Separating adaptation from drift: A cautionary tale from a self-fertilizing plant
Evolution of flowering time in a selfing annual plant: Roles of adaptation and genetic drift
In recent years many studies have documented shifts in phenology in response to climate change, be it in arrival times in migrating birds, budset in trees, adult emergence in butterflies, or flowering time in annual plants (Coen et al. 2018; Piao et al. 2019). While these changes are, in part, explained by phenotypic plasticity, more and more studies find that they involve also genetic changes, that is, they involve evolutionary change (e.g., Metz et al. 2020). Yet, evolutionary change may occur through genetic drift as well as selection. Therefore, in order to demonstrate adaptive evolutionary change in response to climate change, drift has to be excluded as an alternative explanation (Hansen et al. 2012). A new study by Gay et al. (2021) shows just how difficult this can be.
The authors investigated a recent evolutionary shift in flowering time by in a population an annual plant that reproduces predominantly by self-fertilization. The population has recently been subjected to increased temperatures and reduced rainfalls both of which are believed to select for earlier flowering times. They used a “resurrection” approach (Orsini et al. 2013; Weider et al. 2018): Genotypes from the past (resurrected from seeds) were compared alongside more recent genotypes (from more recently collected seeds) under identical conditions in the greenhouse. Using an experimental design that replicated genotypes, eliminated maternal effects, and controlled for microenvironmental variation, they found said genetic change in flowering times: Genotypes obtained from recently collected seeds flowered significantly (about 2 days) earlier than those obtained 22 generations before. However, neutral markers (microsatellites) also showed strong changes in allele frequencies across the 22 generations, suggesting that effective population size, Ne, was low (i.e., genetic drift was strong), which is typical for highly self-fertilizing populations. In addition, several multilocus genotypes were present at high frequencies and persisted over the 22 generations, almost as in clonal populations (e.g., Schaffner et al. 2019). The challenge was thus to evaluate whether the observed evolutionary change was the result of an adaptive response to selection or may be explained by drift alone.
Here, Gay et al. (2021) took a particularly careful and thorough approach. First, they carried out a selection gradient analysis, finding that earlier-flowering plants produced more seeds than later-flowering plants. This suggests that, under greenhouse conditions, there was indeed selection for earlier flowering times. Second, investigating other populations from the same region (all populations are located on the Mediterranean island of Corsica, France), they found that a concurrent shift to earlier flowering times occurred also in these populations. Under the hypothesis that the populations can be regarded as independent replicates of the evolutionary process, the observation of concurrent shifts rules out genetic drift (under drift, the direction of change is expected to be random).
The study may well have stopped here, concluding that there is good evidence for an adaptive response to selection for earlier flowering times in these self-fertilizing plants, at least under the hypothesis that selection gradients estimated in the greenhouse are relevant to field conditions. However, the authors went one step further. They used the change in the frequencies of the multilocus genotypes across the 22 generations as an estimate of realized fitness in the field and compared them to the phenotypic assays from the greenhouse. The results showed a tendency for high-fitness genotypes (positive frequency changes) to flower earlier and to produce more seeds than low-fitness genotypes. However, a simulation model showed that the observed correlations could be explained by drift alone, as long as Ne is lower than ca. 150 individuals. The findings were thus consistent with an adaptive evolutionary change in response to selection, but drift could only be excluded as the sole explanation if the effective population size was large enough.
The study did provide two estimates of Ne (19 and 136 individuals, based on individual microsatellite loci or multilocus genotypes, respectively), but both are problematic. First, frequency changes over time may be influenced by the presence of a seed bank or by immigration from a genetically dissimilar population, which may lead to an underestimation of Ne (Wang and Whitlock 2003). Indeed, the low effective size inferred from the allele frequency changes at microsatellite loci appears to be inconsistent with levels of genetic diversity found in the population. Moreover, high self-fertilization reduces effective recombination and therefore leads to non-independence among loci. This lowers the precision of the Ne estimates (due to a higher sampling variance) and may also violate the assumption of neutrality due to the possibility of selection (e.g., due to inbreeding depression) at linked loci, which may be anywhere in the genome in case of high degrees of self-fertilization.
