions between bacteria and viruses turbid (third fl ask from left). Investigators can compare two strains’ fi tness by culturing them together called bacteriophages. But their systems and then counting colonies (petri dish). One strain carries a mutation that makes its colonies red. were still too complex to get at the question Lenski most wanted to answer: Was Gould right about evolution’s irreproducible nature, in a stable, comfortable environment, graduate student who is 22 today can study or would evolution often repeat itself if given wouldn’t reveal much about how the process populations that come from before they a second chance? unfolds in nature. Yet, as they monitored their were born and use techniques that haven’t So after starting his own lab at the cultures, Lenski and his colleagues saw what existed before,” says Christopher Marx, a University of California, Irvine, Lenski used looked very much like an accelerated process microbiologist at Harvard University and a some of his own money, from a National of evolutionary change. By using bacteria former Lenski postdoc. Science Foundation (NSF) Presidential instead of a slower-growing organism, For the first decade, things hummed Young Investigator Award, to set up a much Lenski compressed time: The E. coli go along. The investigators monitored the simpler system using just bacteria. It was through 6.6 generations in a day, compared bacteria’s fi tness by measuring how fast they “like a physics experiment [where] you try with more than a year for the same number of could multiply relative to their ancestors. At to strip [out] all the complications so you generations in mice. In 25 years, he’s seen the fi rst, their fi tness rapidly increased, but then can isolate the phenomenon that you want equivalent of a million years of evolution in the improvement slowed. Over the whole humans. With that span, “you can examine a experiment to date, fi tness has improved by

CREDITS: BRIAN BAER AND NEERJA HAJELA/MSU (2) *Evolution 2013, Snowbird, Utah, 21 to 25 June. whole range of ideas that there hasn’t been an an average of 70%—meaning the most recent

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descendants undergo 1.7 doublings in the time it took the original microbes to double once. All 12 lines improved by about the same Leenski’snski’s LONG-TERMLONG-TERM amount, showing that, broadly speaking, evolution is reproducible (Science, 25 June EEvolutionvolution EExperimentxperiment 1999, p. 2108). But some lines improved faster than others, and genetic analyses Twelve batches of bacteria, replicating and evolving for 25 years, of the strains showed that they had taken yield some pretty big numbers. different evolutionary paths. For example, six lines developed defects in DNA repair, Lenski’s experiment has and instead of dying out, began sustaining * been running for more than higher mutation rates than their counterparts. 58,000 GENERATIONS “Historically, which mutations arise early changes the subsequent evolution,” explains (*as of June 2013) 25 GENERATIONS Janette Boughman, an evolutionary biologist PER years at Michigan State University. “But you really DAY 6.6 at an get to the same endpoint”: better fi tness in ESTIMATED COST of sugar solutions. Other striking differences emerged 14 $ among the fl asks. At generation 6500, about ROUGH NUMBER OF 4 MILLION 3 years into the experiment, two types 10 BACTERIAL CELLS of E. coli evolved in one of the fl asks: one that made small colonies consisting of Workforce involvement relatively small cells and one that made large REPLICATE equals about colonies, with large cells. Lenski expected POPULATIONS 12 PERSON that eventually one type would take over and 75 YEARS the other would disappear, or both would be All started with identical E. coli, but are now all different ousted by a bacterium with an even more PARTICIPATING benefi cial mutation. But to his surprise, both GRADUATE STUDENTS types have persisted, creating an ecosystem The number of 30 AND POSTDOCS in which competition and other interactions FROZEN VIALS >4000 between the colony types allowed both to that hold ancestral and evolved bacteria OUTSIDE COLLABORATORS be viable. “He created his own Galápagos Islands,” Marx says. LIQUID MEDIA FREEZERS 40 >10,000 6 Going digital LITERS Having done so, Lenski almost gave up on the >50 PUBLICATIONS fl ask experiment. He had caught a glimpse of an experimental evolution system that prom- ised to be even simpler and faster. “He’s a shiny object researcher,” says former student replicated thousands of times faster than his Lenski’s fascination with digital life Paul Turner, now an evolutionary biologist at microbes, and their “mutations” could be hasn’t faded. In 2010, he, Ofria, and others Yale University. “If there’s something interest- tracked in more detail. For the next 6 years, got funding for BEACON, an NSF Center ing to pursue, he will try to pursue it.” Dur- he focused as much on digital evolution as for the Study of Evolution in Action, which ing a squash game, a physics colleague invited on bacteria. includes 400 investigators and students from Lenski to a seminar by California Institute of In 2003, he teamed up with Ofria and fi ve U.S. universities. They collaborate on Technology physicist Christoph Adami. With Adami, both of whom are now at Michigan studies of evolution as it occurs in both real his graduate student Charles Ofria, Adami State, and with Robert T. Pennock, a and digital organisms. had developed software that allowed self- philosopher there, to follow mutation by Fortunately, he did not abandon his fi rst replicating computer programs, aka digital mutation how computer programs that initially love. The weekend that Lenski discovered organisms, to compete with one another for could do no more than replicate evolved the digital organisms, he had told his wife he processing power and evolve new functions. ability to perform complex operations, such might shut down the long-term experiment. Lenski was entranced. “One of the fi rst data as checking whether one numerical string Only her nudging convinced him to keep it slides [Adami] put up looked like data from equals another. They found that many earlier going—and in January 2003, the value of my long-term experiment,” he recalls. mutations—some of them deleterious in the doing so became clear, when the bacteria In 1998, Adami sent Lenski an advance short term—had to accumulate before a fi nal served up yet another surprise. “Digital copy of his book Introduction to Artifi cial “enabling” mutation conferred the new trait. organisms have the advantage that you Life (Science, 8 May 1998, p. 849). Lenski The work demonstrated that complex traits, can get complete information about pretty stayed up for 2 nights straight reading about such as the vertebrate eye, likely come about much all aspects of an experiment,” Lenski the program and trying out simulations he through a series of intermediate steps that explains. “The bacteria, though, are a lot

had designed himself. The digital organisms open the way for future adaptation. more complex and so they have a lot more R. LENSKI SOURCE: DATA

