Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: OXIDATIVE STRESS AS A POTENTIAL TARGET IN ACUTE KIDNEY INJURY
Review round: 1
Reviewer: 2

Output:
Basic reporting: This is a review of oxidative stress in acute kidney injury. However, the title is almost the same published review article ("Role of Oxidative Stress in Drug-Induced Kidney Injury." Int J Mol Sci, 17: E1826, 2016). Then, it is better to change the title such as "The involvement of oxidative stress in acute kidney injury: an overview." The authors should avoid the similar title with published article.

1.The authors should add the ilustration about antioxidant in the kidney (lines 266-337).

2. As for oxdative stress biomarker (lines 339-350), descritpion should be expanded. For example, oxidative renal biomarker, urinary vanin-1, has been reported in animal models with acute kidney injury (J Pharmacol Exp Ther. 2012 Jun;341(3):656-62.).
Experimental design: N/A
Validity of the findings: N/A
Additional comments: This is a review of oxidative stress in acute kidney injury. However, the title is almost the same published review article ("Role of Oxidative Stress in Drug-Induced Kidney Injury." Int J Mol Sci, 17: E1826, 2016). Then, it is better to change the title such as "The involvement of oxidative stress in acute kidney injury: an overview." The authors should avoid the similar title with published article.

1.The authors should add the ilustration about antioxidant in the kidney (lines 266-337).

2. As for oxdative stress biomarker (lines 339-350), descritpion should be expanded. For example, oxidative renal biomarker, urinary vanin-1, has been reported in animal models with acute kidney injury (J Pharmacol Exp Ther. 2012 Jun;341(3):656-62.).


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: OXIDATIVE STRESS AS A POTENTIAL TARGET IN ACUTE KIDNEY INJURY
Review round: 1
Reviewer: 3

Output:
Basic reporting: Authors tried to review the oxidative stress in AKI. I found this article is difficult to read and many contents has already included in reference #21 (
Oxidant Mechanisms in Renal Injury and Disease, Ratliff BB, Abdulmahdi W, Pawar R, Wolin MS. Antioxid Redox Signal. 2016 Jul 20;25(3):119-46).

My recommendation is “Major revision” to improve the followings;
1.	English language.
2.	Text arrangement.
3.	Full name should appear the first time in the text along with abbreviations.
4.	Many references cited are outdated and many are review articles.
5.	Sessions 3 and 4 are very short, only one paragraph each.
6.	Need tables and figures to enhance readership.

I suggest change the title to only cover recent advances (in the past 5-years period) in this field as reference #21 (dated 2016) already have a through and comprehensive coverage.
Experimental design: N/A
Validity of the findings: N/A
Additional comments: My recommendation is “Major revision” to improve the followings;
1.	English language.
2.	Text arrangement.
3.	Full name should appear the first time in the text along with abbreviations.
4.	Many references cited are outdated and many are review articles.
5.	Sessions 3 and 4 are very short, only one paragraph each.
6.	Need tables and figures to enhance readership.

I suggest change the title to only cover recent advances (in the past 5-years period) in this field as reference #21 (dated 2016) already have a through and comprehensive coverage.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: OXIDATIVE STRESS AS A POTENTIAL TARGET IN ACUTE KIDNEY INJURY
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors addressed all my comments.

Minor:
Table 1. Please check sources of nitric oxide. eNOS is mentioned twice.
Experimental design: No comment
Validity of the findings: No comment
Additional comments: The authors addressed all my comments.

