On damaged genes and polar bears

Michael Behe has a new book called Darwin Devolves, published by HarperOne. Nathan Lents, Joshua Swamidass, and I wrote a review of that book for the journal Science. (You can find an open-access version of our review here.) As our review says (in agreement with Behe), there are many examples of evolution in which genes and their functions have been degraded, sometimes yielding an advantage to the organism. Unfortunately, though, Behe largely ignores the ways that evolution generates new functions and thereby produces complexity. That’s a severe problem because Behe uses the evidence for the ease of gene degradation to support his overarching implication that our current understanding of the mechanisms of evolution is inadequate and, consequently, the field of evolutionary biology has a “big problem” and is therefore in scientific trouble.

I hope to accomplish several things in a series of posts. (I initially planned to write three posts, but it will now be more than that, as I delve deeper into several issues.) In my first post, I explained why Behe’s so-called “first rule of adaptive evolution” does not imply what he says it does about evolution writ large. In summarizing, I wrote that Behe is right that mutations that break or blunt a gene can be adaptive. And he’s right that, when such mutations are adaptive, they are easy to come by. But Behe is wrong when he implies these facts present a problem, because his thesis confuses frequencies over the short run with lasting impacts over the long haul of evolution.

In this post, I take a closer look at Behe’s “rule” and how one might decide whether or not a particular mutation is damaging to a particular gene in a particular context. I’ll then describe and discuss the example that Behe chose to illustrate his argument at the outset of his book, calling attention to the fact that his inferences were indirect, and as a result a key conclusion was quite possibly wrong. [These issues came to my attention based on work by Nathan Lents, Art Hunt and Joshua Swamidass. They voiced concerns about this example on their own blogs, here and here. I’ve now done my own reading, and in this post I attempt to provide just a tiny bit of important technical background before addressing the main concern, as I see it.]

II-A. How does one know if a mutation has damaged a gene?

Behe’s first rule of adaptive evolution says this: “Break or blunt any functional gene whose loss would increase the number of a species’ offspring.” Every biologist knows that many mutations break or reduce the functionality of genes and the products they encode. Every biologist also realizes that this can sometime increase an organism’s fitness (i.e., its survival and reproductive success), in particular when two conditions are met. First, the function has to be one that is not—or rather, no longer—useful to the organism. For example, eyes are no longer useful to an organism whose ancestors lived above ground, but which itself now lives in perpetual darkness in a cave. Second, there must be a meaningful cost to the organism (again, in the currency of fitness) of having the functional form of the gene, and that cost must be reduced or eliminated for the mutated version of the gene. This second point means that mutations that break or blunt a particular gene—even one that is useless—are not necessarily advantageous; they might instead be selectively neutral, such as when an encoded protein is still expressed but, for example, has diminished activity on a substrate that isn’t even present. Therefore, compelling evidence for a broken or blunted gene in a particular lineage suggests that the gene’s function is under what evolutionary biologists call “relaxed” selection—relaxed because some capability that was useful during the history of a lineage is no longer important under the organisms’ present circumstances. However, that does not mean that the loss or diminution of the capability necessarily provided any advantage; instead, the gene could have decayed by the random fixation of mutations that were entirely inconsequential for fitness.

Two very important issues center on (i) how an observer can tell whether a particular mutation breaks or blunts a gene; and (ii) how that observer can determine whether the resulting mutation is advantageous. In short, neither inference is ironclad without an in-depth case-by-case investigation, although there are shortcuts that biologists often take because they make sense and are often sound, provided one takes care to understand the potential limitations of the inference. To characterize the biochemical consequences of a mutation, for example, the gold standard would be to perform detailed analyses of the activities of proteins encoded by different forms (alleles) of the same gene. That’s difficult, technical work.

But as I said, there are shortcuts that allow scientists to draw reasonable inferences in some cases. For example, a mutation that generates a premature stop codon (a so-called “nonsense” mutation) usually eliminates the encoded protein’s function. However, there are exceptions, such as when the premature stop is very near the end of the gene. It’s also possible that a truncated protein might even have some new activity and function, or that it might accumulate additional mutations that produce a new activity. That’s unlikely in any one case, but a lot of unlikely things can happen over the vast scales of space and time over which evolution has operated. As the Nobel laureate François Jacob famously wrote years ago, “natural 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.”

