Negative frequency-dependent selection maintains partner quality variation in a keystone nutritional mutualism
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Abstract
Mutualisms, interactions that benefit both partners, are critical for promoting biodiversity and ecosystem resiliency, yet are considered evolutionarily unstable and vulnerable to global change. Understanding how genetic variation in mutualisms is maintained is key to predicting their future persistence and explaining their long evolutionary history. While theory addresses factors maintaining variation in partner quality (the fitness benefits partners provide), few studies have experimentally tested these mechanisms. Here, we experimentally evolved multiple replicate populations of rhizobia, nitrogen-fixing mutualists of legumes, varying in partner quality under contrasting environments: nitrogen (N)-supplemented or N-free conditions, with or without host plants. After one year of rhizobial evolution, we quantified selection on partner quality across environments and evaluated resulting changes in mutualism traits and population-level genetic diversity. Strikingly, selection on partner quality was population-dependent: high quality strains were favoured when initially rare but disfavoured when common, revealing negative-frequency dependent dynamics that can maintain variation. Although neither N-supplementation nor host presence directly imposed selection, both were critical for preserving genetic diversity in rhizobia populations – fuel for ongoing evolution. By demonstrating negative-frequency dependent selection in the legume-rhizobium mutualism, our study reveals a dynamic more akin to antagonistic interactions than traditionally assumed. This overlooked mechanism may be the key to explaining the ecological and evolutionary persistence of mutualisms under changing environments.
Significance Statement
Mutualisms are central to biodiversity, yet their stability is puzzling because the mechanisms thought to stabilize them tend to eliminate the genetic variation needed for continued adaptation, even though partners in nature show striking variation in quality. In an experimental evolution study of a keystone plant-microbe mutualism (legume–rhizobium symbiosis), we found that negative frequency-dependent selection allows high- and low-quality symbionts to coexist: high-quality strains are favoured only when rare. Environmental factors such as nitrogen addition or host presence did not alter which partners were selected, but they helped maintain genetic diversity needed for future adaptation. Our findings reveal an overlooked mechanism that stabilizes mutualisms and explain how these interactions can remain resilient under changing environmental conditions.
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This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/19394552.
General Summary:
The study made observations that contribute to the field of rhizosphere microbial ecology. Isolates for the evolution of rhizobial populations were used from field plots that had fertilizer for N-supplementation isolate populations and from plots without fertilizer for lacking N-supplementation. Experimental evolution was performed on soil seeded with three rhizobial population types: low, medium, and high quality partners, and in the presence of plants. Additionally, the authors performed lab evolution with the medium quality partner population with plants absent. After evolution, plant fitness metrics were collected, and isolates from nodules were streaked out and …
This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/19394552.
General Summary:
The study made observations that contribute to the field of rhizosphere microbial ecology. Isolates for the evolution of rhizobial populations were used from field plots that had fertilizer for N-supplementation isolate populations and from plots without fertilizer for lacking N-supplementation. Experimental evolution was performed on soil seeded with three rhizobial population types: low, medium, and high quality partners, and in the presence of plants. Additionally, the authors performed lab evolution with the medium quality partner population with plants absent. After evolution, plant fitness metrics were collected, and isolates from nodules were streaked out and sequenced. One of their key findings is that lower- and medium-quality Rhizobial populations in clover exhibit stabilizing selection, while higher-quality microbial populations exhibit negative selection. Overall, these results show that the populations tend to become a more medium quality population as they evolve. As well, N supplementation treatments gave interesting results where, in evolved populations, there are more rare strains enriched.
General Assessment:
400 generations show a longitudinal view of selection in mutualism, although the paper reports only the rhizobial community composition at the beginning and end of each condition. N-supplementation and plant presence conditions genetic diversity data gave novel key findings that may lead to future work on mutualism evolution between rhizobia and plants. Moreover, the introduction was organized well and clearly established the background and knowledge gap in the field. Their system showed innovation in their experimental setup, being able to separate N supplementation as an independent variable from other soil factors on the evolution of partner quality.
Another significant finding is that Nitrogen and plant host presence are key drivers of the maintenance of genetic diversity. While statistical power is limited with 3-4 replicates, the observed evolutionary trends gathered from this study could be the foundation of more focused, higher-powered future studies. Inclusion of details referred to in the supplement/appendix will be needed, as references to these materials include important details on experimental design and data handling. The researchers make claims that are incompletely supported by the data shown in the figures which decrease the strength of their conclusions.
Major concerns:
Phenotype changes are unknown if they are caused by genetic factors: Genetic differences among individuals and populations for both the pre-evolved and evolved populations need to be examined to determine if the phenotypes seen in the experiments are driven by population-level dynamics or by small common genetic variation driving the phenotypes. Leverage WGS data to investigate the shift in specific allele frequencies between pre-evolved and evolved populations.
Results are not sufficient to support the primary claim of the paper: The authors make strong claims about selection of high and low quality populations and the impact of population quality and N-availability on plants, but does not acknowledge that the low statistical power of 3-4 replicates and how that might limit the claims they are making, ie confidence intervals are too variable, and p-values are not significant. Language/tone used in this manuscript should be softened.
Negative frequency-dependent is an unsubstantiated claim, as data is not shown for each population of what strains are rarer in the population: Lack of raw data; Are there any specific strains that are selected for? What are their genotypes?
Minor concerns:
Lack of clarity in the description of experimental methods and statistical analysis. The description of the experiment was extremely unclear. They cite a figure where they outline their experimental method, but put it in the supplement. It would be helpful to include this as Figure 1. There are also multiple details they do not specify about their experimental methods, e.g.
L401-402: "We experimentally evolved three rhizobial population types that differed in the initial frequency of high-quality strains within each starting population…" -Needs more information on the composition of these strains and how they were chosen. Currently, it is difficult to understand how high/low quality is defined and what the genotypic composition of these populations is.
L406-407: "… either Nitrogen supplemented (N+) or nitrogen free (N–) environments" – The specific method of N-supplementation (i.e., the type and amount of fertilizer used, and the dosing scheme) should be mentioned when describing the experimental design.
L144, where they describe their experimental scheme, could be reworded, along with this figure, to make the design clearer
Lack of figure clarity. There are several acronyms in the figure titles and captions that are not defined, no figure legends, and values that are dumped on the axes with no explanation.
Figure 3: The legend does not specify what each of the plot titles means. It would be helpful to include a key such as "LQP+: Low quality population in host presence"
Figure 1: Line thickness makes it pretty difficult to distinguish between lines. Additional explanation of what each of the points means might make it more intuitive to readers.
Figure 2: Could be a panel in Figure 1, as it is used to support the claims made in Figure 1.
The proposed mechanism of maintaining low quality strains is unsubstantiated. Please address this by adding caveats or supplement this claim with data.
In Figure 1, a significant portion of N+ supplementation isolates have 0 relative fitness and very varied standardized partner quality. This is unexplained, but the data are very stark in the plot.
Competing interests
The authors declare that they have no competing interests.
Use of Artificial Intelligence (AI)
The authors declare that they did not use generative AI to come up with new ideas for their review.
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