PGENETICS-D-25-01120

Epistasis among clustered lineage-specific adaptive amino acid substitutions in the Drosophila Trio protein.

PLOS Genetics

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Anne Goriely

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Additional Editor Comments:

Thank you very much for your patience with this lengthy review process. Your manuscript has been evaluated by three reviewers, all of whom appreciated the value of your study. Please address the reviewers' comments, in particular consider incorporating the suggested citations from reviewer 2, the potential role of multi-nucleotide mutations in the evolution of the derived haplotype, and the possibility that intramolecular epistasis at divergent sites excluding the three positions investigated here may modulate the deleterious effects of intermediate genotypes.

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At this stage, the following Authors/Authors require contributions: Flora Borne, Andrew M. Taverner, and Peter Andolfatto. Please ensure that the full contributions of each author are acknowledged in the "Add/Edit/Remove Authors" section of our submission form.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Authors:

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Reviewer #1: Essential protein functions require coordinated effects of amino acids, but relatively little is known about the evolutionary dynamics through which such epistatically interacting combinations arise, especially in the in vivo context of multicellular organisms. To address this knowledge gap, this paper identified a putative adaptively evolved cluster of three amino acids in D. melanogaster using a selection test and functionally characterized the effects on organismal viability of intermediates during the formation of this cluster with precisely constructed transgenic lines. They find that the ancestral (VNR) and derived (ASK) combinations of amino acids are functionally equivalent with respect to effects on viability but all intermediate combinations produce deleterious phenotypes when homozygous. From these results, the authors conclude that alleles with the individual derived amino acids initially persisted because their deleterious effects were masked in heterozygotes and then all three were able to recombine to form the derived haplotype, at which point the haplotype containing all three derived amino acid states swept to fixation.

This paper investigates a very interesting question and is well-written with clear figures. However, I have significant concerns about several of its foundations, which may invalidate the downstream inferences about the evolutionary dynamics of epistatically interacting protein residues.

1. Adaptive evolution in the system

The paper seeks to explain lineage-specific adaptive amino acid substitutions but the case for adaptive evolution in this system is at best ambiguous. The ancestral and derived amino combinations of amino acids at the three focal residues appear functionally equivalent. The authors use a site-specific selection test to identify a putative signature of selection based on clustering, but it is worth noting that the D. simulans lineage has experienced almost as many substitutions as the D. melanogaster lineage (5 vs 6 in protein). As the authors note, the substitutions are biochemically conservative. No other types of tests of selection (selective footprints, dn/ds with more phylogenetic sampling, normal MK) are provided. I recognize that no test of selection is perfect, and the number of changes of course does not indicate anything about the functional significance of the changes because a single change can (and often does) drive adaptation. Nevertheless, alongside the absence of any molecular/biological case for why this region might be evolving adaptively in D. melanogaster (and not D. simulans), the substitution patterns present do not make a compelling case for lineage-specific adaptation in this system.

2. Probability of evolutionary scenario.

An interesting, provocative idea put forward in the paper is that mutations that are individually deleterious and recessive segregate in the population until they recombine by chance into the same haplotype. That haplotype then confers some adaptive benefit to the organism and goes to fixation. Such a scenario playing out involving three mutations that are deleterious in isolation and in pairwise combinations seems incredibly unlikely, especially since there is no evidence that the final haplotype is adaptive. The authors could more formally explore this and simulate the likelihood of such a scenario using recombination rates, dominance, and selective coefficients calculated from their data. The authors show that intramolecular interactions are important for maintaining organismal fitness, and they demonstrate that epistasis creates incompatibilities between amino acid states present in orthologous sequences; they do not show how such epistatic clusters arise, in a molecularly or evolutionarily mechanistic framework, to increase organismal fitness.

3. Epistasis beyond the three residues.

The fact that all intermediates are deleterious is surprising and may represent an artifact that impacts downstream inferences. In many of the cited papers that have found intramolecular epistasis, there are potentiating or permissive substitutions that are by themselves neutral which open up evolutionary paths for subsequent adaptive mutations. The evolutionary paths in this paper have no such steps; all intermediate steps are deleterious. While it is possible that each intermediate form was deleterious, it is more probable that epistasis from one of the other 8 fixed differences between the species is shaping the observed patterns. As an example, when any substitution from the D. simulans lineage is introduced into the D. melanogaster allele, it may produce a deleterious effect because the initial fixation of that variant in D. simulans may have required a permissive substitution that is absent when the residues are put into melanogaster. Any of the 8 variants elsewhere in the protein could affect allosteric regulation, sterics of specific conformations, or a more general feature such as stability. Recombinant alleles, in principle, could be used to show that this is not the case. In absence of such data, the presence of epistatic modifiers in the protein beyond the three sites is as or more probable than the evolutionary scenario the authors articulate. Again, it is hard to imagine three different deleterious variants arising, persisting in a population long enough to recombine into the same haplotype (when pairwise combinations are deleterious), and then the haplotype sweeping to fixation.