At the other end of the spectrum with respect to inferred functionality, some mutations change the DNA sequence of a gene, but they have no affect on the resulting amino-acid sequence of a protein. That happens because the genetic code is redundant, with multiple codons for the same amino acid. Such mutations are called “synonymous” and they are generally presumed to be neutral precisely because they don’t change a protein. Once again, however, there are some exceptions to this usually reliable inference; a synonymous mutation could affect, for example, the rate at which the protein is produced and even its propensity to fold into a specific conformation.

In the middle ground between these (usually) clear-cut extremes are the cases where a mutation produces an amino-acid substitution in the encoded protein. Does that mutation change the protein’s activity? If it does, is it necessarily damaging to the protein and/or to the organism with that altered protein? Biochemical and structural studies of proteins have shed light on this issue by identifying so-called “active sites” of many proteins—positions in the structure of a protein molecule where it interacts with a substrate and facilitates a chemical reaction. Mutations in and around active sites are more likely to affect a protein’s activity than ones that are far away. Also, even at the same site in a protein, different mutations are likely to have more pronounced affects on the protein’s activity, depending on whether the substitution affects the charge and/or size of the amino acid at that site.

Computational biologists have developed tools that take into account these types of information, which can be used to draw tentative inferences or make predictions about the likely effect of a specific mutation. Not surprisingly, one application is for understanding possible health effects of genetic variation in humans. For example, are certain variants in some gene likely to affect an individual’s susceptibility to cardiovascular disease?

One such tool is called PolyPhen-2. The website says: “PolyPhen-2 (Polymorphism Phenotyping v2) is a software tool which predicts possible impact of amino acid substitutions on the structure and function of a human proteins using straightforward physical and comparative considerations.” In addition to using structural information described above, it also uses information on whether a given site is highly conserved (little or no variation) or quite variable across humans and related species for which we have information. Why does it use that information? In essence, the program assumes that evolution has optimized a given protein’s activity for whatever it does in humans, related species, and our common ancestors. If a particular site in a protein varies a lot, according to that implicit assumption, the variants probably aren’t harmful because, well, if they were, then those lineages would have died out. If a site is hardly variable at all, by contrast, it’s presumably because mutants at those sites damaged the protein’s important function and led to the demise of those unfortunate lineages.

All that makes a lot of good sense … provided the protein of interest is performing the same function, and with the same optimal activities, in everybody and every species used in the analysis. Let’s look now at a specific case that Behe chose to highlight in his book.

II-B. The APOB gene in polar bears

Behe sets the stage for his rule—“break or blunt any functional gene whose loss would increase the number of a species’ offspring”—by summarizing the results of a study by Shiping Liu and coauthors that compared the genomes of polar bears and brown bears. Their paper examined mutations that distinguish these two species. The authors identified a set of mutations that had accumulated along the branch leading to modern polar bears, and in a manner that was consistent with those changes having been beneficial to the polar bears. One of the mutated genes, which was discussed in some detail both by the paper’s authors and by Behe, is called APOB. As Liu et al. wrote (p. 789), the APOB gene encodes ApoB, “the primary lipid-binding protein of chylomicrons and low-density lipoproteins (LDL) … LDL cholesterol is a major risk factor for heart disease and is also known as ‘bad cholesterol.’ ApoB enables the transport of fat molecules in blood plasma and lymph and acts as a ligand for LDL receptors, facilitating the movement of molecules such as cholesterol into cells … The extreme signal of APOB selection implies an important role for this protein in the physiological adaptations of the polar bear.”

As part of their study, Liu et al. analyzed the polar-bear version of the APOB gene using the PolyPhen-2 computational tool described above. Roughly half the mutations in APOB were categorized by that program as “possibly damaging” or “probably damaging,” and the rest were called “benign.” Behe than concluded that some of the mutations had damaged the protein’s function, and that these mutations were beneficial in the environment where the polar bear now lives. In other words, Behe took this output as strong support for his rule.

So what’s the problem? The PolyPhen-2 program, as I explained, is designed to identify mutations that are likely to affect a protein’s structure and therefore its function. It assumes such mutations damage (rather than improve) a protein’s function because structurally similar mutations are rare in humans and other species used for comparison. It does so because it presumes that natural selection has optimized the protein to perform a specific function that is the same in all cases, so that changes must be either benign or damaging to the protein’s function. In fact, the only possible categorical outputs of the program are benign, possibly damaging, and probably damaging. The program simply cannot detect or suggest that a protein might have some improved activity or altered function.

