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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Lucky in Life, Prologue

I’ve been meaning to write for a long time about the role of chance … luck … whatever you want to call it … in life, from the grand sweep of evolution to our individual existence.

Well, just this morning, I came pretty close to demise by random genetic drift. Almost every weekday, I walk to and from work at Michigan State University. It’s a pleasant walk through pretty neighborhoods and the beautifully landscaped MSU campus.

Today was not much different from most other days. It had been sprinkling lightly, but no wind or anything out of the unusual.

I walked the route I usually take, crossing the streets by habit in more or less the same spots every time, I guess. The only moderately big road I cross is Grand River Avenue, where it intersects with Bogue Street. No problem on Grand River.

I walk down Bogue on the east side or the west side of the street depending on the traffic light, where cars are, on whimsy I guess. I was walking on the east side, though I would have to cross over to the west to get to the building where I work.

At this point in my walk, I’d guess that’s the side I’m still on maybe 80% of the time. Lucky today was one of those days. I heard a loud crack on the other side of the street. A tree limb snapped and crashed hard on the sidewalk.

Maybe half a second from snap to crash? And the limb was big and bifurcating, with two main branches, each maybe a foot in diameter. It came down straight, square and hard against the sidewalk. Even if you had an instant to react, it wouldn’t be clear which way to run to avoid one branch and not get smacked by the other.

I wasn’t the only lucky one. No one was there to get hit. A student was walking toward the spot, maybe 100 feet away. I called out something like “That was crazy, lucky you weren’t there.” He nodded and crossed to my side of the street.

It was only in walking the next couple hundred feet that I realized I had been lucky, too, to be walking this morning on the east side and not the west side of the road.

Indeed, each of us is incredibly lucky just to be here—the product of billions of generations of parents who were not only fit enough to survive and reproduce, but also lucky enough to have escaped the random drift of life and death.

[Both photos: Richard E. Lenski.]

Added November 1:  The second tree to come after me this autumn … or maybe I should say this fall.  This one was much smaller but fell just a few steps behind me on my morning run!

[Photo: Madeleine Lenski]

The LTEE as meta-experiment: Questions from Jeremy Fox about the LTEE, part 3

EDIT (23 June 2015): PLOS Biology has published a condensed version of this blog-conversation.

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This is the 3rd installment in my responses to Jeremy Fox’s questions about the LTEE (my lab’s long-term evolution experiment with E. coli), which he asked at the Dynamic Ecology blog. This response addresses his 2nd and 7th questions, which I’ve copied below. I like all of Jeremy’s questions, but I especially like his 2nd one because it forced me—and many readers, I hope—to think carefully about what experiments are and why we do them.

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• Is the LTEE actually an experiment, and wouldn’t it have been even better if it was? It’s just one “treatment”–12 replicates of a single set of conditions. Wouldn’t it have been even more interesting to have, say, two treatments? Two different culture conditions, two different founding genotypes, two different founding species…?
• Is the LTEE itself now a “model system”? Model systems in biology–systems in which it’s tractable to ask a given question–often are systems that we know a lot about. We can leverage that background knowledge to ask questions that otherwise wouldn’t be tractable. coli of course is a model organism for many purposes, because we know so much about it. But is the LTEE itself now a model system?

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You’re certainly right, Jeremy, that experiments in the fields of ecology and evolutionary biology typically have two or more treatments. But it’s not an essential part of the definition of an experiment that it has that sort of structure. It would have been nice, perhaps, if the LTEE did have two or more environments and/or two or more ancestors, as you suggest—in fact, we’ve run several of those types of experiments over the years in my lab, and I’ll mention a few of them below.

The reason I didn’t do that with the LTEE, though, was because one of my core motivating questions (see part 2 of my response) concerned the repeatability of evolutionary dynamics across replicate populations. That’s a question about the trajectory of variances over time, which is challenging statistically because estimates of variances have large uncertainties. So if the LTEE had two treatments, I might have been able to say something meaningful about the differences between them, but I would have had less power to say anything about the among-replicate variances for either treatment. In other words, with respect to that motivating question, going from 12 replicate populations down to 6 replicates would have been risky.

It certainly would be nice to have more total populations, say, 24 or even more; and nowadays many labs use 96-well plates for evolution experiments, with each well a replicate population and liquid-handling robots to automate the transfers. When I started the LTEE, though, we worked with flasks (albeit small ones); 12 may not seem like too many, but when we run the competition assays to measure fitness, we then have replicate assays for each population and we analyze multiple generations simultaneously, so the students and postdocs running these assays are handling many dozens or even hundreds of flasks.

