Life-history trade-offs explain local adaptation in Arabidopsis thaliana

Reviewer #1 (Public review):

Summary:

As a general phenomenon, adaptation of populations to their respective local conditions is well-documented, though not universally. In particular, local adaptation has been amply demonstrated in Arabidopsis thaliana, the focal species of this research, which is naturally highly selfing. Here, the authors report assays designed to evaluate the spatial scale of fitness variation among source populations and sites, as well as temporal variability in fitness expression. Further, they endeavor to identify traits and genomic regions that contribute to the demonstrated variation in fitness.

Strengths:

With many (200) inbred accessions drawn from throughout Sweden, the study offers an unusually fine sampling of genetic variation within this much-studied species, and through assays in multiple sites and years, it amply demonstrates the context-dependence of fitness expression. It supports the general phenomenon of local adaptation, with multiple nuances. Other examples exist, but it is of value to have further cases illustrating not only the context-dependence of fitness expression but also the sometimes idiosyncratic nature of fitness variation. I commend the authors on their cautionary language in relation to inferences about the roles of particular genomic regions (e.g.l.140-144; l.227)

Weaknesses:

To my mind, the manuscript is written primarily for the Arabidopsis community. This community is certainly large, but there are many evolutionary biologists who could appreciate this work but are not invited to do so. The authors could address the broader evolution community by acknowledging more of the relevant work of others (I've noted a few references in my comments to the authors). At least as important, the authors could make clearer the fact that A. thaliana is (almost) strictly selfing and how this feature of its biology both enables such a study and also limits inferences from it. Further, it seems to me that though I could be wrong, readers would appreciate a more direct, less discursive style of writing, and one that makes the broader import of the focal questions clearer.

As a reader, I would value seeing estimates of the overall fitness of the accessions in the different conditions, i.e., by combining the survival and fecundity results of the common garden experiments.

Reviewer #2 (Public review):

Summary:

The goal of this study was to find evidence for local adaptation in survival and fecundity of the model plant Arabidopsis thaliana. The authors grew a large set of Swedish Arabidopsis accessions at four common garden sites in northern and southern Sweden. Accessions were grown from seed in trays, which were laid on the ground at each site in late summer, screened for survival in fall and the following spring, and fecundity was determined from rosette size and seed production in spring. Experiments were complemented by 'selection experiments', in which seeds of the same accessions were sown in plots, and after two years of growth, plants were sampled to determine fitness from genotype frequencies, providing a more comprehensive evaluation of lifetime fitness than can be gleaned from fecundity alone.

As the main result, southern accessions had higher mortality in northern sites in one of two years, but also suffered more slug damage in southern sites in one year, indicating a potential link between frost tolerance and herbivore resistance. Fecundity of accession was highest when growing close to the 'home' environment, but while accessions from one sand dune population in southern Sweden had among the lowest fecundities overall, they consistently had the highest fitness in the selection experiment. Accessions from this population had large seed size and rapid root growth, which might be related to establishment success when arriving in a new, partially occupied habitat. However, neither trait could fully explain the very high fitness of this population, suggesting the presence of other, unmeasured traits.

Overall, the authors could provide clear evidence of local adaptation in different traits for some of their experiments, but they also highlight high temporal and spatial variability that makes prediction of microevolutionary change so challenging.

Strengths:

A major strength of this study is the highly comprehensive evaluation of different fitness-related traits of Arabidopsis under natural conditions. The evaluation of survival and fecundity in common garden experiments across four sites and two years provides an estimate of variability and consistency of results. The addition of the 'selection experiment' provides an extended view on plant fitness that is both original and interesting, in particular highlighting potential limitations of 'fitness-proxies' such as seed production that don't take into account seedling establishment and competitive exclusion.

Throughout the study, the authors have gone to impressive depths in exploring their data, and particularly the discovery of 'native volunteers' in selection experiment plots and their statistical treatment is very elegant and has resulted in compelling conclusions. Also, while the authors are careful in the interpretation of their GWAS results, they nonetheless highlight a few interesting gene candidates that may be underlying the observed plant adaptations, and which likely will stimulate further research.

