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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:

1. 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.

2. 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.

3. 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:

1. 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.

   1. 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.

   2. 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.

   3. L144, where they describe their experimental scheme, could be reworded, along with this figure, to make the design clearer

2. 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.

   1. 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”

   2. 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.

   3. Figure 2: Could be a panel in Figure 1, as it is used to support the claims made in Figure 1.

3. The proposed mechanism of maintaining low quality strains is unsubstantiated. Please address this by adding caveats or supplement this claim with data.

4. 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.