Reviewer #2: Comments on Borne et al. “Epistasis among clustered lineage-specific…”

In this interesting manuscript the authors investigate a cluster of 3 amino acid substitutions that have occurred along the D. melanogaster lineage. These amino acid substitutions are inferred to have been fixed by positive selection. They recreate all the intermediate states between the inferred ancestral and the Dmel state. They find that all intermediates are deleterious in homozygous form in at least one of their fitness assays. However, the intermediates are recessive, when heterozygous with the ancestral allele. This suggests a model by which the mutations might have risen to moderate frequency in the population and then recombined onto the same haplotype, which then swept to fixation; or one of the mutations rose to moderate frequency and the other mutations occurred on that background.

These are interesting results, and I don’t have any substantial comments.

1. The heterozygous effects are always tested alongside the ancestral allele; this is reasonable since under their model, the ancestral allele may well have been the dominant haplotype until the derived haplotype was formed. However, it would have been interesting to explore the fitness of a haplotype and the haplotype from which it was derived – e.g. what is the fitness of the ASR/VSR individual? This might reveal additional pathways that could have been taken.

2. Lines 207-208 “6 males and 1 female out of 113 adults” is unclear.

3. A central question is how important are these sorts of trajectories? do most mutations fixe sequentially, or is the simultaneous fixation a common occurrence. This is a difficult question to answer.

Reviewer #3: In this manuscript, Borne et al search for evidence of epistatic interactions between clusters of substitutions in D. melanogaster. Specifically, the authors examine the possibility that amino acid changes that are near one another on primary sequence space may interact with one another in such a way that creates fitness valleys that should make a derived haplotype with multiple replacements harder to reach via a series of substitutions. The authors investigate this by first searching for clusters of amino acid replacements that appear to be have been positively selected, and then identifying a subset of these clusters that are tractable for in vivo fitness assays. For a proof of concept, the authors settle on a cluster of three amino acid replacements in the Trio protein, and use genome editing to examine fitness (as measured by viability and locomotion assays) for all 6 intermediate haplotypes. They find that all intermediate haplotypes show fitness defects when homozygous, with most being lethal. However, when heterozygous, all intermediate haplotypes appear to be roughly as fit as both the ancestral haplotype or the derived haplotype. The authors conclude that, rather than through a sequence of fixations, the derived haplotype may have been able to accumulate mutations while segregating at low frequency such that most individuals with these (recessive) mutations were heterozygous. While not being definitive, I think the authors' experimental results certainly are consistent with this explanation, and the paper is well written and fair in its framing of the evidence for their conclusions. However, I do have some comments that I would like to see addressed in a revision.

General comments:

1) The authors' model for how the derived Trio haplotype may have arisen in dmel appears to be identical to the "stochastic tunnel" model (e.g. https://pmc.ncbi.nlm.nih.gov/articles/PMC1470783/). There have been numerous theoretical studies about this model, and the authors should prominently discuss and cite that literature here. Intuitively, it makes sense that this phenomenon would be more likely when the intermediate haplotypes that form the fitness valley are recessive, as the authors find for Trio and include in their proposed version of this model. I note that while most of the papers on the stochastic tunnel phenomenon do not discuss the role of dominance (e.g. https://pubmed.ncbi.nlm.nih.gov/16050095/), some do explicitly examine the case when the deleterious effects of intermediate haplotpyes are recessive (https://pubmed.ncbi.nlm.nih.gov/12028779/).

