Is the LTEE breaking bad?

Michael Behe has written a third book, Darwin Devolves, that continues his quixotic effort to overturn evolutionary biology. Nathan Lents, Joshua Swamidass, and I wrote a book review for Science. (You can find an open-access copy here.) As our short review states, there are indeed many examples of evolution in which genes and their functions have been degraded, sometimes conferring an advantage to the organism. However, Behe’s book largely ignores the ways by which evolution generates new functions. That’s a severe problem because Behe uses the evidence for the ease of gene degradation to support his claim that our current understanding of the mechanisms of evolution is inadequate.

This is my third in a series of posts delving into various issues where I think Behe’s logic and evidence are weak. These weaknesses undermine his position that the known mechanisms of evolution are inadequate to explain life as we see it in the fossil record and in the diversity of living species. Let me be clear: there is still much to learn about the intricacies of how evolution works, both in terms of a better understanding of the general mechanisms and unraveling all the fascinating particulars of what happened along various lineages. However, I don’t see much chance of future research upending the central role of natural selection—operating over vast time along with mutation, drift, and recombination (including various forms of horizontal gene transfer)—in creating new functions that spark the diversification of life. By contrast, Behe accepts that natural selection occurs, but he treats it almost entirely as a degradative process that weakens and destroys functions. To explain all the new functions that have arisen during evolution (and he accepts the fact that evolution has occurred for billions of years), Behe appeals to an “intelligent agent” who somehow, mysteriously has added new genetic information into evolving lineages.

In my first post, I explained why Behe’s “first rule of adaptive evolution” doesn’t imply what he says it does about evolution writ large. In particular, his overarching thesis confuses frequency over the short run with lasting impact over the long haul of evolution. In my second post, and building on the work of others, I examined a specific case involving polar bears, which Behe argued showed adaptations resulting from degradative evolution. He apparently regarded the case as so compelling that he used it as the lead example in his book, but a careful review of the science suggests an alternative explanation, in which gene function actually improved.

In this post, I examine Behe’s interpretation of findings from a long-term evolution experiment (LTEE) with E. coli bacteria that has been running in my lab for over 30 years. In short, the LTEE represents an ideal system in which to observe degradative evolution, and indeed we’ve seen examples of such changes. However, Behe overstates his case by downplaying or dismissing evidence that runs counter to his thesis.

III. Evolution of functionality in the LTEE

Recall what Behe calls “the first rule of adaptive evolution: break or blunt any functional gene whose loss would increase the number of a species’ offspring.” In support of that rule, Darwin Devolves pays considerable attention to the LTEE. Behe skillfully uses it to build his case that unguided evolution produces adaptations (almost) exclusively by breaking or blunting functional genes. The implication is that constructive adaptations—those that do not involve breaking or blunting genes—require an “intelligent agent” who has introduced new genetic information, by some mysterious process, into certain lineages over the course of life’s history.

Am I surprised that Behe uses the LTEE as one of the centerpieces of Darwin Devolves? No, not at all. Does the LTEE provide strong support for his argument? No, it does not. The LTEE fits the bill for Behe because it’s just about the best case possible to showcase his rule. But just as loss of sight in cave-dwelling organisms is a special case that won’t tell us how eyes evolved, one must be careful when extrapolating from this experiment to evolution writ large. (I say this even though the LTEE is my scientific “baby” and has been a useful model system for studying some aspects of evolution.)

The LTEE was designed (intelligently, in my opinion!) to be extremely simple in order to address some basic questions about the dynamics and repeatability of evolution, while minimizing complications. It was not intended to mimic the complexities of nature, nor was it meant to be a test-bed for the evolution of new functions. The environment in which the bacteria grow is extremely simple. The temperature is kept constant at 37C, the same as our colons where many E. coli live. The LTEE “host” is an Erlenmeyer flask, not an animal with an immune system and other defenses. There are no antibiotics present, no competing species, and no viruses that plague bacteria in nature. And the culture medium contains a single source of energy that the ancestral bacteria can use, namely the sugar glucose. In contrast, E. coli lineages have endured and adapted over millions of years to countless combinations of resources, competitors, predators, toxins, and temperatures in nature.

Indeed, the LTEE environment is so extremely simple that one might reasonably expect the bacteria would evolve by breaking many existing functions. That is because the cells could, without consequence, lose their abilities to exploit resources not present in the flasks, lose their defenses against absent predators and competitors, and lose their capacities to withstand no-longer-relevant extreme temperatures, bile salts, antibiotics, and more. The bacteria might even gain some advantage by losing these functions, if doing so saved time, energy, or materials that the cells could better use to exploit the limited glucose supply.

And just as one would expect, the bacteria have diminished or lost various abilities during the LTEE. For example, all 12 populations lost the ability to use another sugar, called ribose, and they gained a small but measurable competitive advantage as a result. Similarly, half of the lines evolved defects in one or another of their DNA repair systems, which led to hypermutability. While hypermutability resulted from a loss of function at the molecular level, it produced a slight gain in terms of the rate at which those lineages adapted to their new laboratory environment. There are undoubtedly many functional losses that have occurred during the LTEE, some that have been described and others not.

If that was all there were to the story, I might say that Behe’s portrayal was correct, but that he had missed the point—namely, that of course evolution often involves the loss of functions that are no longer useful to the organism. Biologists have known and understood this since Darwin.