Minor:
Table 1. Please check sources of nitric oxide. eNOS is mentioned twice.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: LIMITS TO LOAD-LIFTING PERFORMANCE IN A PASSERINE BIRD: THE EFFECTS OF INTRASPECIFIC VARIATION IN MORPHOLOGICAL AND KINEMATIC PARAMETERS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Specific comments follow.
Experimental design: Specific comments follow.
Validity of the findings: Specific comments follow.
Additional comments: L4:  What led to this expectation?
L76:  The authors should either present specific results from other studies, or theoretical reasons for this prediction.
L80:  Delete “flight”.  You may want to frame this in terms of mass-specific and marginal power.
L113:  How would one camera capture amplitude?  Was it perfectly orthogonal to the stroke plane at all body positions?
L116:  What is “interaction frequency?”
L132: 2R^2/S
L133: The prediction that maximum load lifted will be correlated with wing loading is not really a prediction; with wing loading calculated as maximum load lifted divided by wing area, it must be correlated.  Why was ‘load lifted’ included in wing loading?  Why not just a pure morphological parameter body mass/area?
L150: “…[greater] flight muscle masses..”
L154: Again, not sure max wingloading is purely dependent; it contains the independent variable wing area.
L160:  Why an AIC of <6?  Ref.
L227:  “in [flights of short duration], oxygen and….”
L240:  How might thermoregulation and prey selection influence burst lifting performance?
L243:  Are your frame rates insufficient to determine accelerations resulting from leg push before the first downstroke?  You should be able to determine roughly when the resulting upward velocity is lost.  Wind resistance would be small.
L256:  “[correlated with….”]  “Influenced” denotes causality.
Legends in Figure 1 A and B are reversed.
The correlation of bill length:  Insignificant correlation with total lifted load.  Yet it leaps to significance in the model averaged results.  Why?
Incidentally, searching for correlations of something so difficult to precisely measure as bill length (averaged for the two sexes at 8.5 sd <0.5 mm!) invites spurious correlations.
The best part of this study is the finding that frequency and stroke amplitude were not correlated with load lifting.  If true, the only physical parameter left that could explain their greater performance is downstroke force, which would indeed correlate with pectoralis mass.
I think there are some interesting data here, and I’d hate to have this much work be for naught.  The authors should trim this down and focus on those morphological variables that could possibly affect flight performance, and keep them simple and physically relevant.  If more precise kinematics are available, the authors should report them.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: LIMITS TO LOAD-LIFTING PERFORMANCE IN A PASSERINE BIRD: THE EFFECTS OF INTRASPECIFIC VARIATION IN MORPHOLOGICAL AND KINEMATIC PARAMETERS
Review round: 1
Reviewer: 2

Output:
Basic reporting: Please see below my general comments...
Experimental design: Please see below my general comments...
Validity of the findings: Please see below my general comments...
Additional comments: General Comments
Please find below my inquiries and suggestions that must be addressed before I recommend this paper for publication.

I) GENERAL COMMENTS AND SUGGESTIONS

A) Climbing Rate During Ascending
During trials, sparrows seem to be ascending at different rates during load-lifting experiments (from Supplementary Videos in Sun et al. 2016, doi: 10.1242/jeb.142216). Moreover, climbing flight duration seems longer than the very brief ‘hovering’ (Could you please add some supplementary videos?). Thus, the total mechanical power must depend on the climbing speed (i.e. the rate of kinetic energy, see Berg and Brewer 2008, doi: 10.1242/jeb.010413) and not just on the maximal induced power (i.e. maximum lift). For example, consider two birds lifting the same weight, the one climbing at higher speed will experience higher costs.
Without knowing the climbing speed it is hard to understand the “Limits to load-lifting performance in sparrows”. What is the speed rate during ascending? What is the actual ‘hovering’ duration? Please consider including this in your analysis.

B) Jumping from the ground

Bird are using their legs to gain a large initial speed which can impact maximal lift production.
Can you report what is the speed after the birds are not touching the ground? How related is it with the tarsus length?

C) Tail morphology and kinematics
It has been reported that tail in passerines plays an important role during flight (see Su et al. 2012, J R Soc Interface, doi:10.1098/rsif.2011.0737). Can you report relevant tail kinematics and morphology using your videos and include them in your analysis?

D) Down-stroke Duration
In songbirds, aerodynamic forces are mostly generated during down-strokes. Can you include downstroke duration in your analysis?

E) Wall effects in a closed plexiglass chamber
Were birds ascending in the center of the chamber or some were ascending close to the walls? If so, ave Dobirds flying closer to the walls have more load-lifting capacity?

II) SPECIFIC COMMENTS

Line 105: "Briefly, a thread with different plastic beads (each approximately 1.0 g in mass)" .
-Did you include the thread mass in your calculations? In video 2 from Sun et al. 2016 seems like the thread was 1 meter long before the first plastic bead was lifted by the bird.

