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1. Summary

This study aimed to investigate the impact of temperature and species interactions on microbial evolution, addressing the broader question of how climate change may shape eco-evolutionary dynamics. The authors evaluate how a focal population, comprising three genotypes of the protist Tetrahymena thermophila, changes in response to three temperatures (19, 22, and 25°C) and to competition with three protist heterospecifics. In the absence of interspecific interactions, increasing temperatures led to decreased genotypic diversity of the focal population, but the presence of each heterospecific modulated focal population diversity and density in different, temperature-dependent ways. Focal population density and diversity were found to correlate with the growth rate of the heterospecifics.

2. General Assessment

Whereas most microcosm experiments investigate the effects of temperature or biotic interactions on evolution independently, this study was novel in examining their combined effects. Therefore, the principles demonstrated in this work could guide modelling efforts to more accurately predict how organisms respond to climate change by incorporating community contexts. In terms of scientific rigor, the use of microcosms minimized confounding variables and allowed for clear inferences about causal relationships. In addition, the use of fluorescently tagged genotypes to count cells via flow cytometry implies that the data collected was highly accurate.

However, though the authors concede that the generalizability of this study is limited, they did not address other pertinent concerns regarding significance and generalizability. In particular, the temperature range investigated in this study was restricted to what is typically experienced in the protist growth season and does not include extreme temperatures predicted under climate change scenarios. Other possible limitations of the study, such as possible variation within the inoculated bacterial prey communities, are noted as minor revisions. In addition, the seven-day experimental period reflects short-term responses, which may not be translatable to longer-term evolutionary consequences of climate change.

3. Major Concerns

Our foremost concern is that the temperature range used in this study (19-25°C) is relatively limited and does not reflect the full range of environmentally relevant temperatures, especially within the context of climate change. Additionally, the optimal growth temperature for T. thermophila is higher than the temperatures tested, with the fastest reproduction rates around 30°C [1]. Thus, the chosen temperatures may not be realistic for this organism. Performing additional experiments at higher temperatures would be ideal, but not absolutely necessary; as an alternative, we suggest the authors explain their choice of temperatures and acknowledge the limitations of these temperatures, as they cannot confidently extrapolate their results to conditions caused by climate change.

[1] Tarkington, J., Abbott, K. C., & Petchey, O. L. (2021). Temperature affects the repeatability of evolution in the microbial eukaryote Tetrahymena thermophila. Ecology and Evolution, 11(18), 12389–12401. https://doi.org/10.1002/ece3.8036

4. Minor Concerns / Line Edits

L87: “...can in turn influences” – change to “influence”

L107: “…a heterospecific… can either decrease genotype performance… or fundamentally alter the genotype-specific thermal response” – What about increasing genotype performance, as suggested in the last sentence of this paragraph, and also in the results?

L120: “diversity (i.e., evolution)” – It would be good to emphasize rapid evolution here; the paper does so in other places, but this is the first place that clearly lays out what this work intends to measure.

L123: “play a role in it at all?” – “it” is unclear verbiage.

L151: “dionized” – “deionized”

L152: “The media was inoculated with a bacterial community from Duke Forest Gate 9 pond/Wilbur pond” – The manuscript assumes the bacterial communities are identical across conditions, and this assumption is not addressed. The use of an environmental source of bacteria provided a realistic context for protist predation; however, differences in the inoculated communities were not addressed, i.e., it was assumed that the inoculated bacterial communities were the same. It is possible that differences in these communities could have contributed to the effects of heterospecific competition on the frequency and diversity of T. thermophila and could have been used to account for variation amongst replicates.

L156: “Once we account for the complexity of the bacterial community, our microbial communities (protists + bacteria) are much more similar to what might be observed in nature than what is commonly used in the laboratory… and arguably represents an entire complex microbial community, despite its simplicity at the protist level.” – This is overstated; the bacterial community growing under lab conditions may be significantly different from what is present in nature.

L188: “Experiment lasted 7 days, we sampled all microcosms to estimate genotypic frequencies at the end of the experiment” – The seven-day experimental period reflects short-term responses, which may not be reflective of a stable microbial community. We suggest the authors explain why 7 days was chosen. (Also, this is a run-on sentence.)

L251: “Temperature was excluded from the final model because it did not show a significant effect in this analysis.” – The insignificance of temperature here should be addressed/interpreted in the Results or Discussion, since one of the paper’s main claims is that evolutionary outcomes are temperature-dependent.

L281: “there was no difference in growth response across temperatures among genotypes (Table S3)” – When claiming “no difference,” especially for a key result that underlies the interpretations of subsequent findings, the authors should include the statistical significance (Pr(>F) value) inline.

L340: “Specifically, a response that is larger (smaller) than the control along the density axes points to a larger (smaller) ecological response to the treatment… a response that is larger (smaller) relative to the control along the genotypic diversity axis suggests a larger (smaller) evolutionary response to the treatment compared to control. A larger (smaller) response along both axes simultaneously suggests larger (smaller) coupled or joint ecological and evolutionary responses relative to control.” – The purpose of the “(smaller)” here is unclear; also, shouldn’t the size of the response be defined by the distances from the standard deviations of the control, and not the absolute values “along the axes”?

L366: “evolutionary change –characterized as shifts in genotypic frequencies– ” and L373: “ecological interactions, operationally defined here as the net effect of heterospecifics on the focal population density” – It was not entirely clear until this point (the Discussion) how “evolution” and “ecology” were being operationalized for these experiments; this should also be emphasized in the Results.

L391: “evolutionary change across treatments, can result from” – Typo: there shouldn’t be a comma.

L407: “like increased temperatures (Singleton et al., 2021), or grazing” – Typo: there shouldn’t be a comma.

L435: “We hope that understanding why that is may be the subject of future...” – “that” is vague.

L461: “...therefore suggesting that they do not in fact” – remove “therefore” and “in fact”

L485 (Figure 1) – Make the legend for panel B larger (specifically, make the cross-hatch pattern to scale); the pattern gets lost when zoomed out

L486 (Figure 1) – The title for panel D, “(temperature and heterospecific interaction)”, is unclear; it could be parsed as [(temperature) and (heterospecific interaction)], but you likely mean [(temperature and heterospecific) interaction].

L501 (Figure 2) – Label each panel’s x-axis, or stack the plots vertically so the x-axis label reads as applicable to all three plots.

L502 (Figure 2) – We would like error bars for frequencies in panel B.

L510 (Figure 3) – We would like error bars for frequencies in panel A, and a legend explanation of the colors in panel B.

L517 (Figure 4) – We would like a key for the shapes in the figure panels, not just in the legend text.

L533 (Figure 5) – Y-axes on panel A and B are the opposite of what’s written in the legend text and the Results. We would like a key for shapes and colors in the figure, not just the legend text.

Recommendation:

Major revision

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.