But there is more to evolution than that, not only in nature but, as it turns out, even in the simple world of the LTEE. We’ve discovered cases where beneficial mutations evolved in genes that encode proteins that are essential, not dispensable, including ones involved in synthesis of the cell envelope and in structuring DNA so that it can be copied, transcribed, and packed into the tiny space of a cell. We’ve also found genes in which mutations occur repeatedly near key interfaces of the encoded proteins, in ways that imply the fine-tuning of protein functions to the LTEE environment, rather than degradation or loss of those functions.

In Darwin Devolves, Behe asserts (p. 344) that “it’s very likely that all of the identified beneficial mutations worked by degrading or outright breaking the respective ancestor genes.” He includes a footnote that acknowledges our work that suggests the fine-tuning of some protein functions, but there he writes (p. 609): “More recent investigation by Lenski’s lab suggests that mutations in a small minority (10 of 57) of selected E. coli genes may not completely break them but rather, as they put it, ‘fine-tune’ them (probably by degrading their functions).” Why does Behe assert that fine-tuning of genes occurred “probably by degrading their functions”?

Perhaps it’s because this assertion supports his claim, but more charitably I suspect the underlying reason is similar to the problematic inferences that got Behe into trouble in the case of the polar bear’s genes. That is, if one assumes the ancestral state of a gene is perfect, then there’s no room for improvement in its function, and the only possible functional changes are degradative. In my post on the polar bear case, I explained why the assumption that a gene is perfect (or nearly so) makes sense in certain situations. However, that assumption breaks down when an organism encounters a new environment, where the optimal state of a protein might differ from what it was before. Perhaps, for example, a mutation that would have slightly reduced an essential protein’s activity in the ancestral environment slightly improves its activity in the new environment. As I explained earlier, the LTEE environment differs from the conditions that E. coli experienced before being brought into the lab. It would be surprising if some proteins couldn’t be fine-tuned such that their activities were improved under the particular pH, temperature, osmolarity, and other conditions of the LTEE. It is unreasonable to simply assume that fine-tuning mutations “probably” degrade functions when evolving populations—whether of bacteria or bears—encounter new conditions.

The adaptation in the LTEE that has garnered the most public attention, though, is far less subtle. (The attention grew enormously after I had an email exchange with Andrew Schlafly, who runs the “Conservapedia” website.) After more than 30,000 generations, one of the 12 lines evolved the ability to consume citrate in an oxygen-rich environment—something that E. coli normally cannot do. Citrate, it turns out, has been a potential source of carbon and energy in the culture medium ever since the LTEE started. (The citrate is there, despite the inability of E. coli to import it from the medium, because it chelates iron and, in so doing, makes that micronutrient available to the cells.)

Sequencing the genomes of the citrate-using lineage revealed an unusual mutation—a physical rearrangement that brought together regulatory and protein-coding sequences in a new way—and genetic experiments demonstrated that mutation was responsible for this gain of function. In the line that gained the ability to consume citrate, the rearrangement involved duplicating a particular DNA segment; additional experiments showed that other types of rearrangements could also generate this ability. Even now, after more than 70,000 generations, none of the other LTEE populations has managed to evolve this new ability, despite its great benefit to the bacteria. This difficulty reflects several factors: (i) the low rate of occurrence of the necessary rearrangement mutations; (ii) the fact that efficient use of citrate requires certain additional mutations; and (iii) the absence of other, more highly beneficial mutations that could out-compete early, weakly beneficial citrate-using mutants.

To his credit, Behe does write about the lineage that evolved the ability to consume the citrate. However, he dismisses it as a “sideshow” (p. 365), because he refuses to call this new capability a gain of function. Instead, Behe writes (p. 362) that under his self-fulfilling scheme “the mutation would be counted as modification-of-function—because no new functional coded element was gained or lost, just copied.” In other words, Behe won’t count any newly evolved function as a gain of function unless some entirely new gene or control region “poofs” into existence.

But that’s not how evolution works—unless you believe, as Behe apparently does, that God or some other “intelligent agent” intervened to insert new genetic information into various lineages during the course of history. (Suffice it to say that I don’t regard this as a scientifically useful hypothesis, because I don’t think it can be tested.) Evolutionary biology doesn’t require that new genes poof into existence. Instead, old genes and their products are coopted, modified, and used in new ways—a process called exaptation. For example, crystallin proteins in the lenses of our eyes derive from proteins that performed other functions. At a larger physical scale, the wings of birds and bats derive from the forelimbs of their four-legged ancestors, which in turn derive from fins of fishes.

In short, Darwin Devolves presents a biased picture of the LTEE’s findings. Behe is overly confident in asserting that the vast majority of beneficial mutations have degraded functions, when the functional effects of most of these mutations have not been measured under relevant conditions. In any case, the experiment was designed to address issues other than molecular functionality, with the environment deliberated constructed to be as simple as possible. And yet, having closed the door on nearly all opportunities for new functions to evolve, a striking example arose in a tiny flask after a mere decade or two.

[This image shows some of the LTEE populations in their flasks. The one in the center is more turbid because the bacteria have reached a higher density after they evolved the ability to consume citrate in the culture medium.  Photo credit: Brian Baer and Neerja Hajela.]

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A Birthday Sonnet

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

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

 

ODE TO AN LTEE LINEAGE

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

Thou start more humbly, but sure potentiate.

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

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

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

And how was its controlled complex “skin” pinned?

Promoter capture, over some time refined.

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

But thy Cit-minus partner shall not fade

Nor gain possession of the flair of most

C₄ shall Cit^– snag, now spawned by carbon trade

Then on it turns ‘til lines will species now boast

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

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

 

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?”

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