Synonymous mutations in representative yeast genes are mostly strongly non-neutral

Synonymous mutations in protein-coding genes do not alter protein sequences and are thus generally presumed to be neutral or nearly neutral1,2,3,4,5. Here, to experimentally verify this presumption, we constructed 8,341 yeast mutants each carrying a synonymous, nonsynonymous or nonsense mutation in one of 21 endogenous genes with diverse functions and expression levels and measured their fitness relative to the wild type in a rich medium. Three-quarters of synonymous mutations resulted in a significant reduction in fitness, and the distribution of fitness effects was overall similar—albeit nonidentical—between synonymous and nonsynonymous mutations. Both synonymous and nonsynonymous mutations frequently disturbed the level of mRNA expression of the mutated gene, and the extent of the disturbance partially predicted the fitness effect. Investigations in additional environments revealed greater across-environment fitness variations for nonsynonymous mutants than for synonymous mutants despite their similar fitness distributions in each environment, suggesting that a smaller proportion of nonsynonymous mutants than synonymous mutants are always non-deleterious in a changing environment to permit fixation, potentially explaining the common observation of substantially lower nonsynonymous than synonymous substitution rates. The strong non-neutrality of most synonymous mutations, if it holds true for other genes and in other organisms, would require re-examination of numerous biological conclusions about mutation, selection, effective population size, divergence time and disease mechanisms that rely on the assumption that synoymous mutations are neutral.

I knew it wouldn’t be long before this showed up here. Don’t count on Neutral Evolution being overturned just yet. :slight_smile:


I hope some of the biologists will explain the significance of this.

I have already seen anti-evolution people proclaiming the refutation of the neutral theory. But I don’t see the relevance.

I understood neutral theory to say that much of the evolutionary change is due to neutral mutations. But I never took it to say that all mutations were neutral. And this paper does not seem to say that all mutations are non-neutral. So I don’t see that this changes anything with respect to neutral theory.

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Well, you should note that the paper is about synonymous mutations in protein-coding sequences, which make up a small fraction of the eukaryote genome (about 2% in humans).


The problem is we have often treated those particular mutations as neutral or nearly so.

I honestly just skimmed this paper. I came across it earlier today. If I recall correctly they induced different mutations in yeast and followed those variants under lab conditions. About half of those synonymous mutations had deleterious fitness effects which means about half or so didn’t.

I think evograd in another thread mentioned the finding from his blog about the distribution of different mutations with divergence time. I would think there are likely enough neutral substitutions at synonymous sites over time such that the fixed synonymous differences between two taxa should be neutral since all the deleterious synonymous mutations would be lost to selection.

A take home here is you probably shouldn’t be naively be treating all mutations the same way or using mutation rates measured over very short periods of time (a few generations) to draw inferences about deep divergences.

That’s my initial take on this based on skimming the paper early today. Either way it doesn’t help creationism even though I’m sure creationists think that anything they perceive as hurting evolution helps creationism.


Well I guess this means you are now off the Nathaniel Jeanson bandwagon huh?


Then the question becomes, why wasn’t this work done by any of those people?

Correct, and it in no way implies that we won’t find new mechanisms underlying selection. That’s why real biologists are doing experiments and anti-evolution people are doing rhetoric.

The key IMO is this:

Both synonymous and nonsynonymous mutations frequently disturbed the level of mRNA expression of the mutated gene, and the extent of the disturbance partially predicted the fitness effect.

My first guess is that yeast have a very well-tuned mechanism for selectively degrading mRNAs on the basis of their secondary structure.


I’ve seen a few criticisms/caveats floating around in response to this paper already.

First, some people are highly skeptical that the wild-type controls in the study are sufficient, as they weren’t edited in the same way as the mutants so don’t control for any off-target effects etc. As a result, the fitness of all mutants may reduced relative to the WT regardless of their identity, potentially exaggerating fitness effects of synonymous mutations. Indeed, if you set a stricter threshold of fitness reduction to be counted as truly deleterious, more non-synonymous mutations pass it than synonymous ones. In other words, non-synonymous mutations tend to cause larger fitness declines than synonymous mutations.

Second, with regards to generalisability of these results (assuming they’re all perfectly accurate), it seems likely that this kind of noticeable fitness decline from synonymous mutations affecting the transcripts is going to be much more relevant to a unicellular haploid organism growing in rich media than other organisms, such as multicellular diploid organisms in a natural environment.

We know that having a large number of cells buffers organisms against perturbations in transcript abundance in some contexts, for example. Similarly, in a heterozygous setting, non-synonymous mutations are more likely to result in a substantial fitness decline than synonymous mutations acting only through transcript effects because again there would be a buffer in the form of the WT transcript. The rich media environment of the experiment because, to borrow from a commenter on twitter, a little rattle in a car is more likely to cause problems when driving flat out than it is in everyday use. If you push the yeast to operate at maximum, minor differences that usually go unnoticed might suddenly be limiting.

