Elevated temperature and CO2 strongly affect the growth strategies of soil bacteria

The trait-based strategies of microorganisms appear to be phylogenetically conserved, but acclimation to climate change may complicate the scenario. To study the roles of phylogeny and environment on bacterial responses to sudden moisture increases, we determine bacterial population-specific growth rates by 18O-DNA quantitative stable isotope probing (18O-qSIP) in soils subjected to a free-air CO2 enrichment (FACE) combined with warming. We find that three growth strategies of bacterial taxa – rapid, intermediate and slow responders, defined by the timing of the peak growth rates – are phylogenetically conserved, even at the sub-phylum level. For example, members of class Bacilli and Sphingobacteriia are mainly rapid responders. Climate regimes, however, modify the growth strategies of over 90% of species, partly confounding the initial phylogenetic pattern. The growth of rapid bacterial responders is more influenced by phylogeny, whereas the variance for slow responders is primarily explained by environmental conditions. Overall, these results highlight the role of phylogenetic and environmental constraints in understanding and predicting the growth strategies of soil microorganisms under global change scenarios.

The work appears to be well and carefully done and the data and analyses are of great value to the larger microbial ecological community. There are very few studies in which the growth rates of bacterial populations from FACE experiments have been investigated via qSIP and the large quantity of data that comprises the study described in this manuscript, will substantially advance our knowledge of bacterial responses to elevated CO2 and temperature manipulations. However, I think the manuscript will require major changes and a considerable rewrite.
My chief complaint about the manuscript is that it is too long because of redundant information. For instance, L114 describes the soils that were collected in the results section, it is again described in L432 and in the legends of various figures. Similarly, the discussion section has many sentences that restate findings presented in the Results section. Another example is the description of growth strategies (L192 to 204) which occurs repeatedly in the manuscript. It is important to write a concise manuscript in which information is only presented once.
I also find that the discussion section is too long and while it contains presentation of other studies, these findings are often not well related to the growth rates of bacteria measured by the authors. For instance, the authors did not measure the number of rRNA genes on a taxa's genome and so there appears limited need to extensively discuss relationships between rRNA copy number and growth rates. If there are difficulties relating other studies to the authors findings, the discussion of those studies should be removed from the paper. I think the introduction focuses too much on the "Birch effect". While soil wetup effects are well documented, they always occur when liquids, be it water or a glucose solution, are added to soil. I think in soils that routinely dry and wet back up, unlike soils from Mediterranean climates such as California, have minimal Birch effects upon rewetting. The paper is not really about the "Birch effect", it is about the impact of CO2 and temperature on bacterial growth rates in soil and the introduction should focus on that.
I suggest changing the title of the manuscript from "Phylogenetic constraint and environmental acclimation link to soil microbial growth strategies under CO2 enrichment and warming." To something like: "Elevated temperature and CO2 strongly affect growth strategies of soil bacteria." More minor edit suggestions: Abstract: I suggest deleting the first few sentences of the abstract and start with the sentence in Line 25.
L135 insert "the" before "6" L43 Delete "The evidence is overwhelming that" L159 change "the" to "a" before phylogenetic L217 change "shared with three comparisons" to "shared among the three treatments" I struggle with the following argument: "The essential genes for microbial growth are considered to be the core genome, which is generally inherited vertically and reflects phylogeny strictly. This helps explain the observed phylogenetic conservation of the strategies related to growth response." How do the authors know that the core genome allows microorganisms to grow faster or slower (i.e. be more competitive)?
L280 Suggest change of "visually evident based" to "evident". L293 Suggest changing "validated" to "supported" L295 Suggest deleting "This helps grasp the relationship between phenotypic characteristics and the phylogeny of microorganisms. L 309 Does the sentence "Microbes possessed metabolic plasticity for carbon use to suit resource availability or interspecies interactions, which promoted or reduced microbial growth28, 29, 30 refer to microorganisms in this study? If not, that should be clearly indicated. L310 "extremely high intraspecific variation of the genome" It is unclear what genome this sentence refers to L355 suggest changing "for resources under limitation" to "for limited resources". L357 suggest changing "mitigated" to "mitigating" L370 suggest changing "microorganismic" to "microbial".
L442 "The incubations were described in detail previously6." I think this refers to another study and not the actual incubations in this study.
L513 suggest deleting "As 18O labeling occurs during cell growth via DNA replication"  Figure 6 seems to recap information from other figures and could also be removed from the manuscript Reviewer #2 (Remarks to the Author): The manuscript by Ruan et al. describes the growth responses of soil bacteria following rewetting in soils from a long-term global change experiment. The authors used qSIP with 18O water to measure microbial growth rates after 1, 3, and 6 days. Based on the day of maximum growth taxa were classified as "rapid", "intermediate" or "slow" responders. In general, the experimental design and methods appear to be of sufficient quality. The results are likely to be of interest to the microbial ecology community and may help advance our understand of how microbial systems will respond to global change.
Introduction -The introduction does not adequately establish why understanding the influence of phylogeny on function is important.
Line 126 -I suggest you change "adapted" to "acclimated" Line 42 -Remove "conserved" Line 144 - Fig 2 shows proportions but is labeled as (%). The percentages are a little confusing. I think it would help if you reminded the reader how to interpret them. For instance, 29% of new 16S rRNA gene copies.
Line 172 This is incorrect -Blomberg's K can be greater than 1. Make sure you fully understand the statistics you are employing.
Line 176 If a trait is conserved it should have a higher phylogenetic signal than expected under Brownian motion (k>1). This is not the case for the responses observed, there is an influence of phylogeny but that does not indicate it's conserved.
Line 226-236 The Picrust analysis and it's results here and the heat map in Fig 4c are not well integrated with the rest of the manuscript. They feel tacked on and do not meaningfully enhance the manuscript. I suggest they be removed as they detract from the focus of the paper.
Line 237 This section would fit better with the other phylogenetic analysis.
Line 294 "unifying life strategies" , this language is far too strong to described the responses you measured.
Line 307 You have no evidence that environmental section is acting on the trait you measured.