The LTEE as a meta-experiment

Stepping back a bit, I’d like to suggest that the LTEE is a sort of meta-experiment, to coin a term. (This idea echoes the question where you suggested that the LTEE has itself become a model system.) By “meta” I mean the LTEE transcends—goes above and beyond—what one usually considers an experiment because the LTEE enables experimentation at several levels.

Level 1: The LTEE as an experiment

First, it is an experiment in the sense that it set out to measure, under defined conditions and with replication, certain specific quantities, such as fitness trajectories. It may not be typical in having a single treatment, but the temporal dimension coupled with being able to analyze multiple time points simultaneously—that is, the “time travel” enabled by the frozen samples across the generations, including the use of the ancestral strain as an internal control in fitness assays—functions in much the same way from an analysis standpoint.

Level 2: The LTEE as a generator of new questions and experiments to answer them

Second, the LTEE has generated a number of new questions and hypotheses that are themselves amenable to structurally independent follow-on experiments. Let me give two examples. We observed fairly early on that several populations had evolved changes in their DNA metabolism and repair that caused their mutation rates to increase by roughly 100-fold (Sniegowski et al. 1997). Such “mutator” mutations can arise by hitchhiking, albeit only occasionally and stochastically, with beneficial mutations that they cause (Lenski 2004, see pp. 246-251). It wasn’t clear, though, whether they would necessarily increase the rate of fitness improvement, given the large populations and correspondingly large potential supply of beneficial mutations in the LTEE. So we designed a separate, shorter-duration experiment with some 48 populations where we varied the mutation rate, population size, and initial fitness of the founding ancestor, and assessed the resulting fitness gains over 1,000 generations (de Visser et al. 1999).

Another case is the “replay” experiments that Zachary Blount ran after one lineage evolved the ability to grow on citrate in the presence of oxygen, which E. coli generally cannot do (Blount et al. 2008). Zack ran thousands of populations that started from genotypes isolated at different times from the population that eventually evolved this new function, in order to test whether it could have arisen at any time by an appropriate mutation or, alternatively, whether it required first evolving a “potentiated” genetic background, or context, in which the “actualizing” mutation would then confer the citrate-using phenotype.

In both of these examples, the subsequent experiments, though separate and distinct from the LTEE, nonetheless emerged from the LTEE. That is, the questions and hypotheses tested in these later experiments were motivated by observations we had made in the LTEE itself.

Level 3: The LTEE-derived strains as useful ancestors for a variety of experiments meant to address existing questions

The third level of the meta-experiment involves questions that arise outside of the LTEE, but for which the LTEE generates a set of materials—specifically, strains—that are especially useful for experiments to address those questions. Again, I’ll give a couple of examples.

Many ecologists, physiologists, and others are interested in studying adaptation to specific environmental factors—such as resource availability, temperature, etc.—as well as examining possible tradeoffs associated with adaptation to those factors. One difficulty, though, is that by moving organisms from nature into the lab and allowing them to evolve under, say, different temperature regimes, adaptation to the shared features of the lab environments may well outweigh adaptation to the specific variable of interest. If so, that would interfere with one’s ability to identify the mutations and adaptations most relevant to the factor of interest, and it could also obscure tradeoffs that might be important if populations were already well adapted to the other aspects of the environment. With these considerations in mind, Albert Bennett and I took a strain from the LTEE that had evolved in and adapted to those conditions—the resources, pH, absence of predators, etc.—and we used it as the ancestor for a new evolution experiment where 6 replicate populations evolved under each of 4 different thermal regimes: 32C, 37C (the same as in the LTEE), 42C, and daily alternations between 32C and 42C (Bennett et al. 1992, Bennett and Lenski 1993). In that way, we could focus attention on temperature-specific adaptations, which were Al’s main interest, rather than having such changes overwhelmed by adaptation to the lab environment.

My second example where LTEE-derived strains were ancestors for an experiment meant to address an extrinsic question is one of an abstract nature. In this study, we quantitatively partitioned the effects of adaptation, history, and chance on phenotypic evolution by founding 3 replicate populations from 12 different ancestors—each one a genotype sampled from a different one of the LTEE populations—and we then let these 36 populations evolve in a new environment, where we changed the identity of the limiting nutrient (Travisano et al. 1995). By measuring the fitness of the 12 ancestors and 36 derived lines in the changed environment, we were able to disentangle and quantify the relative contributions of adaptation, history, and chance to the observed outcomes (see figure below). That is, adaptation measured the mean tendency for fitness to increase, history reflected the effect of the different starting genotypes on the fitness achieved, and chance the variation in the resulting fitness among the replicates that started from the same ancestor.

