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Wednesday, June 26, 2019

Reactionary fringe meets mutation-biased adaptation. 1. The empirical case

This is the second in a series of guest posts by Arlin Stoltzfus on the role of mutation as a dispositional factor in evolution. The first post was: Reactionary fringe meets mutation-biased adaptation: Introduction.

Reactionary fringe meets mutation-biased adaptation. 1. The empirical case
by Arlin Stoltzfus

Reactionary fringe meets mutation-biased adaptation
1. The empirical case
2. Some objections addressed
3. The causes and consequences of biases in the introduction process
4. What makes this new?
5. Beyond the "Synthesis" debate
    -Thinking about theories
    -Modern Synthesis of 1959
    -How history is distorted
    -Taking neo-Darwinism

    -Synthesis apologetics
6. What "limits" adaptation?
7. Going forward
As noted in the intro to this series, the appearance of an opinion piece on how mutation bias affects adaptation in Trends in Ecology and Evolution (TREE) appears to be a milestone for understanding this interesting new topic. The authors misrepresent a position on dual causation stated consistently in the literature for 20 years, ignore or misinterpret important new empirical work, and blithely repeat a self-serving view of history that my colleagues and I spent years debunking with careful scholarship.

In other words, as a colleague once said, "This is what victory looks like-- everyone butchering your ideas."

But it gets better. Scientists often invoke the cliche that new truths are first ridiculed as impossible, then opposed as unlikely, then claimed as traditional.  QuoteInvestigator has a piece on this "stages of truth" meme, with the following from Dr. J. Marion Sims, 1868:
For it is ever so with any great truth. It must first be opposed, then ridiculed, after a while accepted, and then comes the time to prove that it is not new, and that the credit of it belongs to some one else.
The idea that the course of evolution reflects internal biases in variation-- or, stated differently, that the generation of variation plays a dispositional role in evolution-- has been (1) ridiculed as an appeal to "the vagueness of inherent tendencies, vital urges, or cosmic goals, without known mechanism" (Simpson, 1967), and (2) ruled out by the famous "opposing pressures" argument from Fisher and Haldane that we will address later.

So, it was exciting that the TREE authors, who represent the reactionary fringe of evolutionary biology-- dedicating to shifting the "Synthesis" goal-posts to maintain the illusion that nothing is new--, not only butcher the idea of mutation-biased adaptation, try to minimize its importance, and misrepresent the evidence: they also want to claim it! Yes, they want to appropriate this unoriginal, unimportant, unsubstantiated idea for the legacy of Ronald Fisher, the closeted mutationist!

Seriously, you could not make this stuff up!

To dig out from under this mess will take some time. Let's begin by reviewing the evidence that the changes that occur during adaptation are enriched for mutationally likely changes.

The case for mutation-biased adaptation

First, consider 4 analyses of experimental evolution.

In the first stage of the compound study by Sackman, et al. (2017), Rokyta, et al. (2005) measured fitness for the beneficial changes-- 9 of them-- implicated in 20 replicate episodes of adaptation of phiX174, aka ID11. Because the fittest variant was found only once, yet the 4th most-fit was found 6 times, the authors explored a model of mutational effects, including transition bias and the multiplicity of mutational paths to an alternative amino acid (e.g., an ATG Met codon can mutate to Ile in 3 different ways, but an Ile codon such as ATC has only one mutational path to Met). An origin-fixation model with mutation bias fit the data better than the mutational landscape model of Orr, which considers only fixation probability. Sackman, et al. (2017) carried out this same 20-replicate protocol with 3 closely related phages: the combined results how a strong bias favoring transitions, 29:5 for paths, 74:6 for events (figure below).

Note the terminology. A "path" specifies the starting and ending genotype, e.g., "gene F, site 3665, C → T", and an event is an occurrence of change along a path-- in this case, a replicate culture in which a phage genome changes. Events along the same paths are parallel events.

