In the last post in this series, we discussed how the development of Cit+ bacteria in the Long Term Evolution Experiment (LTEE) took place in three stages: potentiation (necessary mutations that allowed the Cit+ trait to evolve much later), actualization (the mutation step that converted a Cit- cell to a Cit+ cell), and refinement (mutations subsequent to actualization that improved the nascent Cit+ function). We then went on to examine the details of how the actualization step took place, and noted that it was a significant “gain-of-FCT” mutation according to the criteria of Intelligent Design (ID) proponent Michael Behe.
As interesting as the details of the actualization step are, the other steps (potentiation and refinement) are even more significant when considering Behe’s claimed “edge” to what evolutionary processes can achieve. Before we consider the impact of these findings on Behe’s ideas, a thorough investigation of these details is in order.
Potentiation: setting the stage
As we noted back in 2011, the LTEE culture that eventually went on to evolve the Cit+ trait had a mutational event at around generation 20,000 that was necessary for future actualization. One of the challenges for determining the details of the potentiation step is that this mutation (or possibly mutations) does (do) not produce an obvious change in the organism that a researcher can detect at the time. The only change they produce is to allow for future rare events (i.e. conversion to Cit+), making them tricky to identify.
In any other experimental system, it would be pretty much impossible to track down these potentiating steps, or even to conclusively determine that any potentiation steps occurred at all. In the LTEE, however, we have access to the ancestors of the future Cit+ bacteria, since they were frozen down and saved for later studies. This allowed the Lenski group to thaw out selected ancestors from various generations, re-run the evolution experiment from these selected time points, and watch to see if the Cit+ trait would evolve again. In the re-run experiments they observed numerous Cit+ actualization events, allowing for a statistical analysis of the results. These results, combined with extensive sequencing to place the various Cit+ cells from the re-run experiments into groups, demonstrated that at least two separate potentiation mutations occurred in the original LTEE, and that these mutations were separated by a few thousand generations. Once these two potentiating mutations were in place, cells were able to become Cit+ through the actualization mutation event we discussed in detail in the last post in this series. As we noted back in 2011, however, this actualization step produced only a very weak Cit+ ability. Further mutations that improved Cit+ function would soon follow.
Refinement: honing a new function
Once the (albeit very poor) Cit+ ability arose in the LTEE, it provided only a very slight selective advantage. These first Cit+ cells, when placed in competition with their descendants, are easily outcompeted – indicating that an additional mutation, or mutations, improved the nascent Cit+ trait. Work by the Lenski group showed that later, more robust Cit+ cells had duplications of the original tandem mutation that lead to the original Cit+ cell – effectively increasing the copy number of the new citrate / succinate transporter gene with its altered regulatory DNA. As the refinement process proceeded, the Lenski group found cells with three copies, four copies, and even nine copies (!), all of which most likely increased the amount of the citrate / succinate transporter made under aerobic conditions. Cells later on in the refinement process settled on a four-copy system, which the Lenski group hypothesizes to be more stable (based on research on tandem arrays in other systems). This switch from a predominance of high copy number cells (i.e. nine copies) back to cells with a more moderate number (four) predominating suggests that additional mutations are arising that allow the four-copy cells to outcompete the nine-copy cells, and find a balance between (unstable) high copy number and the Cit+ function derived from each copy. Some candidates for these additional mutations identified by the Lenski group include mutations in the citrate / succinate transporter itself, and two enzymes involved in citrate metabolism. Future work will be needed to determine if these mutations were the ones that had a significant effect on the Cit+ refinement process, or if other mutations were responsible.
With these details in hand, we can now improve our accounting for the number of mutations involved in the entire process, from potentiation, through actualization, and on to a certain point of refinement (which remains ongoing in the LTEE):
The first potentiation mutation (total = 1)
The second potentiation mutation (total = 2)
The actualization mutation (total = 3)
Duplication of the Cit+ tandem array (at least once, more likely twice) (total = 4 or 5)
Mutation(s) to improve Cit+ function with moderate copy number (at least one, likely more) (total = 5 or 6, or more)
As we can see, at a minimum the entire process involved at least 5 mutations, and more likely 6 or more. An additional important point to note is that these mutations did not occur simultaneously, but were spread out over thousands of generations.
Implications for Behe’s “edge”
In the last post in this series, we noted ID microbiologist Ann Gauger’s response to Lenski’s results. Two features are important to note: the number of mutations she claims are postulated for the Cit+ transition, and her reasoning that this transition is within Behe’s “edge” of what evolution can accomplish:
“When is an innovation not an innovation? If by innovation you mean the evolution of something new, a feature not present before, then it would be stretching it to call the trait described by Blount et al. in "Genomic analysis of a key innovation in an experimental Escherichia coli population" an innovation…
The total number of mutations postulated for this adaptation is two or three, within the limits proposed for complex adaptations by Axe (2010) and Behe in Edge of Evolution. Because the enabling pre-adaptive mutations could not be identified, though, we don't know whether this was one mutation, a simple step-wise series of adaptive mutations, or a complex adaptation requiring one or two pre-adaptations before the big event.
Presumably, Gauger is counting only potentiation and actualization and omitting refinement altogether. As we have seen, the entire process requires at least 5 mutations, and probably more. Even more interesting, however, is that she generalizes Behe’s “edge” beyond the formation of protein-protein binding sites (the focus of Behe’s claimed limit to evolution) to a number of mutations needed for a new function. This might seem odd, but in fact this generalization is absolutely in keeping with Behe’s model. Behe’s calculation for his “edge” is an estimate based on simultaneous mutations – in other words, Behe proposes a limit to the generation of protein-protein binding sites as a specific application of his general rule that multiple, simultaneous mutations are vanishingly rare. For those interested in a detailed discussion of how Behe’s model is based on an assumption that simultaneous mutations are required for the evolution of new protein-protein binding sites, I have discussed it at length in a previous series. The point here is a simple one: Gauger fails to note that Behe’s edge is based on simultaneous mutations. If indeed all five (or more) mutations needed for this transition to Cit+ in the LTEE were required simultaneously, we could be confident that the trait would never arise.
Put more simply, Behe is right that numerous mutations occurring simultaneously are too rare to expect in evolution. What he has not demonstrated, however, is that evolution must proceed only by numerous mutations occurring simultaneously. With the LTEE, we have direct evidence of what Behe defines as a “noteworthy gain-of-FCT mutation” occurring step by step, without the need for simultaneous mutations.
In the next post in this series, we’ll explore Behe’s ideas further, and examine the second source that Gauger cites as a limit for complex adaptation – the work of ID biologist Douglas Axe.
For further reading:
Blount, Z.D., Barrick, J.E., Davidson, C.J. and Lenski, R.E. (2012). Genomic analysis of a key innovation in an experimental Escherichia coli population. Nature 489; 513- 518.
Michael J. Behe, The Edge of Evolution: The Search for the Limits of Darwinism (New York: Free Press, 2007).
Michael J. Behe (2010). Experimental evolution, loss-of-function mutations, and “The first rule of adaptive evolution”. The Quarterly Review of Biology 85(4); 419-445.