Line 159: "To account for model selection uncertainty, model-average estimates of variable coefficients were computed using the ‘best model set’, defined as the set of models for which delta AIC was less than 6".
-What was the criterion to choose the AIC value?

-Perhaps you can include the following papers on your discussion regarding wing loading.
Lind 2001.Escape flight in moulting Tree Sparrows (Passer montanus).Functional Ecology 15, 29 – 35
Lind and Sven Jakobsson 2001.Body building and concurrent mass loss: flight adaptations in tree sparrows (DOI: 10.1098/rspb.2001.1740)


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: LIMITS TO LOAD-LIFTING PERFORMANCE IN A PASSERINE BIRD: THE EFFECTS OF INTRASPECIFIC VARIATION IN MORPHOLOGICAL AND KINEMATIC PARAMETERS
Review round: 1
Reviewer: 3

Output:
Basic reporting: Some fundamental literature references are missing, e.g. Marden (1987), J Exp Biol
The hypotheses should be better rooted in biomechanics. I did not understand the rational for including many of the variables, such as bill size and internal organ sizes, and these need explanation.
Experimental design: This is a statistical approach to a biomechanics problem, and many variables measured and included in statistical models don't seem to be well motivated. More information is needed about the flight assay, nit simply referring to another paper (that I haven't read).
Validity of the findings: There is a lack of connection between the results and biomechanics (aerodynamic) relationships.
Additional comments: This paper investigated the sprint load lifting capacity of tree sparrows, which were exposed to a load-lifting assay. This assay consists of take-off flight with an attached thread of beads spaced along the thread, and the asymptotic load lifting capacity is measured. This was then analysed statistically against a great number of measures taken on the birds, including many internal organs after that the birds had been sacrificed. Why some of these measured were thought to influence load-lifting capacity was not always clear and not well motivated. I would have preferred a more hypothesis-driven approach where relationships based on aerodynamics are used to derive qualitative/quantitative predictions. It is nowhere mentioned that the assay refer to anaerobic muscle work, while some of the relationships referring to internal organ size (liver, intestine) should rather be expected to influence aerobic flight capacity. Also, what is the rational to belive that bill length will influence load-lifting capacity. Wing area was taken as the area of one wing doubled, while the convention in animal flight research is to include the area of the body between the two wings (there is no motivation for excluding this area). There are also confusion about some measures, where for example wing-related morphology is sometimes area and sometimes length, and even “wing size” is used without defining what it is.
Line 189 That maximum wing loading is related with body mass is a direct consequence from its definition (mg/wing area), and need no statistical analysis.
Line 233 why would migratory species (taxa) have more pronounced interactions among “physiological systems”?
There are some literature omissions, for example Marden (1987) , J Exp Biol.
Line 256 Please note that by the statistical analysis you could not infer that a certain factor is “influencing” load lifting, but only that there is a correlation.
Line 261 “in contrary to our initial hypothesis, size of heart, lungs and digestive organs were unrelated to maximum load-lifting capacity,..”. I did not understand the rational for hypothesising such a relationship in the first place.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: LIMITS TO LOAD-LIFTING PERFORMANCE IN A PASSERINE BIRD: THE EFFECTS OF INTRASPECIFIC VARIATION IN MORPHOLOGICAL AND KINEMATIC PARAMETERS
Review round: 2
Reviewer: 1

Output:
Basic reporting: My detailed comments point out where they fall short in this respect.
Experimental design: Nothing to add.  I still don't understand why they used such a small flight arena, but apparently, according to their reply to other reviews, birds habituated to humans can fly just fine in a small box?
Validity of the findings: I didn't check the supplementary data, so maybe it's there, but they could easily provide a table of all of the primary data.  People could then run any statistical model they saw fit.
Additional comments: This study contains a large sample of short burst load lifting performance data for a single species of bird from a single location, with a set of morphological and kinematic variables. The premise of the study is that some body features are expected to increase performance whereas others should reduce performance.

This version of the manuscript is improved over a previous one I reviewed for a different journal.  There is now a hypothesis and Fig 1 summarizes the main data.  As my comments show, I have some issues with the way they have posed and reflected on their hypotheses, and I’ve offered some constructive comments for them to consider.