Sniegowski, P. D., P. J. Gerrish, and R. E. Lenski. 1997. Evolution of high mutation rates in experimental populations of Escherichia coli. Nature 387:703-705.

Lenski, R. E. 2004. Phenotypic and genomic evolution during a 20,000-generation experiment with the bacterium Escherichia coli. Plant Breeding Reviews 24:225-265.

De Visser, J. A. G. M., C. W. Zeyl, P. J. Gerrish, J. L. Blanchard, and R. E. Lenski. 1999. Diminishing returns from mutation supply rate in asexual populations. Science 283:404-406.

Blount, Z. D., C. Z. Borland, and R. E. Lenski. 2008. Historical contingency and the evolution of a key innovation in an experimental population of Escherichia coli. Proc. Natl. Acad. Sci. USA 105:7899-7906.

Bennett, A. F., R. E. Lenski, and J. E. Mittler. 1992. Evolutionary adaptation to temperature. I. Fitness responses of Escherichia coli to changes in its thermal environment. Evolution 46:16-30.

Bennett, A. F., and R. E. Lenski. 1993. Evolutionary adaptation to temperature. II. Thermal niches of experimental lines of Escherichia coli. Evolution 47:1-12.

Travisano, M., J. A. Mongold, A. F. Bennett, and R. E. Lenski. 1995. Experimental tests of the roles of adaptation, chance, and history in evolution. Science 267:87-90.

[The figure below appeared in Science (Travisano et al. 1995), and it is reproduced here under the doctrine of fair use.]

Science and Religion: Vive la Différence

[This post is the text of a talk that I presented on October 18, 1998, in East Lansing, Michigan, as part of a forum on “Our Evolving World: Challenge to Mind and Spirit.” This document is in the public domain and may be used without charge and without permission, provided the source is acknowledged.]

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Preface

Let me begin by thanking Glenn Johnson and Lars Clausen for inviting me to participate in this forum, and Philip Hefner for providing a thoughtful and thought-provoking view of the relation between religion and science in his book, “The Human Factor: Evolution, Culture and Religion” (Fortress Press, Minneapolis, 1993).

Pastor Clausen suggested that I begin by telling you briefly about my own research. I work in evolutionary biology, a field of study that seeks to understand the history and mechanisms by which life on Earth has changed during the past several billion years. Most of you probably first associate the study of evolution with dusty fossils, many of which demonstrate the existence of species that no are no longer alive today. Certain fossils provide striking evidence for evolution because the fossils have combinations of morphological traits that are no longer present in living organisms, yet were predicted from an evolutionary theory of relationships among modern-day species. For example, scientists have found fossil birds with exquisite feathers and hind-limbs like those of modern birds, but which also have teeth, clawed digits on their fore-limbs, and a long vertebral tail like their reptilian ancestors. Some of you may also know about the evidence for evolution that exists within the genes of all living organisms, including ourselves. The myriad similarities and differences among the genes of different organisms provide a material basis for evaluating the evolutionary relationships among all organisms, from bacteria to humans. These genetic similarities and differences enable scientists to determine which species are more closely related than others, using much the same logic and material that is used to establish paternity in lawsuits. The information in these genes provides independent support for the evolutionary derivation of birds from reptilian ancestors. By digging out fossil bones and sequencing genes using molecular methods, scientists can reconstruct historical events.

But I suspect that few of you think of evolution as an on-going process, one with consequences in our lifetime. And yet, evolution is happening all around us, sometimes with tangible repercussions for human welfare. Consider many disease-causing bacteria that have recently evolved resistance to the antibiotics that we use to treat infections. For example, Staphylococcus aureus, which is often acquired in hospitals following surgery, can cause potentially lethal infections; some strains of this species are now resistant to all but one of the antibiotics that were once available for its treatment. By the same token, many agricultural pests have evolved resistance to pesticides that we began using only in our lifetime. Indeed, while much of the public may regard evolutionary biology as abstract and far-removed from our present lives, in fact a substantial component of the costs of medicine and agriculture reflects an arms-race with our biological enemies. While we seek to control or eradicate diseases and pests using chemicals and other methods, these enemies are evolving genetic defenses against our best weapons. The evolution of these defenses by our natural enemies causes illness and economic devastation, and it forces us to spend more money to develop new means of combat.