MacLean, et al. (2010) repeatedly evolved Rifampicin-resistant Pseudomonas aeruginosa, with results showing a significant correlation between the chance of evolving and the measured mutation rate for 11 changes in rpoB (center panel below). All of these changes are nucleotide substitutions with mutation rates that differ due to unexamined context effects. The lack of correlation in the left panel is not too surprising, given that the calculated probability of fixation ranges only from 0.47 to 0.84, because s is so large (meanwhile, the mutation rate varies 50-fold).

Couce, et al. (2015) evolved cefotaxime resistance repeatedly in 2 different Escherichia coli mutator strains with distinctly different mutation spectra, resulting in two distinct distributions of changes among resistant strains, each with a strong correspondence to the respective parental mutation spectrum.

McCandlish and Stoltzfus (2017) gathered a large set of published cases of laboratory parallel adaptation due to recurrent amino acid replacements (389 events on 63 paths), and found that these data exhibit a substantial excess of transitions, relative to the null expectation for no mutation bias (a 1:2 ratio). Note that this study integrates data from MacLean, et al. (2010) and Rokyta, et al. (2005), but (1) this is a minority of the data (22 %), and (2) the test for transition bias is an independent result from the correlation shown earlier in data from MacLean, et al. (2010).

Next, consider 4 analyses of natural evolution. McCandlish and Stoltzfus (2017), in the same paper just mentioned, gathered data on natural parallelisms from 10 different study systems (231 events on 55 paths), and showed the same kind of transition bias. The summary table below shows that they draw from some famous adaptive stories in molecular evolution, including spectral tuning, resistance to cardiac glycosides (e.g., bird vs. monarch vs. milkweed), foregut fermentation, and echolocation.

The table above represents natural cases from Stoltzfus and McCandlish (2017). Cases 1, 4, 8 and 10 represent recent local adaptation of sub-populations (total 11:10 paths, 69:48 events), while the others represent species divergence (17:17 paths, 63:51 events).

Payne, et al. (2019) examined effects of transition bias in two different curated databases of causative mutations in antibiotic-resistant isolates of Mycobacterium tuberculosis, finding an excess of transition mutations. For instance, they take advantage of the unusual case of Met-to-Ile replacements, which can take place by 1 transition (ATG to ATA) or 2 different transversions (ATG to ATT or ATC). Instead of this 1:2 ratio of possibilities, they see a ratio of 88:49 (Basel dataset) or 96:39 (Manson dataset), roughly 4-fold above null expectations. Because all the replacements are the same type, the bias can not be due to selection preferring some replacements over others.

Storz, et al. (2019) examined changes in hemoglobins using 35 phylogenetically independent comparisons of low- and high-altitude bird populations. They identified adaptive changes in 20 comparisons, implicating 10 different paths and 22 events. (See Table below: Asterisks indicate CpG mutations.) The observation of 6 paths and 10 events associated with CpG mutations was about 6-fold over the null expectation, a statistically significant excess.

Finally, in a completely different type of study, Liu, et al. (2019) explore the emergence of imatinib resistance in leukemia patients, combining clinical data on the frequency of various resistant mutants (of the BCR-ABL oncogene) across 4 continents over 17 years, with laboratory characterization of engineered mutants. In a model for clinical frequency based on drug resistance (measured) and mutation biases (inferred from comparative data), they found that both factors were important, but the mutational factor was more important.

I mention Liu, et al. (2019) to draw attention to a fascinating, biomedically important study. However, I won't include it in future discussions about biological significance, because typically we do not include resistant tumor outgrowths in the category of natural adaptation (and I don't want to confuse people or invite distracting criticisms).

To summarize, various recent studies suggest that the changes that occur during adaptation are enriched for the kinds of changes that are mutationally likely. The spectrum of adaptive changes shows modest biases with the same orientation and magnitude as modest mutation biases (either known or suspected) that range in magnitude from a few-fold (transition bias) to 10-fold (CpG bias) to as large as 50-fold (the range of mutation rates measured by MacLean, et al., 2010).

Going further

Considered on their own, these results are perhaps uninteresting. Mutation biases are secretly influencing the details underlying adaptation. Perhaps this was not expected on classical grounds, but why should we care?

