Repeatability of adaptation in sunflowers: genomic regions harbouring inversions also drive adaptation in species lacking an inversion

Reviewer #1 (Public Review):

Soudi, Jahani et al. provide a valuable comparative study of local adaptation in four species of sunflowers and investigate the repeatability of observed genomic signals of adaptation and their link to haploblocks, known to be numerous and important in this system. The study builds on previous work in sunflowers that have investigated haploblocks in those species and on methodologies developed to look at repeated signals of local adaptations. The authors provide solid evidence of both genotype-environment associations (GEA) and genome-wide association study (GWAS), as well as phenotypic correlations with the environment, to show that part of the local adaptation signal is repeatable and significantly co-occur in regions harboring haploblocks. Results also show that part of the signal is species specific and points to high genetic redundancy. The authors rightfully point out the complexities of the adaptation process and that the truth must lie somewhere between two extreme models of evolutionary genetics, i.e. a population genetics view of large effect loci and a quantitative genetics model. The authors take great care in acknowledging and investigating the multiple biases inherent to the used methods (GEA and GWAS) and use a conservative approach to draw their conclusions. The multiplicity of analyses and their interdependence make them slightly hard to understand and the manuscript would benefit from more careful explanations of concepts and logical links throughout. This work will be of interest to evolutionary biologists and population geneticists in particular, and constitutes an additional applied example to the comparative local adaptation literature.

Some thoughts on the last paragraph of the discussion (L481-497): I think it would be fine to have some more thoughts here on the processes that could contribute to the presence/absence of inversions, maybe in an "Ideas and Speculation" subsection. To me, your results point to the fact that though inversions are often presented as important for local adaptation, they seem to be highly contingent on the context of adaptation in each species. First, repeatability results are only at the window/gene level in your results, the specific mutations are not under scrutiny. Is it possible that inversions are only necessary when sets of small effect mutations are used, opposite to a large effect mutation in other species? Additionally, in a model with epistasis, fitness effects of mutations are dependent on the genomic background and it is possible that inversions were necessary in only certain contexts, even for the same mutations, i.e. some adaptive path contingency. Finally, do you have specific demographic history knowledge in this system that maps to the observations of the presence of inversions or not? For example, have the species "using" inversions been subject to more gene flow compared to others?

Reviewer #2 (Public Review):

In this study the authors sought to understand the extent of similarity among species in intraspecific adaptation to environmental heterogeneity at the phenotypic and genetic levels. A particular focus was to evaluate if regions that were associated with adaptation within putative inversions in one species were also candidates for adaptation in another species that lacked those inversions. This study is timely for the field of evolutionary genomics, due to recent interest surrounding how inversions arise and become established in adaptation.

Major strengths

Their study system was well suited to addressing the aims, given that the different species of sunflower all had GWAS data on the same phenotypes from common garden experiments as well as landscape genomic data, and orthologous SNPs could be identified. Organizing a dataset of this magnitude is no small feat. The authors integrate many state-of-the-art statistical methods that they have developed in previous research into a framework for correlating genomic Windows of Repeated Association (WRA, also amalgamated into Clusters of Repeated Association based on LD among windows) with Similarity In Phenotype-Environment Correlation (SIPEC). The WRA/CRA methods are very useful and the authors do an excellent job at outlining the rationale for these methods.

Major weaknesses

The study results rely heavily on the SIPEC measure, but I found the values reported difficult to interpret biologically. For example, in Figure 4 there is a range of SIPEC from 0 to 0.03 for most species pairs, with some pairs only as high as ~0.01. This does not appear to be a high degree of similarity in phenotype-environment correlation. For example, given the equation on line 517 for a single phenotype, if one species has a phenotype-environment correlation of 1.0 and the other has a correlation of 0.02, I would postulate that these two species do not have similar evolutionary responses, but the equation would give a value of (1+0.02)*1*0.02/1 = 0.02 which is pretty typical "higher" value in Figure 4. I also question the logic behind using absolute values of the correlations for the SIPEC, because if a trait increases with an environment in one species but decreases with the environment in another species, I would not predict that the genetic basis of adaptation would be similar (as a side note, I would not question the logic behind using absolute correlations for associations with alleles, due to the arbitrary nature of signing alleles). I might be missing something here, so I look forward to reading the author's responses on these thoughts.

An additional potential problem with the analysis is that from the way the analysis is presented, it appears that the 33 environmental variables were essentially treated as independent data points (e.g. in Figure 4, Figure 5). It's not appropriate to treat the environmental variables independently because many of them are highly correlated. For example in Figure 4, many of the high similarity/CRA values tend to be categorized as temperature variables, which are likely to be highly correlated with each other. This seems like a type of pseudo replication and is a major weakness of the framework.

Below I highlight the main claims from the study and evaluate how well the results support the conclusions.

* "We find evidence of significant genome-wide repeatability in signatures of association to phenotypes and environments" (abstract)
* Given the questions above about SIPEC, I did not find this conclusion well supported with the way the data are presented in the manuscript.

* "We find evidence of significant genome-wide repeatability in signatures of association to phenotypes and environments, which are particularly enriched within regions of the genome harbouring an inversion in one species. " (Abstract) And "increased repeatability found in regions of the genome that harbour inversions" (Discussion)
* These claims are supported by the data shown in Figure 4, which shows that haploblocks are enriched for WRAs. I want to clarify a point about the wording here, as my understanding of the analysis is that the authors test if *haploblocks* are enriched with *WRAs*, not whether *WRAs* are enriched for *haploblocks*. The wording of the abstract is claiming the latter, but I think what they tested was the former. Let me know if I'm missing something here.
* Notwithstanding the concerns about highly correlated environments potentially inflating some of the patterns in the manuscript, to my knowledge this is the first attempt in the literature to try this kind of comparison, and the results does generally suggest that inversions are more likely capturing, rather than accumulating adaptive variation. However, I don't think the authors can claim that repeated signatures are enriched with haploblock regions, and the authors should take care to refrain from stating the relative importance of different regions of the genome to adaptation without an analysis.

* "While a large number of genomic regions show evidence of repeated adaptation, most of the strongest signatures of association still tend to be species-specific, indicating substantial genotypic redundancy for local adaptation in these species." (Abstract)
* Figure 3B certainly makes it look like there is very little similarity among species in the genetic basis of adaptation, which leaves the question as to how important the repeated signatures really are for adaptation if there are very few of them. (Is 3B for the whole genome or only that region?). This result seems to be at odds with the large number of CRAs and the claims about the importance of haploblock regions to adaptation, which extend from my previous point.

* "we have shown evidence of significant repeatability in the basis of local adaptation (Figure 4, 5), but also an abundance of species-specific, non-repeated signatures (Figure 3)"
* While the claim is a solid one, I am left wondering how much of these genomes show repeated vs. non-repeated signatures, how much of these genomes have haploblocks, and how much overlap there really is. Finding a way to intuitively represent these unknowns would greatly strengthen the manuscript.

Overall, I think the main claims from the study, the statistical framework, and the results could be revised to better support each other.

Although the current version of the manuscript has some potential shortcomings with regards to the statistical approaches, and the impact of this paper in its present form could be stifled because the biology tended to get lost in the statistics, these shortcomings may be addressed by the authors.

With some revisions, the framework and data could have a high impact and be of high utility to the community.