And because evolution is occurring in the world around us, it is possible to perform experiments on evolution, just as one can in the fields of chemistry and physics. What is required for these experiments are organisms, such as bacteria, that have rapid generations and large populations, so that one can observe — on the time-scale of a student’s doctoral dissertation, for example — evolutionary changes that require many generations and that depend on infrequent genetic events. In my laboratory here at MSU, graduate students and I have monitored some 20,000 generations in bacterial populations that have been propagated for about ten years as part of one long-running experiment.

This experimental approach enables us to address certain evolutionary questions that would be difficult to resolve using a retrospective (historical) approach, such as studying fossils or comparing genes of living organisms. For example, on the theoretical side, how repeatable is evolution? That is, what are the relative roles of chance — from random mutation — and necessity — reflecting natural selection — during evolution? To address this question, we measure changes that take place when several initially identical populations of bacteria evolve in parallel in identical laboratory environments. On the applied side, are bacteria that have evolved resistance to antibiotics inferior to sensitive bacteria when they compete for resources, and hence for reproductive success, in the absence of antibiotic? If so, then this suggests that we may prevent, or at least slow, the spread of antibiotic-resistant bacteria by more judicious use of antibiotics.

One of the intriguing and powerful features of bacteria for this evolutionary research is the fact that they can be stored frozen, in a state of suspended animation. These frozen bacteria can be later revived to allow direct comparison, and even competition, with their own evolutionary descendants. Imagine if we could resurrect our own ancestors — from ten-thousand or a million generations ago — and then challenge them to a game of chess, or in the struggle for existence.

My students and I perform these experiments because we find them fascinating, and because evolution is a critically important process in the world in which we live. It is a process that has shaped our own being, yet it can also confound our efforts to shape the world for our well-being.

Response to Hefner’s Theological Theory of the Created Co-Creator

When Dr. Johnson told me that the focus of this forum would be the dialogue between science and religion, I listened politely but cautiously. I was pleased to hear this would not be another debate about evolution versus creation, and therefore would not pit science against religion. But as the magnitude of responding to Dr. Hefner has dawned on me, I’ve almost come to wish that this were a debate about evolution and creation! At least then it would be easy for me to disagree with another speaker’s position, and to feel that I had some expert knowledge to contribute.

Instead, my problem is this: I am a scientist, one with no special knowledge of either theology or philosophy. And yet I must respond to a distinguished theologian who has thought long and hard about the relationship between science and religion, and who has built on subtle philosophical underpinnings. So I begin by admitting that I am in over my head and hoping that I can swim, or at least dog-paddle, across this vast lake. Nonetheless, I do welcome this opportunity to respond, in order to express my admiration for Dr. Hefner’s work, but also to convey my own view of the relationship between science and religion. My view is somewhat different from the harmonious and integrated vision put forward by Dr. Hefner.

A good place to begin my reply is with a cartoon featuring Frank and Ernest, which appeared a few months ago (August 2, 1998). Ernest asks “What do you think of the idea of humans evolving, Frank?” To which Frank replies “I think it’s worth a try.” Instead of the familiar image about life emerging from the primordial ooze, or our descent from apes, this cartoon strikes us as funny because it depicts evolution in a forward-looking fashion, rather than the typical backward view. In fact, it does so at two different levels, at least for me. First, it is forward-looking in the literal sense of suggesting a future course of action. Second, Frank seems to be conveying the progressive view that not all is well with the world as it is, that humans bear some responsibility for the problems, and that therefore a new course of action is necessary.

I think this cartoon captures an important component of Dr. Hefner’s thesis. In his own words, and I quote, “… in the situation to which biocultural evolution has brought us … the life not only of the human species, but of the entire planetary ecosystem is made to depend on a great wager going well. This wager is that the cultural systems of information that the co-creator fashions will interface with the natural systems and with the global human culture so as to promote survival and a wholesome future.” Dr. Hefner then suggests that “… the wager is not going well. The cultural systems of information are not meshing adequately enough with other systems, and calamity is the prospect.” He therefore proposes “…revitalization of our mythic and ritual systems, in tandem with scientific understandings, so as to reorganize the necessary information. This may help us to put our world together …”

In a nutshell, I share Dr. Hefner’s profound concern for the future welfare of our species and planet. I agree with him that our species has evolved the unique ability to make decisions that profoundly influence the future of the world in which we live. And I share his view that this decision-making ability imposes — on all of us — a tremendous responsibility to do what it takes to ensure a wholesome future for our species and planet.

As an academic who values creative synthesis, in a world where narrow specialization has become the norm, I admire Dr. Hefner’s effort to integrate scientific and religious perspectives. But I am also troubled by the idea of integrating two such different ways of knowing. To clarify the reason for my discomfort, I must present my own views of the relationship between science and religion.