The authors of the paper recognized these limiting assumptions and their implications for the evolution of polar bears. In fact, they specifically interpreted the APOB mutations as follows (p. 789): “… we find nine fixed missense mutations in the polar bear … Five of the nine cluster within the N-terminal βα1 domain of the APOB gene, although the region comprises only 22% of the protein … This domain encodes the surface region and contains the majority of functional domains for lipid transport. We suggest that the shift to a diet consisting predominantly of fatty acids in polar bears induced adaptive changes in APOB, which enabled the species to cope with high fatty acid intake by contributing to the effective clearance of cholesterol from the blood.” In a news piece about this research, one of the paper’s authors, Rasmus Nielsen, said: “The APOB variant in polar bears must be to do with the transport and storage of cholesterol … Perhaps it makes the process more efficient.” In other words, these mutations may not have damaged the protein at all, but quite possibly improved one of its activities, namely the clearance of cholesterol from the blood of a species that subsists on an extremely high-fat diet.

It appears Behe either overlooked or ignored the authors’ interpretation. Determining whether those authors or Behe are right would require in-depth studies of the biochemical properties of the protein variants, their activities in the polar bear circulatory stream, and their consequences for survival and reproductive success on the bear’s natural diet. That’s a tall order, and we’re unlikely to see such studies because of the technical and logistical challenges. The point is that many proteins, including ApoB, are complex entities that have multiple biochemical activities (ApoB binds multiple lipids), the level and importance of which may depend on both intrinsic (different tissues) and environmental (dietary) contexts. In this example, Behe seems to have been too eager and even determined to describe mutations as damaging a gene, even when the evidence suggests an alternative explanation.

[The picture below shows a polar bear feeding on a seal.  It was posted on Wikipedia by AWeith, and it is shown here under the indicated Creative Commons license.]

Reply to Michael Behe’s gentle comment

Michael Behe posted a kind, brief comment on my previous post. As I began to write my reply, I realized his comment and my reply would interest many readers, and hence this separate post.

Here is his comment, and my reply follows.

Good day, Mike (if I may): Thank you for your kind words. I do appreciate the fact that you remain upbeat about my lab’s research, and much other work that you describe in your writings, even though I disagree with the “big picture” that you take from the evolution literature.

I find it interesting and personally enjoyable (despite some frustrations as well) that evolution remains such a “hot” topic. That’s true scientifically, with many extraordinary discoveries in recent years—from fossils like Tiktaalik and Archaeopteryx [edit: this one was discovered long ago, but it’s better understood now] to the DNA-based evidence that Denisovans and Neanderthals contributed to the genomes of many of us living today. It’s also the case that evolution remains “hot” for many non-scientists, and that’s wonderful. Whether for secular or religious reasons, we humans are deeply interested in where we came from and how we came about. In my own small way, I take pleasure in knowing that my lab’s research helps people get a glimpse of how evolution works.

I’m concerned, though, when these scientific and religious perspectives get intertwined and confused, even when they concern those big, important questions that interest all of us. I get even more concerned when I see what I regard as non-scientific ideas (such as “intelligent agents” introducing “purposeful design” by unstated and untestable means) being used to undermine the admittedly imperfect (and always subject to revision) understanding of evolution that science provides to those who want to learn. And I am most disturbed when these confusions appear to be part of a deliberate “wedge” strategy with ulterior sociopolitical motives. People will undoubtedly have diverse views about whether scientific explanations are adequate and/or satisfying ways to understand the world, but I see danger in trying to undermine scientific methodology and reasoning to advance religious beliefs and political goals.

Asking for a Skeptic Friend

I sometimes get email from people asking, in one way or another, whether our long-term evolution experiment (LTEE) with E. coli provides evidence of evolution writ large – new species, new information, or something of that sort. I try to answer these questions by providing some examples of what we’ve seen change, and by putting the LTEE into context. Here’s one such email:

Hi Professor Lenski,

I have a quick question. I’m asking because I am having a discussion with someone who is skeptical of evolution. The question is: Over the 50,000 generations of e-coli has any of the e-coli evolved into something else or is it still e-coli?

I am a non-religious person who likes to think of myself as an adherent to science but I am not sure how to respond to my skeptic-friend.

Thank you!

And here’s my reply:

Hello —-,

50,000 generations, for these bacteria, took place in a matter of ~25 years. They have changed in many (mostly small) ways, and remained the same in many other respects, just as one expects from evolutionary theory. Although these are somewhat technical articles, I have attached 3 PDFs that describe some of the changes that we have seen.