Overall, the authors provide a rich new resource that is relevant and interesting both in the context of general evolutionary theory as well as more specifically for molecular biology.

Weaknesses:

While the repetition of the common garden experiments over two years is certainly better than no repetition (hence its mention also under 'strengths'), the very high variability found between the two years highlights the need for more extensive temporal replication. In this context, two temporal replicates are the bare minimum, and more repeats in time would be necessary to draw any kind of conclusion about the role of 'high mortality' and 'low mortality' years for the microevolution of Arabidopsis. It also seems that the authors missed an opportunity to explore potentially causal variation among years, as they did not attempt to relate winter mortality to actual climatic variables, even though they discuss winter harshness as a potential predictor.

The low temporal variation also makes the accidental slug herbivory appear somewhat random. Potted plants are notoriously susceptible to slug herbivory, and while it is certainly nice that slug damage predominantly affected one group of accessions, it nonetheless raises the question whether this reflects a 'real' selection pressure that plants commonly face in their respective local environments.

The addition of the 'selection experiment' is certainly original and provides valuable additional insights, but again, it seems a bit questionable which natural process really has affected this outcome. While the genetic and statistical analysis of this experiment seems to be state-of-the-art, the experimental design is rather rudimentary compared to more standard selection experiments. Specifically, the authors added seeds from greenhouse-grown mothers to experimental plots and only sampled plants two years later. This means that, potentially,y the first very big bottleneck was germination under natural conditions, which may have already excluded many of the accessions before they had a chance to grow. While this certainly is one type of selection, it is not exactly the type of selection that a 2-year selection experiment is set up to measure. Either initially establishing the selection experiment from plants instead of seeds, or genotyping the population over several generations, would have substantially strengthened the conclusions that could be drawn from this experiment. Also, the complete lack of information on population density is a bit problematic. It is not clear if there were other (non-Arabidopsis) plants present in the plots, how many Arabidopsis plants were established, if numbers changed over the year, etc. Given all of these limitations, calling this a 'selection experiment' is in fact somewhat misleading.

Despite these weaknesses, the authors could achieve their main goals, and despite the somewhat minimal temporal replication, they were lucky to sample two fairly distinct years that provided them with interesting variation, which they could partially explain using the variation among their accessions. Overall, this study will likely make an important contribution to the field of evolutionary biology, and it is another very strong example of how the extensive molecular tools in Arabidopsis can be leveraged to address fundamental questions in evolution and ecology, to an extent that is not (yet) possible in other plant systems.

Reviewer #3 (Public review):

Summary:

The manuscript presents a large common garden experiment across Sweden using solely local germplasm. Additionally, there is a collection of selection experiments that begin investigating the factors shaping fecundity in these populations. This provides an impressive amount of data and analysis investigating the underlying factors involved. Together, this helps support the data showing that fluctuations and interactions are key components determining Arabidopsis fitness and are more broadly applicable across plant and non-plant species.

Strengths:

The field trials are well conducted with extensive effort and sampling. Similarly while the genetic analysis is complex it is well conducted and reflects the complexity of dealing with population structure that may be intricately linked to adaptive structure. This has no real solution and the option of presenting results with and without correction is likely the only appropriate option.

Weaknesses:

A significant finding from this study was that fecundity is shaped more by yearly fluctuations and their interaction with genotype than it is by the main effect of location or genotype. Another significant finding is that the strength of selection can be quite strong, with nearly 5x ranges across accessions. It should be noted that there are a number of other studies using Arabidopsis in the wild with multiple years and locations that found similar observations beyond the Oakley citation. In general, the context of how these findings relate to existing knowledge in Arabidopsis is a bit underdeveloped.

The effects of the populations across the locations seem to rely on individual tests and PC analysis. It would seem to be possible to incorporate these tests more directly in the linear modeling analysis, and it isn't quite clear why this wasn't conducted.

I'm a bit puzzled by the discussion on how to find causative loci. This seems to focus solely on GWAS as the solution, with a goal to sequence vast individuals. But the loci that the manuscript discussed were found by a combination of structured mapping populations followed by molecular validation that then informed the GWAS. As such, I'm unsure if the proposed future approach of more sequencing is the best when a more balanced approach integrating diverse methods and population types will be more useful.