Evidence and Faith

What is the difference between science and religion? I think it is fair to say that one important difference is that science is based on evidence, whereas religion depends on faith. From some philosophical quarters, this distinction has been criticized as naïve, because science (like religion) also depends on certain fundamental beliefs that cannot be tested within the scientific enterprise. For example, science presumes that there is some correspondence between the material universe and our sensory perceptions of it. But science cannot actually prove that the world in which we live is “real” as opposed to a phantasm of our befuddled senses. I will admit, grudgingly, that I cannot prove the material reality of that wall behind me. But I invite anyone who doubts this assertion to try walking through the wall during the next break. Therefore, I will cling to the common-sense view that this distinction between science and religion — between reliance on evidence and faith — is an important one. (As a further complication to this distinction, some religious persons also claim evidence for their beliefs, as witnessed in recent days by thousands of pilgrims who visited a farm in Georgia last week to await a message they said was from the Virgin Mary. One of the pilgrims said he had on other occasions personally met both Jesus and God, and that they looked similar, except that God has more white hairs in his beard than does Jesus. But this religious evidence — unlike that required by science — cannot be reproduced or replicated on demand for a skeptic.)

While I hold to this difficult distinction between evidence and faith — indeed because I make this distinction — I would maintain that the existence of a supernatural God lies outside the realm of scientific inquiry. Science can only address natural forces in the material universe. In our work as scientists, we must assume that what we observe obeys natural laws, and that no supernatural force or being plays tricks with our experiments. Otherwise, interpretation of nature becomes arbitrary. This basic assumption applies to all scientific fields, from nuclear physics and inorganic chemistry to molecular genetics and evolutionary biology. Science therefore is unable to prove, or disprove, the existence of a supernatural God. Thus, while science is based on evidence and religion depends on faith, the two can coexist compatibly in our lives precisely because of their essential difference.

Yet despite the compatibility of science and religion at some level, science places constraints on what a religious person can believe about God, if that person also accepts a scientific world view. Let me emphasize that I don’t mean that science is infallible. Scientific claims are always liable to revision as new evidence, and even new ideas, emerge. Nonetheless, those of us who accept the validity of the scientific enterprise assume that science tends to converge toward some objective truth, even if convergence sometimes involves taking a step back before seeing the way to move two steps forward. Despite this inherent uncertainty of science, those who accept both science and religion as two sources of truth generally allow their understanding of science to constrain their personal vision of God the Creator.

For example, if a religious person accepts the scientific evidence that the Earth is several billion years old, and that all organisms including humans have evolved from a common ancestor, then that person must also accept the view that God the Creator was extremely subtle in the acts of creation, much more so than is indicated by a literal interpretation of Genesis or creation stories of most other religions. Within these scientific bounds, and recognizing the inability of science to answer questions about the supernatural, one might still imagine very different reasons for God’s subtle creativity: Perhaps God is playful and has allowed all nature the freedom to follow a path that is unknown even to God; or perhaps God is willful and directed the laws of natural creation so that they would lead to some ultimate purpose. But it would be in striking conflict with the scientific evidence to suggest that God the Creator took such direct actions as the creation stories would have us believe, if we take them as the literal truth.

Thus, one view of the relationship between science and religion is this: Science can say something about what God the Creator has done to bring about creation. This view is certainly not a new one. Over the centuries, and continuing to the present, many scientists have justified their studies on the ground that they are seeking the truth about the universe in which we live, one that God created and ultimately gave us the powers to explore. What better way to understand and even worship God than to investigate and understand God’s creation? The view that science provides insight into God’s creation, and by extension into God, seems to me entirely sensible — provided that one believes in the existence of God the Creator.

Science and Religion: Vive la Différence

I now want to suggest a slightly different view of the relationship between science and religion. It is an evolutionary view of their relationship within our culture that I personally find both plausible and liberating, in contrast to the scientific constraints that are placed on religious belief according to the previous view. As I explain this view, it should be apparent that there is an important shift in what I mean by religion from the way that I have used it previously, a shift from an emphasis on God the Creator to an emphasis on human morality. Importantly, this shift neither confirms nor denies the existence of God; instead, this view simply accepts the scientific impossibility of settling that issue.

While I am not a historian or a theologian, I think the case can be made that many religions have historically (and probably prehistorically) been conflicted between two distinct functions. On the one hand, religions have often sought to provide explanations about the natural world — how it came into being, and especially our own place or purpose in the world. The stories from Genesis of the creation in six days, and of the tower of Babel leading to different languages, are two familiar examples. On the other hand, religions have also sought to direct actions by explaining which behaviors were morally acceptable and which were not, and often prescribing rewards and punishments (in this life or beyond) to encourage moral behavior. The ten commandments and the parables of Jesus are examples in which religion gives moral direction. Thus, many religions, in an intellectual sense, have served two masters — understanding our place in nature and giving moral guidance.