Lines 311 -315 I think this interpretation is too strong. There was not statistical analysis to show that rates were significantly higher or lower across the 3 time points thus while the categorization may have changed it's unclear whether this is a truly meaningful change in the timing of growth. Even if the time of growth did change, this is not necessarily a result of genomic changes, it's more like to be a consequence of altered biotic and abiotic conditions changing the expression of genes.
Line 320-324 This analysis should be presented in the results. Also the r2 values are very low suggesting that if density dependent selection is occurring it's explaining less that 10% of the variation in growth rates.
Line 338 This context about the study site should be provided much earlier in the manuscript because it's highly relevant to understanding the mechanisms of T and eCO2 influence the microbes by changing plant processes and soil resources.
Line 345 -354 This paragraph is much more helpful than many of the previous. I suggest the authors expand this discussion and connect it better the growth responses.
Line 355 Resources are always limiting, perhaps it would be better to highlight the stoichiometric imbalance?
Line 442 Suggest change to "Incubation conditions were similar to those previously described". So it's clear it's not the same experiment.
Reviewer #3 (Remarks to the Author): Summary. The increase of greenhouse gas emissions give rise to a global warming effect that ultimately influences both above-ground and below-ground processes across Earth's ecosystems. Microbial communities, which underpin the biogeochemical processes that often shape ecosystem function, are no exception to this, and although research shows they can adapt to this warming, population dynamics and ecophysiological responses to these disturbances are not currently known. The manuscript reviewed titled "Phylogenetic constraint and environmental acclimation link to soil" set out to describe an integrated, trait-based understanding of these dynamics with their data which includes 16S rRNA sequences, and qSIP. This research evaluates two fundamental hypotheses, that were clearly outlined in the introduction and will be referred to for the rest of this document as H1 and H2. H1: Microbial growth is phylogenetically conserved in response to nutrient pulses following rewetting events. H2: Warming shifts pre-existing life strategies, which would conflict with phylogenetic signal.
Overall-the manuscript was not challenging to read and except for a few spaces I noted below, I could track the framing quite easily without a sizable background in this specific research area. I appreciated the linkage of climate perturbations in the field to wetting events in the lab, especially the use of growth accelerated and growth delayed. There were 4 areas where additional clarification was warranted. These comments are addressed below in more detail but broadly summarized here.
(1) The hypothesis testing framework doesn't seem robustly evaluated in the text as written.
(2) Novelty of this study relative for other literature -While I recognize after skimming these other prior papers (two by Firestone lab, one by Blasewtiz is quite similar) there are novel areas in this new work, this needs to be more clearly reinforced by the authors, especially for a broader readership like in Nature Communications, to appreciate the relevance of this research. More minor, but added up over time, (3) experimental design considerations and (4) language precision with necessary caveats.
[1] Testing of the hypotheses as currently framed and with data reported in the results. a. I didn't see data that supports the hypotheses as they are written. It thus reads like the story is based on assumptions of processes happening, not actual measurements. For example for H1, "Microbial growth is phylogenetically conserved in response to nutrient pulses following rewetting events" I would expect to see measurements of nutrient pulses. There is from what I can see no measurement of the nutrient pulse (DOC, TN, TC, CO2 fluxes) as other studies on this topic have done. Thus the authors seem to assume the mechanism without showing it. I would also expect to see metric of rewetting which would have initial, final water content. I note both these types of data are in used in Blacewisz, but also it seems glaring given how strongly the hypothesis was framed. Key data is missing that would be necessary to support this hypothesis as written. b. For H2-what is meant by life strategy? I didn't see this defined in the introduction, nor in the results where this hypothesis was evaluated. Can the authors add more clarity/specifics here-How was life strategy explicitly defined here, how wasa it measured, what was the data? c. Stylistic suggestion, remove italics on the hypotheses in the intro. d. Can the authors put the timing of the original field experiment in the methods and or figure 1 experimental design, apologies if I missed it (line 439 in methods and line 85 intro discusses long term but I have no idea how this was done by content in this manuscript). This is important for H2 evaluation and only in line 126 do I infer that experiment that these soils were derived from was a decade? Please add this text clearly to fig 1 and methods so readers do not have to find another journal to understand basics of this story nor wait until results to understand experimental design (also apologies again if I missed it!). e. Lastly given the authors go to great lengths to call out their hypotheses (italics) I wanted to the results to better link to these in the text. On revision I suggest more clear links would be helpful as it is clear I had a tough time tracking exactly what the authors were testing to support these claims.
[2] Novelty of this work relative to prior work. I am loosely familiar with work that is cited in this manuscript (Placella et al) that evaluated a similar concept in grassland soils, published in PNAS 2012. "Rainfall induced CO2 pulses result from sequential resuscitation of phylogenetically clustered microbial groups". This research aligns very closely with this study and uses nearly identical methods (qSIP), "taxon specific microbial growth and mortality patterns reveal distinct temporal population responses to rewetting in California grassland soils" Blacewisz et al, 2020. I found myself wondering what sets this work apart from the prior work and the new insights that were provided here. I think the final take-away and why we should care also could be better communicated for relevance to this journals readership.
[3] Experimental design (1) I have some concerns about the experimental set up. From what I read in figure 1 I did not see a pre-wet treatment (please confirm this), everything was compared to samples collected after the treatment. As cited in the intro, and other papers have all shown, a rapid response is already observed within 1 hour thus growth rate needs to be measured from prewet condition. How long is "immediately"-was the entire experiment set up and them samples removed after? If accurate, address concerns with experimental design, or if I missed it add more clarity to Fig 1. I believe this first since not a true control should be T1 (after treatment) as T0 implies before effect and the papers cite a rapid effect following this treatment. This comes important right away as I found myself wondering if on line 122 the differences in richness were due to wetting event or prior historical artifact. Note prior study by Blasewitz that authors reference often used these treatments and responder categories. a. Prewet {important control} b. Primary 3 hours c. Secondary 24 hours d. Tertiary 72 hours e. Delayed 168 hours (2) Prior studies used fresh soils-collected within a week of incubation. However this study used soils that had been stored in the fridge or at 4C. For how long prior to the study being done-I was confused by language at the end of line 435 and the beginning of line 443 (fresh). Why were the soils sieved as that disrupts structural features? (3) Line 508: What version of RDP Classifier was used? (4) Line 511: The accession numbers of the deposited 16S rRNA data is stated as "not available until 2023/06/01". This needs to be updated on the resubmission as we could not download the data. (5) Line 159 The authors said they used same growth classification as in Blascewisz (which I appreciated linking to prior papers), but I didn't see that manuscript used language of rapid, intermediate, or slow. In fact I couldn't find intermediate in that manuscript -on a quick search. This becomes an issue and I found myself unsure if their calls for "slow / medium / fast" responders are appropriate as this wasn't entirely easy for me to follow. For example, looking at Figure 2, I can see that abundances for some of the groups are very similar across all days, and so calling an organism as "rapid" because it is ever so slightly higher in sample days 0-1 than in sample days 0-6 seems imprecise. However, when they show Fig 3 and Z-Score those values I can definitely see some distinctions by the growth rate categories. Can the authors clarify how this was done here, as this is important to understand so figures/results can be interpreted.