All this to say, even if their results are flawless, the experiment is still very likely to have limited generalisability and therefore relevance to evolutionary biology.

Finally, as this paper was partially brought up as a response what I said about the substitution rates of non-synonymous/synonymous mutations in another thread ("I'm treating the mutation rate as a substitution rate" - Dr. Nathaniel Jeanson - #68 by evograd), I will point out that this paper, even assuming it’s accurate and that it can be generalised to all contexts in all organisms, doesn’t actually change my overall point.

The explanation the authors of this new article propose for why we see more synonymous mutations than non-synonymous mutations is still natural selection, just operating on a larger scale (timeline) than the traditional model. To quote the paper:

We explored whether the low d N/d S could also be caused by a difference between synonymous and nonsynonymous mutants in their fitness variation among environments40,41. Considering this variation is relevant, because the fixation of a neutral mutation takes 4N e generations42 on average, a period during which the environment is highly likely to have changed many times. In addition to influencing the mRNA level and/or mRNA folding strength that can exert a fitness effect, nonsynonymous mutations also alter the protein sequence and potentially alter function, which synonymous mutations do not. Because each of the molecular phenotypic effects could be environment-dependent, nonsynonymous mutants may naturally have a larger across-environment fitness variance than synonymous mutants, especially given recent reports that amino acid substitutions often show environment-specific fitness effects43,44,45. Under the most extreme scenario, the fraction of deleterious mutations is identical between synonymous and nonsynonymous mutations in each environment, but the specific deleterious mutations vary across environments for nonsynonymous but not synonymous mutations. Consequently, when the environment of a population fluctuates within the typical fixation time, some synonymous mutations are never deleterious so may be fixed, whereas nearly every nonsynonymous mutation is deleterious under some environments so cannot be fixed, resulting in d N/d S much lower than 1.

Selection would still be the cause of dN/dS<1, as was proposed before.


Do YECs realize that if neutral theory is wrong (it isn’t, for the record), then genetic entropy is hogwash? Selection is going to take care of those deleterious mutations. And Jeanson’s math is toast? Can’t use a mutation rate as a substitution rate if there’s selection!

Pretty sure most YECs won’t realize the problem, but this is not a road they’re gonna want to go down.

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It’s similar to how the ID Creationists see no problem with their beliefs that all DNA is functional and that functional mutations are so prohibitively rare in sequence space. If these beliefs were true, than no known species would be able to survive for more than a few generations given the observed mutation rate. But do you see any of the creationists in this very discussion giving this problem the slightest consideration? They are the people paying the bills for the creationist organizations, so the latter have no real reason to resolve such problems.

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This also seems likely.

I doubt it. @thoughtful, do you see the massive contradiction here?

Or conversely, nearly any DNA sequence has function given the sequence diversity of non-coding DNA and its ability to accumulate mutations at a rate consistent with neutral drift. So much for specified complexity.

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Not looking incredibly massive from where I’m sitting. I think their theories are more compatible than yours at present. This paper mostly amused me because the DFE was finally (?) tested, and it’s not what neutral theory predicted.

Maybe respond to more later. I’ve been a little under the weather and have a busy weekend ahead of me so it may be a while.

I was going to point out this feature too. The key is that the reduction in fitness looks to occur at the level of mRNA production, and so far not on whatever happens to the protein. Your suggestion as to why that could be seems a good one.
The paper is still an important result in that it calls to task the tendency to overly simplify the effects of synonymous mutations.


Then you’ve placed yourself looking straight into a concrete wall.

If most synonymous mutations are easily measurably strongly deleterious, then they’re also strongly visible to selection- obviously, and GE goes out the window.

Let’s recall this:
A Great Mystery

But the paper you linked says (contrary to GE) most mutations in coding regions are in that red area.

“Three-quarters of synonymous mutations resulted in a significant reduction in fitness, and the distribution of fitness effects was overall similar—albeit nonidentical—between synonymous and nonsynonymous mutations.”

That means the area under curve for visibly deleterious mutations outside the no selection zone should be about 3 times the area in the “no selection zone” that Sanford asserts most mutations fall in.

You need to start thinking instead of just fawning over “this result appears to contradict neutral theory”.


Btw I’d like a copy of that paper if anyone has access.

Who? Perhaps someone who has spent decades designing and performing coisogenic experiments?

Do you not see why outbreeding and diploidy would make small effects much harder to detect?

What do you think that neutral theory predicted, and how?

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Is Sanford talking about protein-coding sequences?

He’s talking about all mutations in all organisms, including viruses.

If these results approximate to the human genome, about what % of the genome are they relevant to?

That will give everyone a good idea of the magnitude of the implications, and be a bit of a reality check to the YECs I’ve seen promoting this without realizing 1) that it doesn’t help their case in the slightest, and 2) how much of the genome it actually applies to, if it’s applicable to humans.