A problem that still remains is that they only examined the total load lifted (body plus added mass).  This is a fine start, but to better understand the result, it would be helpful if they extended the analyses to evaluate some sub-hypothesis.  For example, the results are consistent with prior literature showing that burst flight performance depends strongly on the mass of flight muscles and, and that the added load capacity depends strongly on the ratio of flight muscle mass to total body mass.  Between species, the total load relationship scales as flight muscle mass^1.0 or very nearly so (isometry).  We know less about this scaling within species.  Might they show their result?  Using that result, they can evaluate the residual variation to see if e.g. a larger stomach or smaller beak can explain a significant portion of the residual variation in load lifted.  Clearly this is different from their approach that puts all variables on equal footing and has flight muscle mass counted twice (by its inclusion in body mass), but flight muscle mass was the expected key variable prior to their analysis and is the key variable after their analysis.  Hence, why not use an analysis in which it is the major causational variable and then show how the other variables contribute?  Doing so may provide much clearer insights.  As an example, consider total load and body mass.  More massive birds likely have larger flight muscles, which increase total load capacity.  But larger body mass can result from many different tissues.  Anything they can do to tease apart the size dependencies within their data would be helpful.  Perhaps that is what their only figure aims to do but I find it uninformative.

For example, a specific hypothesis they could address is whether a small beak is an indicator of a relatively small non-muscle body mass. Larger muscles in a bird with a relatively small beak might indicate large muscles in relation to skeletal size of the anterior central body core.  Similarly, if they sum standardized variables for the digestive mass/size (stomach mass, intestine length, liver mass), would they get a stronger result that better supports their (not yet very clearly stated) hypothesis that gathering nutrition co-varies the energy and/or condition of flight muscles and thereby affects short-burst performance?

Specifics:

L. 24.  I’m not convinced that the paper reveals how/why these parameters are “constrained”, so I find this opening sentence to be a bit misleading.  “Interrelated” would be a more accurate term for the approach this study uses.

L. 28.  As multiple reviewers have pointed out, any prediction about organ size needs to consider the difference between short bursts of anaerobic effort vs. sustained effort.  Short burst performance might predictably be related to smaller hearts, lungs and stomachs, i.e. a bird that is nearly all muscle+wing and little else.  The paper they cite on the small heart size of  Tinamous is an excellent example of authors distinguishing between burst and sustainable performance.  Similarly, I recall a paper showing that palatable butterflies that need to evade predatory birds have larger flight muscles and their gut and ovarian mass are reduced compared to chemically protected butterflies that have little or no need to evade birds.

L. 28 There are two other problems with this sentence.  First, if bigger muscles, hearts, stomachs, and lungs are predicted to be favored, then why not intestines, eyes, blood, brains and beaks – all of which could contribute to the gathering and assimilating of food to provide energy for flight?  Second, if bigger is better for all of these things, how can you simultaneously predict “relatively” smaller body size?  Relative to what?  The only way this makes sense is if body parts unrelated to supporting mechanics and energetics of flight are reduced.  I suppose this would have to be bones, feathers, kidneys and reproductive organs, but certainly bones and feathers support muscles and aerodynamic forces, so perhaps that list is shorter.  The broader point is that the hypothesis as posed is poorly formed and needs to consider the physiological role of different tissues/organs in relation to different modes of flight (burst vs. sustained).

L. 59:  What does “upregulated” mean in this context?  Do you mean hypertrophied?

L. 62: “As major metabolic engines, sizes of heart and lung also underpin capacity for both burst power and endurance flight performance (see Bishop and Butler, 1995; Tobalske et al., 2003; Wright et al., 2014).”   The Tobalske paper is about power vs. speed relationships; the  words “heart” and “lung” do not appear in that paper.

More importantly, why expect heart and lung effects in an experiment in which flight bouts lasted only seconds?  Is this enough time for O2 to get from the lung surface to the mitochondria and thereby affect burst performance?

L. 78: There is no reason to specify a method in these references or otherwise differentiate them as it has been shown that the two methods produce indistinguishable results.

L. 91: “including the body”.  Where in the analyses is body mass evaluated?  I don’t see an allometric equation relating total load or added load to unladen body mass.  The positive correlation between total load lifted and body mass in not informative on this point.