[4] Language precision (1) Mainly, the link between microbial taxonomy and microbial metabolism is known to not be straightforward, especially in diverse microbial communities like soils that lack good reference genome databases. I do appreciate that the authors introduce caveats to the end of discussion paragraph 2 very well -and so I am aware they know the limitations of their methods. However, it would be helpful to start framing those caveats earlier within discussion paragraph 1 as well as throughout the results section when the piecrust was introduced. For context, as a reader who does metabolisms in soils I found myself somewhat disappointed when piecrust data was introduced and not properly caveated. Particularly, it might be helpful to introduce briefly at lines 226-236 in the results and lines ~278. These caveats are necessary to not potentially overstate the author's findings, and to make sure the reader knows the authors understand the very clear limitations of these methods.
https://microbiomejournal.biomedcentral.com/articles/10.1186/s40168-020-00815-y With this approach, we found reasonable performance for human datasets, with the metagenome prediction tools performing better for inference on genes related to "housekeeping" functions. However, their performance degraded sharply outside of human datasets when used for inference. Also more: https://academic.oup.com/gigascience/article/doi/10.1093/gigascience/giab090/6505123 (2) In the discussion section there must be some additional caveats introduced to some of the claims the authors are making. Some of the language that is used within discussion paragraphs require data that the authors do not have (e.g., whole-genome sequencing or metagenomics), or that are somewhat contradictory to their results (e.g., "we identified major forces of variation" when only 20% of the data is explained by these metrics). As such, softening of the language is recommended-lines are noted below-and these will not adjust the outcomes of this study, but support more precise interpretation. Line 274-275: Please be mindful and introduce some caveats to the statements made on these lines. What else other than phylogeny and "core genes" could possibly drive these differences?
Line 330: Consider removing this final sentence The explanation in the upcoming paragraphs is both helpful and needed prior to making the (valid!) claim you are making on line 352.
Line 366-368: Careful with the phrase "…we identified the major forces…". Changing this to "we identified some of forces…" is prudent given we do not know all factors. Specifically, there is less than 20% variation explained by these variables in both the rapid and intermediate responders. In other words, 80-89%% of the variation is not explained by these data, and you cannot call them major drivers. Further, please add a strong caveat into this section that speaks to what the other 90% of variability in Rapid and Intermediate responders might relate to, as it is clearly not phylogenetic or environmental acclimation.
Line 374: Consider softening this language. There are possibilities not accounted for here. "…consisting of numerous proteins, evolved more slowly…" with "…consisting of numerous proteins, usually evolve more slowly…" Line 377: Consider softening this language. Change "…microbial trait of rapid growth was inherited vertically…" to "…microbial trait of rapid growth was likely inherited vertically…".
Line 389-390: The claim that they are governed by specific environments is somewhat misleading. They are primarily governed by environmental variables that may or may not be conserved across an environment. Please rewrite this sentence to reflect that.
Line 392: Consider adding in "…the significance of phylogeny and their respective functional traits…" Line 393: Per your results, this was not always the case. Please soften this language to reflect your results. "…response to wet-up events are mostly phylogenetically conserved…" Line 398-399: This claim needs a citation -as you do not have "global" evidence for this. Alternatively, the claim needs to soften the language. "…within a given species locally, and, potentially, globally." [5] Specific line comments: please not just 16S all throughout the manuscript-it should be "16S rRNA gene" to signal primers were used to amplify this gene or similarly "16S rRNA amplicon".
Line 178 tone down claim-suggesting that "under the conditions evaluated here" vertical inheritance was essential for the distribution of growth rate traits in response to rewetting. I think functional traits is a bit oo broad.
Line 60: I believe maybe the intended word was "mediated" instead of "medicated"?
Line 111: Consider making this title reflect the major result of the section as you did with the rest of the titles. It helps the reader focus on the message.
Line 133: according to (6). Consider just writing to "according to a previous publication (6)" as the formatting will look better.
Line 159: Change "The phylogenetic tree…" to "A phylogenetic tree…" and consider joining the sentence after for clarity. "A phylogenetic tree including all responders … was constructed, and 3 phylogenetic indices were used to estimate the …".
Line 170: Consider adding the word additional to "We used two additional indices…", as you mention above you have 3, and these are the other two.
Lines 226-236: Please add a sentence here regarding the caveat that microbial phylogeny is not always representative of microbial metabolism.
Line 239: Are you trying to say that all categories, with the exception of intermediate responders in eT, were clustered at phylogenetic branches? If so, please consider writing this sentence like that to enhance clarity.
Line 247: Consider adding the word ultimately to: "…that a species expresses, which ultimately weakened the strength of phylogenetic patterns." to make your closing statement more powerful.
Line 281: How many are "most" members? It would be helpful to add in exactly how many.
Line 315: The tense for this should read: "which is ubiquitous in the microbiome and considerably shapes the structure and functions…". Style wise I generally avoid the term ubiquitous as it means in everything and since we cannot measure to exhaustion in microbes…but that is my thing I realize.
Line 325-328: Please reword this so that it is understood that these findings are from another study.
Maybe start the sentence with "A general concept is that these…, and previous works have shown that under resource limitation, per capita resource…". Line 332: Consider removing "and elevated CO2 concentration" from this first sentence, as this paragraph is on warming specifically, and the following one is about CO2 concentration.