Wiser et al. (2013) document the process of adaptation by natural selection, which has led to the improved competitive fitness of the bacteria relative to their ancestors.

Blount et al. (2012) describe the genetic changes that led one population (out of the 12 in the experiment) to evolve a new capacity to grow on an alternative source of carbon and energy.

Tenaillon et al. (2016) describe changes that have occurred across all 12 populations in their genomes (DNA sequences), which have caused all of them to become more and more dissimilar to their ancestor as time marches on.

Best wishes,

     Richard

Although these articles were written for other scientists, they make three big points that I hope almost anyone with an open mind can understand.

• We see organisms adapting to their environment, as evidenced by increased competitiveness relative to their ancestors.
• Against this backdrop of more or less gradual improvement, we occasionally see much bigger changes.
• And at the level of their genomes, we see the bacteria becoming more and more different from their ancestors.

In these fundamental respects, evolution in these flasks works in much the same way that evolution works in nature. Of course, the scales of time and space are vastly greater in nature than they are in the lab, and natural environments are far more complex and variable than is the simple one in the LTEE. But the core processes of mutation, drift, and natural selection give rise to evolution in the LTEE, just as they do (along with sex and other forms of gene exchange) in nature.

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.

* * * * *

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?”

* * * * *

Representing Science to My Representative

My research is funded by the National Science Foundation, including the BEACON Center for the Study of Evolution in Action. BEACON is one of a dozen or so NSF Science and Technology Centers. Today, our Representative in the US Congress, Mike Bishop, came to BEACON for 40 minutes to discuss our center—what we do, what impacts our work has, and so forth.

It was something of a “fire hose” for Mr. Bishop, with several presenters trying to convey a lot of information very quickly.  However, he was engaged and asked thoughtful questions.  I think he left with an understanding of the importance of scientific and engineering research, including how fundamental curiosity-driven research can lead to applications.

I had 10 minutes to show him my lab and explain what we do and why.  When I make a short presentation like this one, I often write out a version in advance.  I don’t read it or memorize it by any means. However, writing it out helps get my thoughts in order—removing details that aren’t important, ordering ideas into a narrative, reminding me of what I most want to convey.

I’m sure I was not as clear or coherent as the text that follows.  I offer it here because it conveys the points I tried to make in the few minutes that I had as a representative of science speaking with a representative of the people.

~~~~~~~~~~~

I want to show you one of the experiments in my lab.  We call it the long-term evolution experiment. It’s an unusual experiment because it’s been running for over 27 years.  And we keep it going because it’s been a scientific goldmine leading to new discoveries about how bacteria change over time.

It’s important that we understand bacteria and how they evolve for many reasons. Bacteria are best known because some of them can cause dangerous infections. But many of them protect us against infections—if our guts were not filled with harmless bacteria, then the dangerous ones would have a much easier time getting established in our bodies. Some bacteria also provide nitrogen to plants and perform other essential functions in the environment, including degrading some of the wastes that we produce.  And some bacteria are the workhorses of biotechnology.

To give one example of why bacterial evolution is so important:  If bacteria didn’t evolve, we would have defeated nearly all the pathogenic bacteria on Earth with antibiotics.  But they do evolve and become resistant to our drugs, and so the pharmaceutical industry has to spend billions of dollars trying to keep up with the evolving bacteria and viruses by developing new drugs to treat infections.

It’s possible to see evolution-in-action in bacteria, like we do here, for several reasons.

• Their populations are huge.  The number of bacteria in just one of these little flasks is comparable to number of people in the United States.
• They grow really fast.  Every day, there are about 7 generations of bacteria in each of the flasks.  So each day we see the great-great-great-great-great grandkids, so to speak, of the bacteria that were in our flasks yesterday. After 27 years, the experiment has run for over 63,000 generations.
• And one more important thing about bacteria. We can freeze them and bring them back to life, and so we’ve got a frozen fossil record of the experiment.

When I started the experiment in 1988, there was no human genome project, and not even a single bacterial genome had been sequenced.  Now we go into our freezers and sequence the bacterial genomes to see how their DNA is changing over time.

The work we’ve done in this curiosity-driven experiment has inspired others who are using similar ideas and approaches to understand the rates and mechanisms of how bacteria evolve.

I’ll give two quick examples that show how our NSF-supported fundamental science gets translated into applications that are important for security and health.

First, you remember the anthrax letter attacks on Congress that occurred right after the 9/11 attacks. In the first few days after the anthrax attacks, I was contacted by the Defense Threat Reduction Agency for advice on how to identify the source of the strain used in that bioterrorism, and how to distinguish it from other related strains. And in the months that followed, I was asked for and provided advice to the FBI and other agencies investigating the attacks. Tracking the source of microbes in outbreaks—whether natural or terroristic in origin—requires understanding how they change over time.

Second, my colleague Prof. Martha Mulks studies bacteria that colonize the lungs of people with cystic fibrosis (CF).  There are about 30,000 people with this disease in the US alone.  It’s an inherited disease that makes people susceptible to lung infections and, unfortunately, those infections kill many kids and young adults with CF.  Some of the bacteria that infect the diseased lungs are not pathogens to most of us—they’re bacteria that live in soil and on plants, but when they get into the lungs of CF patients they evolve and adapt to that new environment. They also evolve resistance to the antibiotics that are meant to get rid of them. How exactly the various bacteria change to become better adapted to the CF lung environment is not known. Luckily, though, Martha Mulks and other foresighted scientists and clinicians have kept frozen samples of these bacteria over the years—just like we’ve done with the long-term experiment I described a moment ago. Now the BEACON Center is supporting work by a graduate student, Elizabeth Baird, who will analyze the DNA from old and new samples and apply some of the same approaches and methods that we’ve used and developed for the laboratory experiment to see how the bacteria have changed—how they have become resistant to antibiotics and otherwise adapted to the environment of the lungs of people who suffer from cystic fibrosis.

The bottom line is that the fundamental, curiosity-driven research that the National Science Foundation supports is also an engine for future applications—often ones that we may not even have dreamed of—as well as a training ground for the talented and dedicated young people who you can see working all around us in this lab and throughout the BEACON Center.