These results are important because they demonstrate a principle not previously accepted: modest quantitative biases in the generation of variation may impose predictable biases on evolution, without a requirement for absolute constraints, neutral evolution or high mutation rates, contradicting the classic logic of the opposing-pressures argument.

If this new principle is general, it would apply to other kinds of mutational biases, as well as to other types of biases, e.g., developmental biases induced by the structure of a genotype-phenotype map. That is, these results provide proof-of-principle for ideas long discussed in evo-devo. As argued by Stoltzfus (2019), the same results add plausibility to key ideas in the self-organization literature, regarding what Cowperthwaite and Ancel (2007) call "the large-scale patterns of mutational connectivity within genotype spaces."

Before exploring these implications in future posts, we need to take a more critical look at the evidence. The authors at TREE claim that nothing has been shown, and that the results are more likely to be due to selection. What is the status of this alternative hypothesis?

Cowperthwaite, M.C., Meyers, L.A. (2007) How Mutational Networks Shape Evolution: Lessons from RNA Models. Annual Review of Ecology, Evolution, and Systematics 38:203-230.[]

Couce A., Rodríguez-Rojas A., and Blázquez J. (2015) Bypass of genetic constraints during mutator evolution to antibiotic resistance. Proc. Biol. Sci. Apr 7;282(1804):20142698 [doi: 10.1098/rspb.2014.2698]

Liu, C., Leighow, S., Inam, H., Zhao, B., and Pritchard, J.R. (2019) Exploiting the 'survival of the likeliest' to enable evolution-guided drug design. bioRxiv 557645; [doi: 10.1101/557645]

MacLean R.C., Perron G.G., and Gardner A. (2010) Diminishing returns from beneficial mutations and pervasive epistasis shape the fitness landscape for rifampicin resistance in Pseudomonas aeruginosa. Genetics 186: 1345-1354. [doi: 10.1534/genetics.110.123083]

Payne J.L., Menardo F., Trauner A., Borrell S., Gygli S.M., Loiseau C., et al. (2019) Transition bias influences the evolution of antibiotic resistance in Mycobacterium tuberculosis. PLoS Biol 17(5): e3000265. [doi: 10.1371/journal.pbio.3000265]

Rokyta DR, Joyce P, Caudle SB, Wichman HA. (2005) An empirical test of the mutational landscape model of adaptation using a single-stranded DNA virus. Nat Genet 37:441-444. []

Sackman, A.M., McGee, L.W., Morrison, A.J., Pierce, J., Anisman, J., Hamilton, H., Sanderbeck, S., Newman, C., and Rokyta, D.R. (2017) Mutation-Driven Parallel Evolution during Viral Adaptation. Mol. Biol. Evol. 34:3243-3253. [doi: 10.1093/molbev/msx257]

Simpson GG. (1967) The Meaning of Evolution. New Haven, Conn.: Yale University Press.

Stoltzfus, A. and McCandlish, D.M. (2017) Mutational Biases Influence Parallel Adaptation, Molecular Biology and Evolution 34:2163–2172, [doi: 10.1093/molbev/msx180]

Stoltzfus, A. (2019) Understanding bias in the introduction of variation as an evolutionary cause. []

Storz J.F., Natarajan C., Signore A.V., Witt C.C., McCandlish D.M. and Stoltzfus A. (2019) The role of mutation bias in adaptive molecular evolution: insights from convergent changes in protein function. Phil. Trans. R. Soc. B [doi: 10.1098/rstb.2018.0238]


Mikkel Rumraket Rasmussen said...

//"Considered on their own, these results are perhaps uninteresting. Mutation biases are secretly influencing the details underlying adaptation. Perhaps this was not expected on classical grounds, but why should we care?"//

Seems rather straightforward to me. Perhaps this is just me falling victim to the "knew it all along" rationalization, but assuming transitions are not inherently more likely to be adaptive or deleterious than transversions, then in so far as adaptation occurs due to mutation, the one that is more likely to occur will generally predominate over the other.

I have a hard time seeing why one would object to something like that. Am I missing something?