Line 345: Consider adding in the removed "and elevated CO2 concentration" from the comment above here. This then frames this paragraph as the CO2 paragraph.
Line 370: Add the word "and" to "…former, AND slow responders by the latter" for clarity.
Lines 373-378: Careful with the statements in these last few sentences. Some caveats and softening of language here are necessary. The growth-related genes do not need to be located on the core genome, as the referenced paper (27) states. It is possible it is related to environmental adaptations, and some phylogenies are more prone to that than others due to a myriad of factors. Softening the tone with words like "tend to be located" instead of "are located" (line 374).
Line 541: What happened with the taxa that did not fit into the 3 delimited strategies? Were they removed? Kept? Please elaborate here.  Figure S3: Change "birth rates" to "growth rates" to keep consistent with axes label. Also -the legend seems to be missing from this supplementary figure. Please add. Also -from the manuscript text: "Taxa with fast growth rates were conserved within several 143 bacterial phyla ( Fig. 2 and Fig. S3)". Could you label these within Figure S3 as you did for Figure  2? What are the smaller bar plots inside some of these bar plots?

Reviewer #1 (Remarks to the Author):
The authors determined microbial population-specific growth rates by 18O-DNA quantitative stable isotope probing (18O-qSIP) in soils subjected to a free-air CO2 enrichment (FACE) combined with warming. The soils were sampled from the FACE experiment in Kangbo village Guli Township, Changshu municipality in Jiangsu Province, China, and the experiment started in 2010. The authors found that bacterial taxa could be separated into three distinct groups with different growth strategies which were phylogenetically conserved in soils that were not manipulated with higher CO2 or temperature environments but when these manipulations were implemented the phylogenetic signal disappeared.
The work appears to be well and carefully done and the data and analyses are of great value to the larger microbial ecological community. There are very few studies in which the growth rates of bacterial populations from FACE experiments have been investigated via qSIP and the large quantity of data that comprises the study described in this manuscript, will substantially advance our knowledge of bacterial responses to elevated CO2 and temperature manipulations. However, I think the manuscript will require major changes and a considerable rewrite.
Response: We appreciate the encouragement and positive comments. We have followed the suggestion to considerably rewrite the manuscript in the Introduction, Result and Discussion sections, shown in our point-to-point responses below.
My chief complaint about the manuscript is that it is too long because of redundant information. For instance, L114 describes the soils that were collected in the results section, it is again described in L432 and in the legends of various figures.
Response: We have carefully gone through the manuscript to remove the redundant sentences. We have also sought help from native English speakers to polish the language. We apologize for not listing all of those changes in this response letter since there are too many changes, which are marked in red in the revised manuscript. Using the example pointed out by the reviewer, we have only retained the information about soil collection at the beginning of the Results section (Line 116-118) after deleting similar sentences elsewhere in the main text and figure legend.
Similarly, the discussion section has many sentences that restate findings presented in the Results section. Another example is the description of growth strategies (L192 to 204) which occurs repeatedly in the manuscript. It is important to write a concise manuscript in which information is only presented once.
Response: Since the comment is related to the preceding one, please see our response I also find that the discussion section is too long and while it contains presentation of other studies, these findings are often not well related to the growth rates of bacteria measured by the authors. For instance, the authors did not measure the number of rRNA genes on a taxa's genome and so there appears limited need to extensively discuss relationships between rRNA copy number and growth rates. If there are difficulties relating other studies to the authors findings, the discussion of those studies should be removed from the paper.
Response: We have substantially shortened the Discussion by deleting the contents of other studies that have little relevance to the growth rates in the discussion section, including the discussion about the relationship between rRNA copy number and growth rates, and the discussion about the variations of genome caused by climate change.
Below are some, but not all, examples that have been deleted in the Discussion, 1) "Members of the class Bacilli generally have high copy numbers of rRNA operons, enabling them to produce ribosomes rapidly and assimilate substrates immediately following nutrient addition"; 2) "In addition, the extremely high intraspecific variation of the genome also promotes such fast adaptations"; 3) "Finally, these shifts in life strategies within species may have emerged through horizontal dissemination of genes, which is ubiquitous in the microbiome and considerably shaped the structure and functions of global ecosystems"; 4) "Slow responders, with low maximum growth rates and low rRNA operon copy number, are thought to have growth advantages under limiting resource conditions". I think the introduction focuses too much on the "Birch effect". While soil wetup effects are well documented, they always occur when liquids, be it water or a glucose solution, are added to soil. I think in soils that routinely dry and wet back up, unlike soils from Mediterranean climates such as California, have minimal Birch effects upon rewetting.
The paper is not really about the "Birch effect", it is about the impact of CO2 and temperature on bacterial growth rates in soil and the introduction should focus on that.
Response: We have followed the suggestion to minimize the introduction of "Birch effect". As a result, we have deleted the sentences "This phenomenon is known as the birch effect", and deleted the phrases "response to soil wet up" and "to the resource pulse" to better focus on climate effects (For example, Line 89-91).
We have measured the CO2 production rates during the incubation according to the suggestion of the reviewer #3. Compared with the previous study in Californian soils (Placella et al. 2012), there were lower CO2 production rate and longer time for reaching peak CO2 production rate in our experiment, verifying the reviewer' prediction that the soil used in our study has minimal Birch effects upon rewetting. L135 insert "the" before "6" Response: Done (Line 134).
L43 Delete "The evidence is overwhelming that" Response: Done.
L217 change "shared with three comparisons" to "shared among the three treatments" Response: Thanks. Sorry for the unclear statement. Based on your suggestion, we have revised this sentence to "shared among the three treatment comparisons" (Line 223).
I struggle with the following argument: "The essential genes for microbial growth are considered to be the core genome, which is generally inherited vertically and reflects phylogeny strictly. This helps explain the observed phylogenetic conservation of the strategies related to growth response." How do the authors know that the core genome allows microorganisms to grow faster or slower (i.e. be more competitive)?