There is thus no definite answer to the question of whether or not the observed changes in flowering time in this population were driven by selection. The study sets high standards for other, similar ones, in terms of thoroughness of the analyses and care in interpreting the findings. It also serves as a very instructive reminder to carefully check the assumptions when estimating neutral expectations, especially when working on species with complicated demographies or non-standard life cycles. Indeed the issues encountered here, in particular the difficulty of establishing neutral expectations in species with low effective recombination, may apply to many other species, including partially or fully asexual ones (Hartfield 2016). Furthermore, they may not be limited to estimating Ne but may also apply, for instance, to the establishment of neutral baselines for outlier analyses in genome scans (see e.g, Orsini et al. 2012).
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Evaluation round #2
DOI or URL of the preprint: https://www.biorxiv.org/content/10.1101/2020.08.21.261230v2
Version of the preprint: 2
Author's Reply, 21 May 2021
Decision by Christoph Haag, 19 Apr 2021
Dear Dr Gay,
Your revised preprint "Evolution of flowering time in a selfing annual plant: Roles of adaptation and genetic drift" has now been reconsidered by two of the original reviewers. As you will see, while one of them is satisfied with the new version, the other is positive but recommends an additional round of minor revision. From my own reading, I agree that the suggestions by the reviewer will likely further strengthen the manuscript. Therefore, before reaching a final decision, I would like to ask you to consider these suggestions, and to revise the manuscript accordingly. When you submit the revised version, please include a letter in which you describe how you have responded to each of the referees comments.
Best wishes, and many thanks for submitting to PCI Evol Biol,
Reviewed by Jon Agren, 16 Apr 2021
Reviewed by Stefan Laurent, 20 Mar 2021
Evaluation round #1
DOI or URL of the preprint: https://doi.org/10.1101/2020.08.21.261230
Version of the preprint: 1
Author's Reply, 03 Feb 2021
Decision by Christoph Haag, 26 Oct 2020
Dear Dr Gay, Thank you for submitting your preprint "Evolution of flowering time in a selfing annual plant: Roles of adaptation and genetic drift" to PCI Evol Biol. Your work has now been considered by three reviewers, whose comments are enclosed. As you will see, the reviews are largely positive, and, based on these reviews as well as my own reading, I am happy to further consider your preprint for recommendation. However, before reaching a final decision, I would like you to revise your manuscript according to the recommendations by the reviewers. Besides the more minor points (which also should be considered carefully), I think there are two main issues that need particular attention: - First, the introduction (and perhaps also some other sections) would profit from some streamlining. In my opinion, this does not mean that you should entirely drop the discussion of the effects of selfing on the efficacy of selection. But this section should be reduced in length and care should be taken to clearly state the objective of the study early on without raising issues (e.g., comparison between selfers and outcrossers) that are not subsequently addressed. Incidentally, from my own reading, I also think that the last part of page 1 (where you give some more detail on the different possible approaches to investigate the influence of selection on phenotypic change) would profit from some reformulation: I found this part difficult to follow and its purpose is not entirely clear to me: Do you want to provide details on some of the approaches or do you want to explain why you used only some bot not others in your study? Moreover, the statement that natural populations cannot be replicated may also need to be nuanced (replication might in principle be possible across different populations or using independent samples from the same population). - Second, the analysis of the frequency changes of the multilocus genotypes needs some clarification, both in terms of potential effects of excluding rare genotypes and in terms of confidence intervals given (likely) non-normal distribution of residuals. If you submit a revised version, please include a letter in which you describe how you have responded to each of the referees’ comments. Best withes, and apologies again for the delayed decision, Christoph Haag **Additional requirements of the managing board**: As indicated in the 'How does it work?’ section and in the code of conduct, please make sure that: -Data are available to readers, either in the text or through an open data repository such as Zenodo (free), Dryad or some other institutional repository. Data must be reusable, thus metadata or accompanying text must carefully describe the data. -Details on quantitative analyses (e.g., data treatment and statistical scripts in R, bioinformatic pipeline scripts, etc.) and details concerning simulations (scripts, codes) are available to readers in the text, as appendices, or through an open data repository, such as Zenodo, Dryad or some other institutional repository. The scripts or codes must be carefully described so that they can be reused. -Details on experimental procedures are available to readers in the text or as appendices. -Authors have no financial conflict of interest relating to the article. The article must contain a "Conflict of interest disclosure" paragraph before the reference section containing this sentence: "The authors of this preprint declare that they have no financial conflict of interest with the content of this article." If appropriate, this disclosure may be completed by a sentence indicating that some of the authors are PCI recommenders: “XXX is one of the PCI XXX recommenders.”