Author response:

Reviewer #1 (Public review):

Summary:

As a general phenomenon, adaptation of populations to their respective local conditions is well-documented, though not universally. In particular, local adaptation has been amply demonstrated in Arabidopsis thaliana, the focal species of this research, which is naturally highly selfing. Here, the authors report assays designed to evaluate the spatial scale of fitness variation among source populations and sites, as well as temporal variability in fitness expression. Further, they endeavor to identify traits and genomic regions that contribute to the demonstrated variation in fitness.

Strengths:

With many (200) inbred accessions drawn from throughout Sweden, the study offers an unusually fine sampling of genetic variation within this much-studied species, and through assays in multiple sites and years, it amply demonstrates the context-dependence of fitness expression. It supports the general phenomenon of local adaptation, with multiple nuances. Other examples exist, but it is of value to have further cases illustrating not only the context-dependence of fitness expression but also the sometimes idiosyncratic nature of fitness variation. I commend the authors on their cautionary language in relation to inferences about the roles of particular genomic regions (e.g.l.140-144; l.227)

Weaknesses:

To my mind, the manuscript is written primarily for the Arabidopsis community. This community is certainly large, but there are many evolutionary biologists who could appreciate this work but are not invited to do so. The authors could address the broader evolution community by acknowledging more of the relevant work of others (I've noted a few references in my comments to the authors). At least as important, the authors could make clearer the fact that A. thaliana is (almost) strictly selfing and how this feature of its biology both enables such a study and also limits inferences from it. Further, it seems to me that though I could be wrong, readers would appreciate a more direct, less discursive style of writing, and one that makes the broader import of the focal questions clearer.

we agree that connecting the paper better to the broader field is desirable, and will try to do this in the revision. As for how selfing matters, there certainly are some things we can discuss, but a general discussion is probably a suitable topic for a review/opinion article!

As a reader, I would value seeing estimates of the overall fitness of the accessions in the different conditions, i.e., by combining the survival and fecundity results of the common garden experiments.

Combining estimates would be possible in the common garden experiments, and would bring us somewhat closer to total fitness estimates, although as noted by another reviewer (and also emphasized by us), the time scale of our experiment is not sufficient to evaluate the trade-off between survival and fecundity. Furthermore, we would still be missing the establishment component of fitness, which we found to be extremely important. Therefore little would be gained by combining the estimates, while at the same time losing resolution to disentangle the fitness components. We thus decided to focus on the individual fitness components and leave consideration of their joint effect for the Discussion.

Reviewer #2 (Public review):

Summary:

The goal of this study was to find evidence for local adaptation in survival and fecundity of the model plant Arabidopsis thaliana. The authors grew a large set of Swedish Arabidopsis accessions at four common garden sites in northern and southern Sweden. Accessions were grown from seed in trays, which were laid on the ground at each site in late summer, screened for survival in fall and the following spring, and fecundity was determined from rosette size and seed production in spring. Experiments were complemented by 'selection experiments', in which seeds of the same accessions were sown in plots, and after two years of growth, plants were sampled to determine fitness from genotype frequencies, providing a more comprehensive evaluation of lifetime fitness than can be gleaned from fecundity alone.

To clarify, fecundity was determined from total plant area using photos of the mature stems, not the rosettes or direct counting of seeds. That said, it is true that our fecundity estimate was well correlated with rosette area. Furthermore, we validate our fecundity estimates by showing they were highly correlated with seed production estimated by measuring and counting siliques on a separate set of plants grown under common garden conditions in one of our sites (Brachi et al.2022).

As the main result, southern accessions had higher mortality in northern sites in one of two years, but also suffered more slug damage in southern sites in one year, indicating a potential link between frost tolerance and herbivore resistance. Fecundity of accession was highest when growing close to the 'home' environment, but while accessions from one sand dune population in southern Sweden had among the lowest fecundities overall, they consistently had the highest fitness in the selection experiment. Accessions from this population had large seed size and rapid root growth, which might be related to establishment success when arriving in a new, partially occupied habitat. However, neither trait could fully explain the very high fitness of this population, suggesting the presence of other, unmeasured traits.