But these two functions of religion sometimes come into conflict with one another, especially with the emergence of science as another way of explaining the natural world. This conflict has run both ways, with religious groups sometimes challenging scientific findings as heretical, and scientists (or individuals who usurped science) sometimes suggesting that their knowledge gave them special authority over issues of morality. As examples of the former, consider the trial and imprisonment of Galileo by the church for stating that the Earth revolved around the Sun; and the effort today of some fundamentalist groups to impose their creationist beliefs on the science curriculum in public schools. As examples of the latter, industrialists of the Victorian era sought to use Darwin’s principle of natural selection to justify their exploitation of the poor and weak. And Nazis borrowed pseudoscientific theories of racial differences as a supposed rationale for the irrational genocide of the Holocaust.

By viewing science and religion as two descendants of ancient religion, we can take comfort from the fact that these two interwoven realms of ancient religion — explanation of the natural world, and moral direction — continue to be present in our lives today. Moreover, by separating these two realms of understanding, each is freed from the binding constraints of the other. No longer must science be squeezed through the filter of any religious doctrine; and no longer must religion depend on justification in the natural world, which is often ruthless and unforgiving of mistakes.

Let me make it clear that I don’t believe these two realms must be kept absolutely separate. For example, we may use scientific data to inform the ethical course of action in medical practice. So, too, we need scientific information about our impact on nature, in order to weigh the moral consequences of alternative behaviors with respect to the health of our planet and all its inhabitants. And religious persons may wish to integrate a scientific understanding of the natural world into their religious framework in order to promote morality that is maximally consistent with a wholesome future, as exemplified by Dr. Hefner. By the same token, scientists ought not ignore the moral implications of their work, for example, with regard to methods of warfare or the impact of discoveries on the environment. Moreover, scientists may investigate the evolutionary origins of certain moral and religious beliefs, such as prohibitions against incest or dietary laws. And scientists may take spiritual pleasure in expanding our knowledge of the universe, whether to satisfy curiosity or to promote a wholesome future by informing effective decisions within a moral framework.

But my important point is this: Our understanding of the material world no longer depends on its agreement with any religious faith. At the same time, our moral dimension has the freedom to develop, perhaps enlightened by — but without fear of contradiction by — the natural world, which seems usually to favor selfishness over true altruism.

Conclusion

My discomfort with Dr. Hefner’s theological theory boils down to this: He weaves science and religion together so tightly as to blur the boundary between them, at least in my own reading. In a sense, he uses scientific evidence to support his religious faith; he uses what is known to support the unknowable. But this could be a hazardous enterprise. I think the same facts of evolution — with the perpetual struggle for existence and genetic rewards for selfishness — could just as easily be used to support a religion that both Dr. Hefner and I would find repugnant.

So I respect Dr. Hefner’s faith, and I admire his inclusive religious tone, even as he holds fast to the tenets of his faith. And I applaud his use of science and religion together to promote a more wholesome future for our species and our planet. But I cannot endorse his theological theory of the created co-creator, just as I could not agree with any theological theory that seeks scientific support for a matter of religious faith. Science depends on internal consistency, whereas religions span an enormous range of mutually incompatible beliefs. Some believe that God is embodied in nature, whereas others believe that God exists outside the material universe. Some believe that morality evolved from within nature, whereas others believe that morals are transcendent. Some preach tolerance, while others claim divine support for intolerance toward outsiders. Some look forward to life after death, others fear life after death, while still others view this life as the only one we have. Some even welcome the idea of an apocalypse, while others hope for generations without end.

Science can never settle these differences in faith. While individual scientists may hold diverse religious beliefs, or none at all, science is a way of knowing about the material universe only. Having evolved into two distinct cultural functions — two different ways of knowing — we can hope that science and religion together promote a wholesome future. Let the dialogue continue between science and religion. Mais vive la différence.

Postscript

I’ve spoken about the roles of science and religion in providing explanation of our place in nature and giving moral direction, but I’ve not said much about spirituality. The following poem is from a book that was written by my late mother and published a few years ago. To me, it shows the deep spirituality that can arise out of a material view of life.

---

THE FIRST GIFT

—being comprised of DNA and shared in the

first cell division, 3 to 4 billion years ago.

Take this. It’s part of me

and everything I know

about this emergent art

of getting by.