Response:  showed that the growth-related essential genes are mainly belong to the core genome, which is strongly influenced by phylogeny. This explains why phylogenetic signals were detected for all three growth responders (Table 1). To clarify it, we have revised this sentence to "The growth-related genes mainly belong to the core genome, which is mostly inherited vertically and reflects the phylogeny (Tamames et al. 2016). This explains the phylogenetic signals of the three growth strategies (Table 1) L280 Suggest change of "visually evident based" to "evident".
L295 Suggest deleting "This helps grasp the relationship between phenotypic characteristics and the phylogeny of microorganisms.
Response: Deleted. L 309 Does the sentence "Microbes possessed metabolic plasticity for carbon use to suit resource availability or interspecies interactions, which promoted or reduced microbial growth28, 29, 30 refer to microorganisms in this study? If not, that should be clearly indicated.
Response: As this sentence is dispensable, we have deleted it from the Discussion. L310 "extremely high intraspecific variation of the genome" It is unclear what genome this sentence refers to Response: As we provided no evidences for the variations of the genome (a comment from reviewer #2), we have deleted this sentence in the discussion section.
L355 suggest changing "for resources under limitation" to "for limited resources".
L442 "The incubations were described in detail previously6." I think this refers to another study and not the actual incubations in this study.
Response: We have revised this sentence into "The incubation conditions were similar to those reported in a previous study (Blazewicz et al. 2020)" (Line 396).

References:
Blazewicz et al. (2020) Taxon-specific microbial growth and mortality patterns reveal distinct temporal population responses to rewetting in a California grassland soil. The ISME Journal 14, 1520-1532 L513 suggest deleting "As 18O labeling occurs during cell growth via DNA replication" Response: Deleted.

Figures:
I am unclear what "low growth" means in bacterial phyla list of Figure 2 Response: The "low growth" refers to the bacterial phyla with low growth rates.
To clarify it, we have replaced "low growth" with "Others", and revised the legend of   The manuscript by Ruan et al. describes the growth responses of soil bacteria following rewetting in soils from a long-term global change experiment. The authors used qSIP with 18O water to measure microbial growth rates after 1, 3, and 6 days. Based on the day of maximum growth taxa were classified as "rapid", "intermediate" or "slow" responders. In general, the experimental design and methods appear to be of sufficient quality. The results are likely to be of interest to the microbial ecology community and may help advance our understand of how microbial systems will respond to global change.
I do have some significant concerns about some of the data analyses, the framing of the paper, and the interpretation of the results.
Response: Thanks for your encouragement! We have followed your suggestions to considerably rewrite the manuscript, shown in our point-to-point responses below. 1) Why are the treatments grouped together for the phylogenetic dispersion, Blomberg's K, and Pagel's lambda analysis but not the NTI analysis?
Response: We have added the NTI analysis in the revised manuscript (Table 1). Both rapid and slow responders exhibited phylogenetic clustering (p < 0.001), consistent with the results of other phylogenetic analyses (Table 1). However, only the intermediate responders had a random distribution on the phylogenetic tree (p > 0.05).
This difference may be due to the different algorithms. We have added the description of NTI in Line 186-187 of the revised manuscript as "The nearest taxon index (NTI) showed a clustered distribution of rapid and slow responders at the end of the branches of the phylogenetic tree". For the analyses in table 1 how did you treat OTUs that were present in more than one treatment and had differing responses? Were the data converted into binary (1s and 0s) before all the phylogenetic analyses? These details should be explained in the manuscript.
Response: Yes, the data were converted into binary (1s and 0s) before all the phylogenetic analyses. For the OTUs that were present in more than one treatment and had differing responses, we performed the following processing: an OTU was classified as a corresponding growth responder when it exhibited a certain growth strategy in any treatment. For the phylogenetic analyses of three growth strategies (combined four climate treatments), 1) We obtained the 0 / 1 matrix based on the distribution of the responder in four climate treatments (a matrix with four columns of data); 2) We identified one OTU as a "potential" responder based on its growth response in any treatment. As a result, an OTU can be divided into one or more kinds of "potential" growth responders. Among 1017 OTUs, 53% OTUs were divided into only one kind of growth strategies, 45% OTUs have two, and less than 2% of OTUs were classified into all three types of growth strategies.
We have added the details in the Methods section "The data were converted into binary matrices (1s and 0s) before all phylogenetic analyses. For the OTUs that were present in more than one treatment and had differing responses (in the analyses when all climate treatments are combined, i.e., in Table 1 and Fig. S6), the OTU was classified to the respective growth responder when that taxa exhibited that specific growth strategy in any treatment" (Line 520-525).
2) The authors frequently conflate the categorized growth responses they observed with ecological or life strategies. This study focuses on one trait the timing of maximum growth after rewetting. One trait cannot be used to assign an ecological strategy as ecological strategies are defined by a suite of traits that covary due to eco-evolutionary tradeoffs.
Response: We have replaced the "life strategy" or "ecological strategy" with the "growth strategy" since we focused on microbial growth dynamics throughout the text.
3) The authors seem to confuse adaptation, acclimation, and plasticity in their interpretation of changes in the timing of maximum growth following rewetting.
Response: To our knowledge, 1) acclimation has been defined as the modification of Therefore, we have modified the terminology throughout the text. We think that "acclimation" fits well in most cases. And this terminology also meets your suggestion in the following comment. As with "ecological strategy" these terms are too often used and abused in the field of microbial ecology perhaps contributing to their misuse in this paper.
Response: To reduce the too often use of ecological strategy, we have replaced the "life strategy" or "ecological strategy" with the "growth strategy".
They suggest that communities and OTUs have adapted to the global change factors via changes in their genome but provide no evidence for this.
Response: Thanks for your suggestion. Since this statement is not so accurate and it is not an indispensable sentence, we have deleted the sentences about "microbial acclimation to climate via changes in microbial genome" in the manuscript. In addition, we removed the word "genomes and" from the original version and rephrased the sentences (check Line 286 and Line 337); We also deleted similar sentences "In addition, the extremely high intraspecific variation of the genome also promotes such fast adaptations" and "Finally, these shifts in life strategies within species may have emerged through horizontal dissemination of genes …" from the original version.