Overall, the authors could provide clear evidence of local adaptation in different traits for some of their experiments, but they also highlight high temporal and spatial variability that makes prediction of microevolutionary change so challenging.

Strengths:

A major strength of this study is the highly comprehensive evaluation of different fitness-related traits of Arabidopsis under natural conditions. The evaluation of survival and fecundity in common garden experiments across four sites and two years provides an estimate of variability and consistency of results. The addition of the 'selection experiment' provides an extended view on plant fitness that is both original and interesting, in particular highlighting potential limitations of 'fitness-proxies' such as seed production that don't take into account seedling establishment and competitive exclusion.

Throughout the study, the authors have gone to impressive depths in exploring their data, and particularly the discovery of 'native volunteers' in selection experiment plots and their statistical treatment is very elegant and has resulted in compelling conclusions. Also, while the authors are careful in the interpretation of their GWAS results, they nonetheless highlight a few interesting gene candidates that may be underlying the observed plant adaptations, and which likely will stimulate further research.

Overall, the authors provide a rich new resource that is relevant and interesting both in the context of general evolutionary theory as well as more specifically for molecular biology.

Weaknesses:

While the repetition of the common garden experiments over two years is certainly better than no repetition (hence its mention also under 'strengths'), the very high variability found between the two years highlights the need for more extensive temporal replication. In this context, two temporal replicates are the bare minimum, and more repeats in time would be necessary to draw any kind of conclusion about the role of 'high mortality' and 'low mortality' years for the microevolution of Arabidopsis. It also seems that the authors missed an opportunity to explore potentially causal variation among years, as they did not attempt to relate winter mortality to actual climatic variables, even though they discuss winter harshness as a potential predictor.

We agree that two years is insufficient to understand how variation in selective pressures compound over time to generate micro-evolutionary change. The eight-year data in Oakley et al. (2023), which we discuss in the paper, support this. Our results are nonetheless sufficient to demonstrate the idiosyncratic nature of selection. In the revision, we will further emphasize that far longer time series would be needed for definitive conclusions.

Our short time series is also why we do not try to correlate with climate data, as this would amount to doing statistics with four data points (mostly two groups of accession N vs S, with mostly homogenous climates within groups, and two years).

The low temporal variation also makes the accidental slug herbivory appear somewhat random. Potted plants are notoriously susceptible to slug herbivory, and while it is certainly nice that slug damage predominantly affected one group of accessions, it nonetheless raises the question whether this reflects a 'real' selection pressure that plants commonly face in their respective local environments.

We agree with this point as well. The evidence for selection on glucosinolates by generalist herbivores such as slugs is fairly strong, but the precise agent is not known, and probably varies over time and space. Our results merely demonstrate one possibility (and we will clarify this in the revision).

The addition of the 'selection experiment' is certainly original and provides valuable additional insights, but again, it seems a bit questionable which natural process really has affected this outcome. While the genetic and statistical analysis of this experiment seems to be state-of-the-art, the experimental design is rather rudimentary compared to more standard selection experiments. Specifically, the authors added seeds from greenhouse-grown mothers to experimental plots and only sampled plants two years later. This means that, potentially,y the first very big bottleneck was germination under natural conditions, which may have already excluded many of the accessions before they had a chance to grow. While this certainly is one type of selection, it is not exactly the type of selection that a 2-year selection experiment is set up to measure. Either initially establishing the selection experiment from plants instead of seeds, or genotyping the population over several generations, would have substantially strengthened the conclusions that could be drawn from this experiment.

We agree that more data would have been beneficial, and we do not make strong claims about the nature of selection. Among other phenotypes, we mention dormancy, and note that existing dormancy estimates do not predict fitness in our selection experiments. In addition the same seed batches germinated uniformly in the common-garden experiments with minimal stratification (we will note this in the revision).