Arlin said...

We will get to this issue in about a week. You are absolutely correct that (1) this seems obvious and (2) you are falling victim to the 'knew it all along' fallacy. If you want an intellectual challenge, here is my challenge to you. Fisher, Haldane, Wright, Huxley, Ford, Simpson, Mayr and others all argued that mutational biases were unlikely to influence the outcome of evolution, on the grounds that mutation rates are small and easily opposed by selection. What were they thinking, and what did they get wrong?

Arlin said...

Also, another response to having "a hard time seeing why one would object" is to read the arguments of people who definitely object to this, namely the authors of TREE's hatchet piece

Jass said...

What are you suggesting?

Organisms know in advace which mutations will be most beneficial and have a mechanism to stimulate them?

"Nonadaptive mutations occur on the F' episome during adaptive mutation conditions in Escherichia coli.

P L Foster

DOI: 10.1128/jb.179.5.1550-1554.1997


One of the most studied examples of adaptive mutation is a strain of Escherichia coli, FC40, that cannot utilize lactose (Lac-) but that readily reverts to lactose utilization (Lac+) when lactose is its sole carbon source. Adaptive reversion to Lac+ occurs at a high rate when the Lac- allele is on an F' episome and conjugal functions are expressed. It was previously shown that nonselected mutations on the chromosome did not appear in the Lac- population while episomal Lac+ mutations accumulated, but it remained possible that nonselected mutations might occur on the episome. To investigate this possibility, a second mutational target was created on the Lac- episome by mutation of a Tn1O element, which encodes tetracycline resistance (Tetr), to tetracycline sensitivity (Tets). Reversion rates to Tetr during normal growth and during lactose selection were measured. The results show that nonselected Tetr mutations do accumulate in Lac- cells when those cells are under selection to become Lac+. Thus, reversion to Lac+ in FC40 does not appear to be adaptive in the narrow sense of the word. In addition, the results suggest that during lactose selection, both Lac+ and Tetr mutations are created or preserved by the same recombination-dependent mechanism."

John Harshman said...

This seems simple enough. Fisher, Haldane, etc. didn't know about transitions and transversions, much less their frequencies. Since we know about transition/transversion bias in mutation, it should be obvious to us as it wasn't to them that mutation frequency can influence evolution. Old dead guys are not the current state of evolutionary biology.

Arlin said...

As I said, the classic position is that mutation-induced trends require either neutral evolution or abnormally high mutation rates. I could give you dozens of quotations on this. Here are 4.

"Mutation therefore determines the course of evolution as regards factors of negligible advantage or disadvantage to the species [i.e., neutral ones]. It can only lead to results of importance when its frequency becomes large." (Haldane, 1927)

"For mutations to dominate the trend of evolution it is thus necessary to postulate mutation rates immensely greater than those which are known to occur, and of an order of magnitude which, in general, would be incompatible with particulate inheritance." (Fisher, 1930, Ch. 1)

"For any evolutionary tendency which is supposed to act by favouring mutations in one direction rather than another, and a number of such mechanisms have from time to time been imagined, will lose its force many thousand-fold, when the particulate theory of inheritance, in any form, is accepted; wheras the directing power of Natural Selection, depending as it does on the amount of heritable variance maintained, is totally uninfluenced by any such change." (Fisher, 1930, Ch. 1)

"The whole group of theories which ascribe to hypothetical physiological mechanisms, controlling the occurrence of mutations, a power of directing the course of evolution, must be set aside, once the blending theory of inheritance is abandoned. The sole surviving theory is that of Natural Selection" (Fisher, 1930, Ch. 1)

Arlin said...

Could you please explain which sentence or paragraph in the above post suggests this interpretation to you?

Arnaud Martin said...

Jass, of course "Organisms know[ing] in advance which mutations will be most beneficial and have a mechanism to stimulate them" - would be teleological and I don't think it is the point of the blog posts here to defend the existence of such adaptive / directed mutations. This article (cited in the TREE piece) makes a thorough rejection of the concept of adaptive mutation:
And in the intro of this blog series, there is a mention of "the archaic theory of orthogenesis" (a synonym for directional/teleological mutationism), which is accepted here as invalid in its premises.