It's far more likely that the global change factors altered the soil environment by changing plant and microbial processes that impact water, carbon, and nitrogen availability) which in turn impact the timing of microbial growth following soil    Response: Figure 5 has become Fig. S6 in the revision, based on the suggestion from Reviewer #1. The environmental data used for its analysis are three distribution matrices (binary) of three growth responders in the four climate treatments.
We have added this description in the methods section as "The distribution matrices (binary) of three growth responders in the four climate treatments were used to represent the impact of CO2 and temperature on bacterial growth" (Line 530-532). Line 126 -I suggest you change "adapted" to "acclimated" Response: We have followed the suggestion (Line 125).
Line 144 - Fig 2 shows proportions but is labeled as (%  Line 294 "unifying life strategies" , this language is far too strong to described the responses you measured.
Response: We have replaced the "life strategy" by "growth strategy" throughout the text. We have rephrased this sentence to "which supports the first hypothesis that phylogeny influences the growth strategies of bacterial species after soil wet-up" (Line

273-274).
Line 307 You have no evidence that environmental section is acting on the trait you measured.
Response: Thank you for your comments. We have deleted this sentence.
Lines 311 -315 I think this interpretation is too strong. There was not statistical analysis to show that rates were significantly higher or lower across the 3 time points thus while the categorization may have changed it's unclear whether this is a truly meaningful change in the timing of growth. Even if the time of growth did change, this is not necessarily a result of genomic changes, it's more like to be a consequence of altered biotic and abiotic conditions changing the expression of genes.
Response: Agreed. As a result, we have deleted this sentence.
Line 320-324 This analysis should be presented in the results. Also the r2 values are very low suggesting that if density dependent selection is occurring it's explaining less that 10% of the variation in growth rates. Response: Agreed. We have revised this sentence to "The ability to grow rapidly is crucial in competing for limited resources, especially under stoichiometric imbalance" (Line 327-328).
Line 442 Suggest change to "Incubation conditions were similar to those previously described". So it's clear it's not the same experiment.
Response: Done. We have changed this sentence to "The incubation conditions were similar to those reported in a previous study" (Line 396).

Reviewer #3 (Remarks to the Author):
Summary. The increase of greenhouse gas emissions give rise to a global warming effect that ultimately influences both above-ground and below-ground processes across Earth's ecosystems. Microbial communities, which underpin the biogeochemical processes that often shape ecosystem function, are no exception to this, and although research shows they can adapt to this warming, population dynamics and ecophysiological responses to these disturbances are not currently known. The manuscript reviewed titled "Phylogenetic constraint and environmental acclimation link to soil" set out to describe an integrated, trait-based understanding of these dynamics with their data which includes 16S rRNA sequences, and qSIP.
This research evaluates two fundamental hypotheses, that were clearly outlined in the introduction and will be referred to for the rest of this document as H1 and H2.
H1: Microbial growth is phylogenetically conserved in response to nutrient pulses following rewetting events.
H2: Warming shifts pre-existing life strategies, which would conflict with phylogenetic signal.
Overall-the manuscript was not challenging to read and except for a few spaces I noted below, I could track the framing quite easily without a sizable background in this specific research area. I appreciated the linkage of climate perturbations in the field to wetting events in the lab, especially the use of growth accelerated and growth delayed.
There were 4 areas where additional clarification was warranted. These comments are addressed below in more detail but broadly summarized here.
(1) The hypothesis testing framework doesn't seem robustly evaluated in the text as written.
(2) Novelty of this study relative for other literature -While I recognize after skimming these other prior papers (two by Firestone lab, one by Blasewtiz is quite similar) there are novel areas in this new work, this needs to be more clearly reinforced by the authors, especially for a broader readership like in Nature Communications, to appreciate the relevance of this research.
More minor, but added up over time, (3) experimental design considerations and (4) language precision with necessary caveats.
Response: We appreciate the reviewer's positive comments. We have supplemented more key data and made extensive revisions to make the hypothesis tested robustly, reinforce the novelty of our work and increase the readability of our paper. Below is our point-to-point response in more detail.
[1] Testing of the hypotheses as currently framed and with data reported in the results.
a. I didn't see data that supports the hypotheses as they are written. It thus reads like the story is based on assumptions of processes happening, not actual measurements.
For example for H1, "Microbial growth is phylogenetically conserved in response to nutrient pulses following rewetting events" I would expect to see measurements of nutrient pulses. There is from what I can see no measurement of the nutrient pulse (DOC, TN, TC, CO2 fluxes) as other studies on this topic have done. Thus the authors seem to assume the mechanism without showing it. I would also expect to see metric of rewetting which would have initial, final water content. I note both these types of data are in used in Blacewisz, but also it seems glaring given how strongly the hypothesis was framed. Key data is missing that would be necessary to support this hypothesis as written.
Response: Thank you for your comments. As also mentioned by reviewer #1, soil wet- To verify the nutrient pulse after soil rewetting, we have collected soil samples again, re-conducted the incubation experiments in the laboratory, and measured soil biochemical metrics (DOC, DN, TN, TC, CO2 fluxes and the hydrolase activity of fluorescein diacetate) at four incubation time points. We found that the rate of CO2 production increased significantly after water addition and peaked at 9 h of incubation ( Fig. S1). The FDA hydrolase activity increased significantly after water addition and peaked at 3d of incubation. The content of dissolved organic carbon (DOC) decreased significantly and dissolved nitrogen (DN) increased gradually along the 6-d incubation, indicating the decomposition of organic carbon and nitrogen accumulation. These results indicated that there were nutrient pulses after rewetting dry soil. We have added the description at the beginning of the Results section (Line 109-113). The data of other measured characteristics are from 4 different incubation time points, i.e., before water addition (prewet soil), incubations for 1 d, 3 d, and 6 d, with destructive sampling. Letters (black lowercase): significant differences between sampling times. Error bars: standard deviation (SD). Significance levels: ***, p < 0.001; **, p < 0.01; *, p < 0.05; ns, not significant.

References:
Birch HF ( Response: It is not appropriate to use a life strategy here because one trait (i.e., microbial growth dynamics in our study) cannot be used to assign a life strategy. Based on the suggestion from reviewer #2, we have changed the "life history" and "ecological strategies" in the text to "growth strategies".