Since what I am survived

this long, this place,

my information may enable you

to live a little.

— Jean Lenski (Genesis, 1993, St. Andrews College Press)

---

This cartoon is © by Thaves  (http://frankandernest.com/cgi/view/display.pl?98-08-02). It is shown here under the doctrine of fair use.

“Ham on Nye” Debate, Follow-up #2

Ken Ham spoke about the research in my lab at two points during the “Ham on Nye” science versus creation debate. The first segment also includes a video clip in which Dr. Andrew Fabich, a “biblical creationist” and microbiologist at Liberty University, talks about our work.

Dr. Zachary Blount is a postdoctoral researcher in my lab who performed most of the work that was discussed.  Zack produced these transcripts of Ham’s and Fabich’s presentations while preparing his response to their distortions and misrepresentations of our work.

First segment, beginning at ~44 minutes:

Ken Ham:

“Let me introduce you to another scientist, Richard Lenski of Michigan State University.  He’s a great scientist.  He’s known for culturing E. coli in the lab, and he found there were some E. coli that actually seemed to develop the ability to grow on substrate, um, on citrate substrate.  But, Richard Lenski is here mentioned in this book [Microbiology: An Evolving Science] and it’s called ‘Evolution in the Lab’.  So, the ability to grow on citrate is said to be evolution, and there are those who say, ‘Hey! This is, this is against the creationists.’  For instance, Jerry Coyne from the University of Chicago says, ‘Lenski’s experiment is also yet another poke in the eye for anti-evolutionists.’  He says, ‘The thing I like most is it says you can get these complex traits evolving by a combination of unlikely events.’  But is it a poke in the eye for anti-evolutionist?  Is it really seeing complex traits evolving?  What does it mean that some of these, uh, bacteria are able to grow on citrate?

Let me introduce you to another biblical creationist who is a scientist.”

[via video] Andrew Fabich:

“Hi, my name is Dr. Andrew Fabich.  I got my Ph.D. from the University of Oklahoma in Microbiology.  I teach at Liberty University, and I do research on E. coli in the intestine.  I’ve published in secular journals from the American Society for Microbiology, including Infection and Immunity, uh, and Applied and Environmental Microbiology, as well as several others.  My work has been cited even in the past year in the journals Nature, Science Translational Medicine, Public Library of Science, Public Library of Science Genetics.  I, um, it’s cited regularly in those journals, and while I was taught nothing but evolution, I don’t accept that position, and I do my research from a creation perspective.  When I look at the evidence people cite of the E. coli supposedly evolving over 30 years or over 30,000 generations in the lab, and people say that it is now able to grow on citrate.  I don’t deny that it grows on citrate, but it’s not any kind of new information. It’s …  The information’s already there, and it’s just a switch that gets turned on and off, and that’s what they reported in there.  There’s nothing new.”

Ken Ham: 

“See, students need to be told what’s really going on here.  Certainly there’s change, but it’s not change necessary for molecules to man.”

Second segment, beginning at ~2 hours, 30 minutes:

Ken Ham: 

“What Bill Nye needs to do for me is to show me example of something …uh, some new function that arose that was not previously possible from the genetic information that was there.  And I would claim and challenge you that there is no such example that you can give.  That’s why I brought up the example in, uh, my presentation of Lenski’s, uh, experiments in regard to E. coli.  And there were some that seemed to develop the ability to exist on citrate, but as Dr. Fabich said from looking at his research, he’s found that that information was already there.  It’s just a gene that’s switched on and off.  And so, uh, there is no example because, you know, information that’s there in, in the genetic information of different animals, plants, and so on.  There’s no new function that can be added.  Certainly great variation within a kind, and that’s what we look at, but you’d have to show an example of brand new function that never previously was possible.  There is no such example, uh, that you can give anywhere in the world.”

The Ten Commandments of Statistical Inference

As Handed Down to Lenski by Sir Ronald Fisher

1.  Remember the type II error, for therein is reflected the power if not the glory.

2.  Thou shalt not pseudoreplicate or otherwise worship false degrees of freedom.

3.  Respect the one-tailed test, for it can make thine inferences strong.

4.  Forget not the difference between fixed treatments and random effects.

5.  Thou shalt not commit unplanned comparisons without adjusting the rate of type I error for thy transgressions.

6.  Honor both thy parametric and thy nonparametric methods.

7.  Consider not the probability of a particular set of data, but rather the probability of all those sets as or more extreme than thine own.