I agree based on this post here that adaptive evolution seems more mutation-limited than assumed under standard models. The transition biases cited above are quite strong... personally I feel this is important and hope to see more data and meta-analyses.

Arlin said...

OK @John_Harshman and @Mikkel_Rumraket_Rasmussen, the answer has been posted in

Haldane, Fisher, Wright, Huxley, Ford, Mayr, Simpson and others invoked the opposing-pressures argument to conclude that biases in variation cannot be important except in the special cases of neutral evolution or unusually high mutation rates.

Now I'm hearing crickets chirp. Comments?

John Harshman said...

Did any of that happen after we both understood the structure of DNA and knew about the transition/transversion bias?

Mikkel Rumraket Rasmussen said...

I don't understand the what Haldane and Fisher is saying in your quotes.

Ultimately, genetic variation must arise by mutation. Assuming nobody of relevance would disagree with that, then I simply fail to understand how someone would argue against what you've been proposing. I can't make sense of the objection. But that leads me to suspect you're not really talking about the same thing.

I have to agree with John, are they actually talking about something like transition bias? It's not really clear to me what they're talking about.

When Haldane speaks about "the course of evolution", what is he referring to specifically? The course of evolution in what sense? Is he talking about mutations with particular phenotypic or developmental effects, mutations with particular effects on protein structure, transition vs transversion bias? It's not obvious which one, if any.

When Fisher talks about "any evolutionary tendency" and "mutations in one direction than another", what is the tendency he speaks about? What is the "direction" he speaks about? A directionality to the kind of morphological adaptations that mutations result in? Again it's not clear he's talking about actual transversion/transition bias.

And it's not clear to me that if they had known about transition/transversion bias, that they would have objected to the straightforward inference that if there is a bias in the rate of introduction, and if there is no bias in the magnitude or distribution of selection coefficients between them, that the most frequent to occur would also be the most frequent to fix.

Arlin said...

@John_Harshman and @Mikkel_Rumraket_Rasmussen, I try to avoid personalizing scientific questions, and turning them into questions about whether certain people hypothetically might have understood something if they had known something else that they did not know. I am interested in theories, not heroes. "They would have agreed with us if they knew what we knew" is not a scientific argument, but an attempt to justify the righteousness of dead predecessors.

The prevailing theory used to reason about the consequentiality of variation biases before Yampolsky and Stoltzfus 2001 was the opposing pressures theory. I have documented this. Obviously this was invoked many times after 1953 when the structure of DNA was known. Maynard Smith, et al. 1985 cited the Fisher-Haldane argument after specifically citing cases of mutation bias such as GC bias. To this day, molecular evolutionists still think of mutational explanations for patterns as neutral models. I don't know what more evidence you need.

Arlin said...

@John_Harshman and @Mikkel_Rumraket_Rasmussen, let me clarify that further by saying that, if you look at

this includes the quotation from Maynard Smith, et al. (1985), which cites Cox and Yanofsky, which is about a mutator that favors GC base-pairs.

Jeffrey Townsend said...

would not limit one's thinking to "only" molecular changes such as in transition / transversion bias. If we can take mutation accumulation data as indicative (and we have little else to go on), then there are massive differences in mutation bias in phenotype as well. Not only that, but it makes a huge difference to observed selective outcome—to be ignored at the evolutionary biologist's inferential peril. See e.g. and

Arlin said...

Thanks for the sources, @Jeffrey_Townsend. Does this framework provide a way to estimate the effectiveness of mutational biases on evolution?

There is small body of evo-devo literature regarding the idea that evolutionary change reflects biases in discrete phenotypic variation, following work on Alberch and Gale on taxon-specific patterns of digit loss in salamanders vs. frogs. A more recent example would be Baldi, et al. regarding sperm size in hermaphrodite worms ( But most of this literature is inconclusive and does not offer a quantitative framework for systematic testing of hypotheses.