Microbial growth strategies refer to the growth dynamics throughout the whole incubation. Growth strategies were classified as rapid, intermediate, and slow responders based on the timing of the peak growth rates. The data were the growth rates of species (mean of three biological replicates) at three incubation intervals (0-1, 0-3, and 0-6 d). To estimate population growth rates, we adopted the qSIP technique with steps of incubating soils with 18 O-labeled water and using density gradient were testing to support these claims.
Response: To better link the hypothesis and the corresponding results, we have added the sentences in the discussion section: "This explains the phylogenetic signals of the three growth strategies (Table 1), which supports the first hypothesis that phylogeny influences the growth strategies of bacterial species after soil wet-up" (Line 272-274); and "These results confirm our second hypothesis that warming and CO2 enrichment shift the pre-existing growth strategies of certain species" (Line 291-292).
[2] Novelty of this work relative to prior work. We found that phylogeny and environment jointly influence microbial growth strategies, whereby rapid responders were primarily governed by the former and slow responders were primarily governed by the latter. As a result, we have explicitly indicated it as "Our results add new evidence that phylogeny and environmental acclimation are essential to understand the evolution of growth traits of soil microorganisms and their response to climate change" (Line 365-368).
We did use the same method as Blacewisz et al (i.e., qSIP), because qSIP technique can capture the population growth dynamics well (Koch et al. 2018;Sokol et al. 2022).
The two articles published in PNAS and ISME J mainly focused on the metabolic or growth response of microbes after rewetting the extremely dry soils (i.e., one ecological process, the "Birch effect"). Different from these two studies, we integrated the ecological responses of soil microbes at different time scales: Firstly, the CO2 increase in the atmosphere and climate warming are slow, thus soil microorganisms are well conditioned by these global change components (i.e., long-term time scales); Secondly, the response of decomposers to sudden events, e.g. nutrient pulses by rewetting dry soil (i.e., short-term processes), after the long-term acclimation. treatments (i.e., incubation for 1, 3, and 6 days), we added water (400 μl) to the dry soil in the initial incubation treatment. Then, the destructive sampling and soil DNA extraction were conducted (~30 s after water addition). Since the time interval is very short, microbial community profiles in the initial incubation treatment should be the same as that in pre-wet treatment because the prewet soil will also become wet during DNA extraction. Therefore, this could be a minor adjustment but will not affect the experimental results.

References
To clarify it, we have revised the "initial incubation" to "Prewet soil (0 d)" in Fig.   1 and added the explanatory notes "DNA was extracted from the soils of 0 d incubation treatment immediately after water addition (~30 s interval), representing the prewet treatment" (Line 402-403).

Fig. 1 Field and laboratory experimental design.
To examine the effects of CO2 enrichment (500 ppm CO2) and warming (+2 °C) for a typical rice-wheat rotation system, an open-air experimental field was started in 2010 (A). Four treatments with twelve 50 m 2 circular blocks (each treatment with three biological replicates) were set (B). A qSIP incubation experiment was performed to determine the response strategies of active microbes in each climate change treatment (C). Briefly, 2 g air-dried soils with 400 μl of natural abundance water (H2 16 O) or 98 atom% H2 18 O were incubated in the dark at room temperature, with harvests at 0 day, 1 day, 3 days, and 6 days. We calculated the taxon-specific growth rates and determined which of the three pre-defined growth strategies an OTU exhibited in each simulated climate change treatment (D).
I believe this first since not a true control should be T1 (after treatment) as T0 implies before effect and the papers cite a rapid effect following this treatment. This comes important right away as I found myself wondering if on line 122 the differences in richness were due to wetting event or prior historical artifact.
Response: As mentioned above, soil DNA was extracted ~30 s after water addition for the initial incubation treatment. Therefore, it is highly unlikely that microbial community composition was affected by water addition. Because water was added to four climate change treatments simultaneously, we believe that the differences in richness were due to prior historical artifact rather than wetting event.
Note prior study by Blasewitz that authors reference often used these treatments and responder categories. pointed out that microbial response to water addition in agricultural soils (used in our study) may be slower and weaker than in extremely dry soils (e.g., soils from Mediterranean climates), whose soil respiration rate and the activity of partial taxa peak Our classification criteria of growth strategies could also be valid for identifying rapid responders (e.g., species had maximum growth rates within 3 h), despite the lack of a 3-hour culture treatment. If the growth rate of a species reached a maximum within 3 h and decreased over time, this species can be detected as a rapid responder based on our pipeline because we estimated the average growth rates of species within 1 day, which included the growth within 3 hours. (2) Prior studies used fresh soils-collected within a week of incubation. However this study used soils that had been stored in the fridge or at 4C. For how long prior to the study being done-I was confused by language at the end of line 435 and the beginning of line 443 (fresh).

References
Response: We are very sorry for our negligence of the explanation. A portion of the soil samples (~ 60 g) were air dried at room temperature after transport to the laboratory (not stored at 4 ℃), used for qSIP incubation. The remaining soil samples were stored in a 4 ℃ refrigerator.
To clarify it, we have revised the description of the experimental procedure, "approximately 60 g fresh soil of each treatment were sieved (2 mm) and air-dried (24 h at room temperature) immediately after transport to the laboratory" (Line 397-398).
Why were the soils sieved as that disrupts structural features?
Response: The purpose of sieving is to remove the roots and large plant fibers, which can influence microbial activity. Sieving facilitates the homogenization of the soil to avoid differences due to soil heterogeneity. We did it to follow the same experimental This becomes an issue and I found myself unsure if their calls for "slow / medium / fast" responders are appropriate as this wasn't entirely easy for me to follow. For example, looking at Figure 2, I can see that abundances for some of the groups are very similar across all days, and so calling an organism as "rapid" because it is ever so slightly higher in sample days 0-1 than in sample days 0-6 seems imprecise.
Response: We apologize for the unclear expression at Line 143 in the prior version, "Taxa with fast growth rates were conserved within several bacterial phyla". We want to describe which phyla have relatively larger growth rates compared to other phyla during the incubation, rather than which phyla had rapid growth responses to wet-up.
We have revised this sentence to "Taxa with relatively high growth rates belonged to several bacterial phyla ( Fig. 2 and Fig. S4), including Actinobacteria …" (Line 142).