8.  Thou shalt confuse neither manipulation and observation, nor causation and correlation.

9.  Thou shalt not presume statistical significance to be of scientific importance.

10.  Thou shalt not be fearful of paying homage to a Statistician or His Holy Book, especially before planning an experiment; neither shalt thou be fearful of ignoring the Word of a Statistician when it is damnable; for thou art alone responsible for thine acceptance or rejection of the hypothesis, be it ever so false or true.

The Golden Rule:  Review unto others as you would have them review unto you.

***

Notes:  I wrote this for a graduate course on quantitative methods in ecology and evolutionary biology that I taught in Spring, 1989, at UC-Irvine.  The course focused on experimental design and frequentist methods for drawing inferences.

Telliamed: Indian Philosopher or French Missionary?

Telliamed was written by a French diplomat, Benoît de Maillet (1656-1738).  It circulated as an unpublished manuscript in the 1720s but was not published until 1748, ten years after de Maillet’s death.  (During those years, his text was edited by others, apparently to bring it into conformity with church dogma.  According to the Wikipedia article about de Maillet, the translation of Telliamed published in 1968 by the University of Illinois Press provides the best reconstruction of the text as de Maillet wrote it.)

The book attracted sufficient attention that it was soon translated and published in English in 1750 as Telliamed: Or, Discourses Between an Indian Philosopher and a French Missionary, on the Diminution of the Sea, the Formation of the Earth, the Origin of Men and Animals, And other Curious Subjects, relating to Natural History and Philosophy.

De Maillet used Telliamed to present provocative ideas about the history of our planet and its inhabitants – long before James Hutton (1726-1797), Comte de Buffon (1707-1788), Georges Cuvier (1769-1832), Jean-Baptiste Lamarck (1744-1829), Charles Lyell (1797-1875), Robert Chambers (1802-1871), Alfred Russel Wallace (1823-1913), and Charles Darwin (1809-1882) wrote on these subjects.

The basic thesis of Telliamed is that our planet was once entirely covered with water, but the seas have been slowly receding into a void.  This physical explanation makes no sense today.  Nonetheless, Telliamed was an attempt to understand the natural world based on observations and questions – not based on religious texts and dogma.  In other words, we should try to read from the book of nature itself.

Consider, for example, that Telliamed (1750 edition, pp. 106-107) has seen and wondered about fossil seashells on high mountains far from the sea:

“In a Word, if it was not so ; if the Waves in every Part of our Globe had not been, at least, equal to the Tops of our highest Mountains, how could we in the Composition of the most elevated Places find the same Substances, which at present she produces on her Shores ? … How could they be inserted in the Stones of the Mountains in these Places ?”

Many of the observations and interpretations in Telliamed are wrong, and some seem a bit crazy today.  For example, Telliamed (pp. 220-221) proposes that birds came from flying fish:

“Who can doubt that from the volatile Fish sprung our Birds, which raise themselves in the Air ;”

– although the rest of this sentence brings to mind Neil Shubin, Tiktaalik, and Your Inner Fish –

“… and from those which creep in the Sea, arose our terrestrial Animals, which have neither a Disposition to fly, nor the Art of raising themselves above the Earth ?”

But these insights and errors about nature, while fascinating, are not the reason that this book is one of my all-time favorites.  Rather, I admire the author’s voice, calling out across nearly three centuries, that we should keep religious prejudices and dogma out of science.  This appeal comes at the start of the book (p. 2), when the “Indian Philosopher” responds to the queries of the “French Missionary”:

 “I asked him concerning his Country, his Name, his Family, his Religion, and the Motives of his Travelling ; he accordingly spoke to me nearly in the following Manner :”

Link to the Indian Philosopher’s Reply

Understanding the history of the world based on evidence, rather than religious dogma, was a radical idea in its day.  De Maillet probably feared ridicule and persecution. The printers of dangerous books also wanted protections, lest they be charged with blasphemy.  And so de Maillet took precautions.  He dedicated the book (image below)

“To the illustrious Cyrano de Bergerac, Author of the imaginary Travels thro’ the Sun and Moon.”

De Bergerac had written L’Autre Monde ou les États et Empires de la Lune – an early work of science fiction, and so this dedication might have allowed the defense that de Maillet’s work, too, was intended as fiction.  Also, the French Missionary always speaks of nature through his conversations with the Indian Philosopher, thus merely repeating the speculations of another person rather than offering the ideas as his own.

At the same time, de Maillet managed to be provocative.  Giving the Indian Philosopher his own name spelled backwards hardly hid his identity.  And the book is written as a six-day conversation that seems intended to mirror the six days of creation in Genesis.

Most importantly, Benoît de Maillet was eager to convince his audience that an understanding of nature should be based on observations and questioning rather than religious authority.