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.]