Also, the complete lack of information on population density is a bit problematic. It is not clear if there were other (non-Arabidopsis) plants present in the plots, how many Arabidopsis plants were established, if numbers changed over the year, etc. Given all of these limitations, calling this a 'selection experiment' is in fact somewhat misleading.

Seeds were introduced into sites that appeared appropriate for A. thaliana, leaving the background community intact. We provided information on sowing density; the density of plants (A. thaliana and other species) that we obtained during the course of the experiments varied considerably between sites, much like in natural populations, although we lack systematic measurements. We will provide more information (including photos) in the revision.

Despite these weaknesses, the authors could achieve their main goals, and despite the somewhat minimal temporal replication, they were lucky to sample two fairly distinct years that provided them with interesting variation, which they could partially explain using the variation among their accessions. Overall, this study will likely make an important contribution to the field of evolutionary biology, and it is another very strong example of how the extensive molecular tools in Arabidopsis can be leveraged to address fundamental questions in evolution and ecology, to an extent that is not (yet) possible in other plant systems.

Reviewer #3 (Public review)

Summary:

The manuscript presents a large common garden experiment across Sweden using solely local germplasm. Additionally, there is a collection of selection experiments that begin investigating the factors shaping fecundity in these populations. This provides an impressive amount of data and analysis investigating the underlying factors involved. Together, this helps support the data showing that fluctuations and interactions are key components determining Arabidopsis fitness and are more broadly applicable across plant and non-plant species.

Strengths:

The field trials are well conducted with extensive effort and sampling. Similarly while the genetic analysis is complex it is well conducted and reflects the complexity of dealing with population structure that may be intricately linked to adaptive structure. This has no real solution and the option of presenting results with and without correction is likely the only appropriate option.

Weaknesses:

A significant finding from this study was that fecundity is shaped more by yearly fluctuations and their interaction with genotype than it is by the main effect of location or genotype. Another significant finding is that the strength of selection can be quite strong, with nearly 5x ranges across accessions. It should be noted that there are a number of other studies using Arabidopsis in the wild with multiple years and locations that found similar observations beyond the Oakley citation. In general, the context of how these findings relate to existing knowledge in Arabidopsis is a bit underdeveloped.

We shall remedy this in the revision (see also comments by Reviewer #1).

The effects of the populations across the locations seem to rely on individual tests and PC analysis. It would seem to be possible to incorporate these tests more directly in the linear modeling analysis, and it isn't quite clear why this wasn't conducted.

The fecundity estimates were modelled for all experiments simultaneously and the results are presented in Figure 6 to explore the relative importance of genotype effects and interaction terms including genotypes. For survival and fecundity, the BLUPS are generated from linear mixed models fitted for all experiments simultaneously including a random intercept effect for the genotypes within experiments. A principal component analysis is used to explore the pattern of accession effects (BLUPS) on fecundity (Figure 7); this will be explained in the Methods.

I'm a bit puzzled by the discussion on how to find causative loci. This seems to focus solely on GWAS as the solution, with a goal to sequence vast individuals. But the loci that the manuscript discussed were found by a combination of structured mapping populations followed by molecular validation that then informed the GWAS. As such, I'm unsure if the proposed future approach of more sequencing is the best when a more balanced approach integrating diverse methods and population types will be more useful.

We are puzzled by this comment in return. Our statement about more sequencing (penultimate sentence of discussion) was referring to achieving a better understanding of the history of migration and selection rather than identifying causative loci. Happy for clarification!

References

Brachi, Benjamin, Daniele Filiault, Hannah Whitehurst, Paul Darme, Pierre Le Gars, Marine Le Mentec, Timothy C. Morton, et al. 2022. “Plant Genetic Effects on Microbial Hubs Impact Host Fitness in Repeated Field Trials.” Proceedings of the National Academy of Sciences of the United States of America 119 (30): e2201285119.

Oakley, Christopher G., Douglas W. Schemske, John K. McKay, and Jon Ågren. 2023. “Ecological Genetics of Local Adaptation in Arabidopsis: An 8-Year Field Experiment.” Molecular Ecology, June. https://doi.org/10.1111/mec.17045.