~~~~~~~~~~~

Rep. Mike Bishop (MI-08) and me in the lab.  [Photo: Danielle Whitaker, MSU.]

A Day in the Life of …

Today was a great day – busy and wonderful. Pretty typical, I’m happy to say, though a bit busier than usual but all of it great.

Woke up to beautiful Spring day in East Lansing and walked 1.7 miles to work at MSU.

Did the usual email stuff.

Worked on getting ready for teaching for a class on evolutionary medicine taught by my colleague Jim Smith. Today’s focus will be the paper by Tami Lieberman et al. on the evolution of Burkholderia dolosa in cystic fibrosis patients during an outbreak in Boston. Last night I re-read the paper for the umpteenth time, and I still enjoyed it. Today I organized a series of questions for the students – a very interactive and smart group – around three parts.

Part I: Some background about CF, the inheritance of this disease, the frequency of the disease, how that frequency allows one to estimate the frequency of carriers, why the allele might be so common (not understood), side questions about sickle-cell anemia and why it’s so prevalent, and why, if it’s inherited, the paper we read is all about infections.

Part II: Preparing slides so we could work our way, figure by figure and panel by panel, through all of the main points in Lieberman et al.  (Reminder: Explain to students how scientific papers are often written around figures.  Once the figures and tables are there, then start on the results, etc.)

Part III: Follow up questions about the paper, the system, the interface of epidemiology and evolutionary biology, prospects for the future of this field and the students’ careers (most in this class are premed, many with a research bent), etc. And whatever questions they might want to ask of me.

Sometime in the middle of doing all that: Chatted with second-year grad student Jay Bundy, who is reading some of Mike Travisano’s terrific earlier papers on the LTEE. Specifically, why do we sometimes express fitness as a ratio of growth rates (measured in head-to-head competitions) and sometimes as a difference in growth rates?

Also in the middle of doing all that: Had phone conversation with former Ph.D. student Bob Woods, now also an M.D. specializing in infectious disease, about a faculty job offer he has (congrats, Bob!), some of the issues he needs to clarify or negotiate, and some of the amazing work he’s now doing on the population dynamics and evolution of nasty infections.

Email from grad student Mike Wiser that our paper, submitted to PLOS ONE, has been officially accepted. We had posted a pre-submission version at bioRxiv – now it’s gone through peer-review and revisions and is accepted for publication. Congrats, Mike!