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tricks up their sleeves, a lot more potential that went viral. “He immediately became a glucose, and melding the citrate users with the to evolve in ways that one cannot anticipate.” rock star of science,” Adami says. parent strain produces a less fi t hybrid, Lenski One morning, Lenski and his colleagues Meanwhile, the citrate work continued. reported at the June evolution meeting. noticed that the medium in one flask had The cost of sequencing microbial genomes grown turbid, a sign that it was unusually thick dropped dramatically, enabling Blount to Evolving endlessly with bacteria. They suspected contamination look at the specifi c genetic changes involved. Lenski no longer thinks about ending his but could not confirm it, so they dug out He found that citrate users had undergone experiment. “It’s become more and more the most recent frozen sample of Ara-3, as a duplication of a 2933-base piece of DNA apparent that it’s not good to just do short- that population was called, and restarted it. that activated an otherwise silent gene coding term experiments,” he says. “Any micro- Three weeks later, the turbidity reappeared. for a citrate transporter. Subsequent genetic biology lab with enough people should be This time, they thoroughly tested the culture changes then tuned up the effi ciency of that thinking about doing a 20-year experiment.” and ruled out contamination. By growing transporter, he and Lenski reported in 2012. Funders agree. The project, once rejected by the Ara-3 bacteria on different the National Institutes of Health, types of media, they discovered now has ongoing NSF support: that the bacteria in that fl ask had a 10-year Long Term Research evolved a new way to nourish in Environmental Biology grant. themselves. Instead of relying And it is continuing to pay off, on scarce glucose, they drew on Lenski, Wiser, Ribeck, and their a different energy source in their colleagues report online this medium, citrate, which enabled week in Science (http://scim.ag/ them to reach much higher MWiser). Researchers have long densities than in other flasks. assumed that when organisms “This was the biggest event in encounter a new environment, the entire E. coli experiment,” they will adapt very quickly at Adami says. “To have a complex fi rst, then, as long as conditions new function develop seemingly are stable, ultimately reach an from scratch is a big deal and adapted state. At that point— quite remarkable.” a “fitness peak”—adaptive It fell to graduate student evolution should virtually Zachary Blount to figure out stop. Wiser tested the fi tness of what happened. Was a single all 12 populations at 41 time mutation responsible? And if so, points during their evolution, why hadn’t this ability appeared traveling back in time with earlier, since the bacteria had the frozen samples. At 10,000 already had plenty of time to generations—about 5 years— experience mutations in every it seemed the bacteria were possible gene? Or were multiple reaching that fi tness peak. But mutations required, and if so, now, after 50,000 generations, when and in what order did the improvement has slowed they arise? To find answers, down but not leveled off as Blount rewound the tape of life, expected, Lenski’s team reports. unfreezing earlier generations “The notion of a fi tness peak is and letting them evolve again in more elusive than I anticipated,” the same medium. Citrate use Pileup. Zachary Blount used all these petri dishes to study how one fl ask of bac- Lenski says. “I think fitness emerged in four of 72 cultures, teria evolved the ability to consume citrate. may well continue to increase always in ones derived from more for a million years.” Evolution recently frozen samples. That suggested These citrate users are enabling Blount, is endless, it seems, even in a stable that multiple mutations underlie the trait. now a postdoc in the lab, and Lenski to look environment. “That’s a profound insight,” Populations from those later generations had at another aspect of evolution: the formation Boughman says. already acquired the predisposing mutations of new species. The usual test of separate It’s also an encouraging portent for the and so could more readily take the next step. species is that they are unable to interbreed future of Lenski’s cultures, which are now Blount and Lenski’s results, published successfully, a criterion that can’t be applied to passing 58,500 generations. “If you asked in 2008, drew lots of attention, and not just bacteria because they don’t mate. But because me 20 years ago, I thought [the researchers] from the media and scientific community. one of E. coli’s defining characteristics is were running out of new things to learn,” On his website, Conservapedia, conservative the inability to use citrate for energy in the Levin says. Now, “I think they should go Christian Andrew Schlafly questioned the presence of oxygen, the citrate-consuming on indefi nitely.” Lenski agrees. During his conclusion that the bacteria had evolved this bacteria could be seen as a new species. And presidential address at the recent evolution striking new ability and requested Lenski’s they may even meet the traditional defi nition. meeting, he made a plea: “If you know original data. Lenski referred him to the Researchers can’t interbreed bacteria, but anyone who would like to endow a million- original paper, but Schlafl y persisted. Lenski they can mix the genomes of separate strains. year experiment, have them get in touch

CREDIT: BRIAN BAER/MSU CREDIT: responded with a sharply worded response Bacteria that thrive on citrate do poorly on with me.” –ELIZABETH PENNISI

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