However, when they show Fig 3 and  They feel tacked on and do not meaningfully enhance the manuscript. I suggest they be removed as they detract from the focus of the paper". Therefore, we have followed the suggestion to remove all of the results from the Picrust analysis.
(2) In the discussion section there must be some additional caveats introduced to some of the claims the authors are making. Some of the language that is used within discussion paragraphs require data that the authors do not have (e.g., whole-genome sequencing or metagenomics), or that are somewhat contradictory to their results (e.g., "we identified major forces of variation" when only 20% of the data is explained by these metrics). As such, softening of the language is recommended-lines are noted below-and these will not adjust the outcomes of this study, but support more precise interpretation.
Response: We have removed a large number of redundant and contradictory sentences to improve readability. We have also involved native English speakers to polish the language. Our revisions are marked in red in the revised manuscript.
Line 274-275: Please be mindful and introduce some caveats to the statements made on these lines. What else other than phylogeny and "core genes" could possibly drive these differences?
Response: We have added some caveats as "The effects of environmental selection on microbial strategy, however, cannot be ignored (Arnold et al. 2022;Yang et al. 2021).
Phenotypic plasticity is crucial in the physiological performance of microorganisms to acclimate to broad and varied environments ( Line 366-368: Careful with the phrase "…we identified the major forces…". Changing this to "we identified some of forces…" is prudent given we do not know all factors.
Specifically, there is less than 20% variation explained by these variables in both the rapid and intermediate responders. In other words, 80-89%% of the variation is not explained by these data, and you cannot call them major drivers. Further, please add a strong caveat into this section that speaks to what the other 90% of variability in Rapid and Intermediate responders might relate to, as it is clearly not phylogenetic or environmental acclimation.
Response: We have revised this sentence to "We identified how these two factors shape microbial growth dynamics" (Line 337-338). We have also added the caveat at the end of this paragraph: "Unexpectedly, less than 20% of variation is explained by these variables for the rapid and intermediate responders, which could reflect other factors shaping the growth strategies. For instance, historical contingencies may affect microbial community composition (Ge et al. 2008), which further affected the current community functions as well as species' trait expressions. The metabolic processes could be influenced by the co-limitation of multiple resources, which complicates the prediction of microbial physiological performance (Harpole et al. 2011;Ma et al. 2019).
Lastly, the undetected changes of soil micro-environment and stochastic processes (e.g., genetic drift), may equally contribute to the observed unexplained components" (Line 347-355).
Response: We are very sorry for our unclear writing. Based on our classification method, all detected active species (with growth rates significantly greater than 0) were classified as one of the three growth strategies. We have revised this sentence to "The taxa with growth rates significantly greater than zero can be divided into one of three strategies in each treatment" (Line 495-496). Response: We have changed them to be "intermediate" throughout the revised manuscript. Figure S3: Change "birth rates" to "growth rates" to keep consistent with axes label.
Also -the legend seems to be missing from this supplementary figure. Please add.
Also -from the manuscript text: "Taxa with fast growth rates were conserved within several 143 bacterial phyla ( Fig. 2 and Fig. S3)". Could you label these within Figure   S3 as you did for Figure 2? What are the smaller bar plots inside some of these bar plots?
Response: We have revised this sentence to "Taxa with relatively high growth rates belonged to several bacterial phyla ( Fig. 2 and Fig. S4), including Actinobacteria…" (Line 142-143). We have labeled these phyla with bold in Figure S4.
Since the growth rate differences among treatments were not easily visible for certain taxa (e.g., Deltaproteobacteria and Chloroflexi), we inserted the smaller bar plots with smaller axis orders of magnitude to manifest the differences. We have added this explanation in the legend of Fig. S4.

Fig. S4.
Cumulative population growth rates of dominant phyla or classes. Error bars: standard deviation (n = 3). Different letters: significant differences among treatments. For the phyla Deltaproteobacteria and Chloroflexi (relatively low growth rates), the smaller bar plots with smaller axis orders of magnitude were inserted to better highlight the variation of growth rates among treatments. Bold: the top five phyla with relatively high growth rates. Response: Done.

REVIEWERS' COMMENTS
Reviewer #1 (Remarks to the Author): The authors have substantially improved the quality of their manuscript and it is, in my opinion, acceptable for publication. I do have some minor suggestions for edits: L43 suggest changing "pursuits" to "objectives" and inserting "of microorganisms" after "strategies" L55 suggest inserting a comma before "featuring" and after "response" and inserting "achieve" before "maximum" L61 I suggest rewriting the sentence starting with "The phylogeny is crucial…." I find the sentence awkward. L64 suggest changing "firstly" to "first" L67 suggest deleting the first sentence of this paragraph. L89 suggest replacing "trace" with "characterize" L92 suggest deleting "using" L115 suggest replacing" enabled to trace the bacterial assimilation of water and" with "was used" L145 Explain more clearly what the term "growth dynamics" refers to. L155 delete the first sentence of this paragraph L207 Use the past tense in this sentence L242 and L243 delete "the" before "phylogenetic" L262 -265 delete these sentences L281 delete "the" before "bacterial" L293 delete "the" before "negative" L299 add "values" after "R2" L312 -318 Use the past tense in these sentences L345 It is unclear to me what the accessory genomes are Reviewer #2 (Remarks to the Author): In general the authors addressed my concerns and the manuscript is significantly improved! I do take issue with the presentation of the new biochemical data (Line 106-113 and Figure S1). It's misleading to present data from two separate soil collections two years apart as occurring at the same time. It should be clear that the nutrient flux data is not on the same samples as the qSIP data.
Line 70 Make sure the citations are still appropriate when you change important language such has 'adaptation' and 'acclimation'. Citation '17' may no longer be appropriate.
Line 188 The word "driven" seems too strong for relatively weak K and lambda values, perhaps change to "influenced by phylogeny". 244-246 -The phylogenetic organization is only consistently weaker with climate change for the 'rapid reponders' as currently written it seems like this statement applies to all the groups which is not correct.
Methods -need details on exactly when soil samples were collected for the 18O incubations and need to note that the nutrient flux measurements where taken on samples collected roughly two years later.