Got a draft of the fourth and final chapter of Caroline Turner’s dissertation. The first three chapters are in great shape. Congrats, Caroline! With teaching looming, I had only time to review the figures, tables, and legends on this one, and made some small suggestions. On to the text tomorrow … It’s a beautiful body of work on two fascinating aspects of the interplay between ecology and evolution that have emerged in the LTEE and another evolution experiment that Caroline performed. Stay tuned for these papers!

Took a phone call from an MSU colleague who has friend with a child in high school who is interested in microbiology, who is visiting MSU, and who wanted to see the lab. Yikes, I gotta run teach! But postdoc Zack Blount kindly agreed to give a guided tour as I headed off to teach.  Thanks, Zack!

Beautiful day continues as I walk to teach in another building. Touch base with Jim Smith about what I plan to cover.

Two straight hours of teaching (one 5-minute break) in an overly hot room. Almost all of it interactive, with me asking questions and the students conferring in small groups and then responding. Very interactive, very bright students! The two hours were nearly up, with little time for my third, post-paper set of questions. But all of the students stayed (despite the beautiful weather, hot room, and the dinner hour at hand) an extra 15-20 minutes for a couple of my questions and some great ones from them about the LTEE and the future prospects for microbial evolution in relation to medicine.

It’s 6:20 pm: I’m mentally exhausted but equally invigorated. Beautiful Spring day continues as I walk home. I’m greeted by our lovely hound, Cleopatra. Exercise and feed her. Then an even more lovely creature, Madeleine, returns home and I greet her.

Check email before dinner. Find that paper with grad student Rohan Maddamsetti and former postdoc Jeff Barrick has been provisionally accepted, pending minor revisions, at Genetics. We posted a pre-submission version of that paper, too, at bioRxiv. Though we still need to do some revisions, I think it’s fair to offer congrats to Rohan and Jeff, too!

Time to crack open a bottle of wine and have some dinner. Fortunately, some of the pre-packaged dinners are pretty tasty and healthy, too, these days ;>)

Refill wine glass. Sit down and start to write a blog on a day in the life of …

Favorite Examples of Evolution

When the cold bites, When the review stings, When the news is sad, I simply remember these evolving things, And then I don’t feel so bad! — with apologies to Rodgers and Hammerstein

Over on Twitter, the biology students from George Jenkins High School in Lakeland, Florida, asked me and many others: “What’s your favorite example of evolution?”  There are so many fascinating examples that it’s hard for me to pick just one. So, here are half a dozen examples that are among my favorites.

• The discovery by Neil Shubin and colleagues of Tiktaalik, an extinct fish (pictured below) from the Devonian that was poised to give rise to terrestrial vertebrates. You can read about this work in Shubin’s award-winning book, Your Inner Fish, which was also made into a PBS show.
• The discovery by Svante Pääbo and colleagues of the Denisovans, an extinct lineage of humans, based on sequencing a complete genome from the finger bone of a girl who lived tens of thousands of years ago.
• The analysis by Tami Lieberman, Roy Kishony, and colleagues of the genetic adaptation of an opportunistic species of bacteria to the lungs of patients with cystic fibrosis. I’ve blogged about that paper here.
• Here’s one from the long-term experiment in my own lab — the evolution of the ability to use citrate that arose in just one of the 12 populations and after more than 30,000 generations. There are nice summaries of this work in Carl Zimmer’s blog here and here.
• A study by Hod Lipson and Jordan Pollack on the evolution of robots. I remember hearing about this paper and being shocked: “Wait a second. Robots are expensive, and most things go extinct during evolution. How could they even afford do this?” I had to read the paper to realize they were evolving virtual robots in a physical simulation of the real world. They then built and tested the winners in the physical world. And indeed, the robots worked as they had evolved to do.
• Applying the mechanisms of evolution to artificial systems is a fascinating approach useful for both biology and engineering. One of my favorite basic-science uses of this approach was a paper where we used digital organisms – computer programs that self-replicate, mutate, and compete for resources – to show how very complex functions could evolve if simpler functions were favored along the way. These simpler functions provided building blocks for the more complex functions, illustrating how evolution works by tinkering and borrowing already existing structures and functions and using them in new ways. Incidentally, this work involved collaboration between a computer scientist (Charles Ofria), a philosopher (Rob Pennock), a physicist (Chris Adami), and a biologist (me).

Readers: Please feel free to add your own favorite examples of evolution in the comments section below.

[The picture below shows the Tiktaalik fossil discovered by Neil Shubin and colleagues.  It was posted on Wikipedia by Eduard Solà, and it is shown here under the indicated Creative Commons license.]