2) The authors make the point that amino acid replacements are independent events. However, when mutations are tightly clustered, a possibility that needs to be considered is multinucleotide mutation, when a single event results in multiple simultaneous mutations. Multinucleotide mutation may not be the cause of the derived Trio haplotype, but it could have played a partial role (e.g. jumping directly to an intermediate haplotype with more than one mutation. This process could also have been involved in some of the other 900+ clusters of mutations identified by the authors. For some of these clusters, some or all of the mutations may have occurred simultaneously, which would allow for another way to cross (at least part of) any fitness valley. The authors should discuss all of this and cite the relevant literature. Here are just a few examples of papers on this topic: https://pubmed.ncbi.nlm.nih.gov/21636278/, https://pubmed.ncbi.nlm.nih.gov/23447710/, https://pubmed.ncbi.nlm.nih.gov/25079859/, and https://pmc.ncbi.nlm.nih.gov/articles/PMC6093625/

3) There are several places where the authors claim that they identified clusters of positively selected mutations. I think that, multinucleotide mutations aside, the results presented in this manuscript suggest an alternative possibility: the derived haplotype does not confer higher fitness than the ancestral haplotype, but the occurrence of compensatory substitutions creates a false signal of positive selection. It would be worth noting that this could be happening in some of their clusters, and the authors may wish to rephrase when referring to their clusters as "adaptive substitutions" (e.g. "putatively adaptive substitutions" or similar). Here is one paper I could find that is relevant to this possibility: https://academic.oup.com/mbe/article/37/11/3353/5867919

Specific comments:

Line 100: The phrasing here implies that pleiotropy is limited to multicellular organisms.

Lines 283-285: Here the authors state that they are the first to test the effects of purportedly adaptive substitutions in multicellular organism. While their exact phrasing might make this statement technically true, I think they should cite this paper and point out that it did something fairly similar: https://www.nature.com/articles/s41559-016-0025

Lines 311-320: In the context of dominance reversals, the authors could cite this paper which has implications for the rate of adaptation: https://pubmed.ncbi.nlm.nih.gov/35213740/

Lines 338-341: Here the authors are discussing how the simultaneous fixation of multiple alleles might cause one to incorrectly conclude that the reduction in diversity caused by these fixations was cumulative from multiple independent substitutions rather than the fixation of a single haplotype. I think I know the type of study the authors are referring to here but the description is somewhat vague, so it would be helpful to cite some example studies.

Lines 394-399: To clarify, the authors are saying that there were 940 clusters of at least 2 substitutions within a 20-codon window (call this set A), and then some subset of these had only 2 or 3 substitutions within the 20-codon window (call this set B). The clusters in set B were found in 679 genes. Is this interpretation correct? If so, can the authors share here 1) how many genes are account for the clusters in set A, and 2) how many clusters are in set B?

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: None

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Dear Dr Borne,

We are pleased to inform you that your manuscript entitled "Epistasis among clustered lineage-specific amino acid substitutions in the Drosophila Trio protein." has been editorially accepted for publication in PLOS Genetics. Congratulations!  We encourage you to consider the final comment from Reviewer #1 about the word "adaptive" in the legend of Figure 3 and suggest "evolutionary" or "non-deleterious" as alternative descriptions of these paths.

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Comments from the reviewers (if applicable):

Reviewer's Responses to Questions

Comments to the Authors:

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Reviewer #1: The authors have made numerous substantive changes throughout the manuscript that have enriched it and have addressed the issues previously raised. The added data for heterozygous genotypes, in particular, strengthen inferences about the plausible evolutionary paths from the ancestral VNR to the derived ASK allele.

My only remaining critique concerns Figure 3. I think it is difficult to make the case that Figure 3 shows an adaptive path; fitness remains flat for the heterozygous genotypes with intermediate alleles. I would simply relabel this as “evolutionary paths.” There may indeed be conditions under which the seemingly neutral or deleterious variants were adaptive, and it is reasonable to speculate about this. However, the data show that no allele increases fitness relative to VNR; as such, they seem more consistent with a neutral or compensatory “rewiring” of functional residues, akin to observations from regulatory circuit evolution (e.g., Johnson et al. 2017, https://doi.org/10.1016/j.gde.2017.09.004). The evolutionary path(s) the authors uncover are interesting regardless of whether the amino acid changes were adaptive. The findings --and testing of molecular hypotheses at the organismal level -- raise important issues for how insights from protein epistasis studies—largely drawn from haploid or in vitro contexts—should inform our understanding of the population genetic processes through which alleles segregate and fix.

Reviewer #2: The authors have satisfactorily addressed my questions. This is an interesting manuscript which I look forward to seeing in print.

Reviewer #3: The authors have thoroughly addressed my comments.

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Large-scale datasets should be made available via a public repository as described in the PLOS Genetics data availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.

Reviewer #1: None

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #3: No

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