L. 141: methods describing flight muscle dissection: The pectoralis mass shown in Table 1 strikes me as too low (12.8% of body mass).  Might you have done the dissection on only one side and are reporting only half of the total pectoral muscle mass?

L. 167: cite the method used for centralizing and standardizing

L. 221: For reasons I described in a previous review of an earlier version of this paper submitted to a different journal, you should also analyze the product of wingbeat frequency and amplitude, or at least state explicitly why your video method does not allow you to resolve these variables at the critical time point (i.e. I now understand from your responses to other reviews that you may be generally missing wingbeat amplitude at the max load lifted).  The reader should know the limitations of the data and understand why this product is not being presented, particularly since you are reporting the two component variables of this product.

L. 232.  Why does this sentence begin with “In hummingbirds”?  This has been known for birds in general, including Passer, since 1987.  This strikes me as an egregious example of self-citing.

L. 247: “aerodynamic force production by the wings can constrain whole-animal vertical load-lifting”.  This is always true; force output is limited by force production.  The informative thing to say is why force production during short bursts is NOT limited by aerobic capacity.

L. 252: “Larger digestive organs may thus enhance burst flight performance in non-migratory avian species.”  Finish the thought.  You earlier cited literature suggesting that digestive capacity may affect the condition of the flight muscles.  Use this possibility to say why a continuous-time process (protein and energy assimilation) may relate to motor performance, even during short bursts, perhaps by affecting the glycogen and protein content of the flight muscles.  Thinking hard about your results in this fashion may help you devise you next project and/or get your next grant.

L. 253: I encourage expanding a bit your discussion of bill length effects.  You could start by estimating the mass difference of different size bills, which will undoubtedly by trivial.  This sets you up to say that it instead may reflect foraging differences.  Perhaps you are familiar with the famous studies of Galapagos finches showing that drought and wet years affect the abundance of seeds of different sizes and the fitness of finches with different beak sizes.  Perhaps you know something about what seeds were abundant in the days & weeks preceding your study.  You could speculate that, if foraging success underlies the beak size result, that it could change seasonally as different plants produce seed and this species’ diet changes.  Perhaps you know something about beak sizes in different populations and could suggest that there is gene flow for which beak size may be a marker and that genetic differences between populations may include different burst performance capability, in which case it may not be beak size per se that is affecting load lifting but rather the associated genetic differences.  Biology always challenges us to think broadly!!

L. 278:  We certainly know that burst accelerations are used by birds such as sparrows to evade predatory birds.  This can be observed every day at the bird feeders in my yard when Cooper’s Hawks come by looking for a meal.  You needn’t state complete lack of knowledge/insight here.  See for example https://royalsocietypublishing.org/doi/10.1098/rspb.1996.0244,
https://jeb.biologists.org/content/218/2/212?version=meter+at+null&module=meter-Links&pgtype=article&contentId=&mediaId=&referrer=&priority=true&action=click&contentCollection=meter-links-click, https://www.jstor.org/stable/4163245?seq=1#metadata_info_tab_contents,
https://academic.oup.com/auk/article/117/4/1034/5147010,
https://www.sciencedirect.com/science/article/pii/S0022519304002899,
and others by Lind and co-authors.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: LIMITS TO LOAD-LIFTING PERFORMANCE IN A PASSERINE BIRD: THE EFFECTS OF INTRASPECIFIC VARIATION IN MORPHOLOGICAL AND KINEMATIC PARAMETERS
Review round: 2
Reviewer: 2

Output:
Basic reporting: More information is needed about the methods for obtaining wingbeat amplitude, and the previous recommendation by a reviewer to report downstroke ratio (percent time in downstroke) should be revisited.

In lines 116-118 it is written that the top camera (250 Hz) was used to measure amplitude, but on line 122 it is written that wing-stroke amplitude was derived from lateral video (50 Hz).  Looking at the supplemental videos, I am concerned that there is excessive blur due to the use of low shutter speeds.  First, it is important to clarify which camera view was used to measure amplitude.  Then the specific methods, more clear than in the Sun et al. 2016 JEB paper, would be helpful.  Was the maximum excursion of the wing assessed from shoulder to the tip of the primaries.  If so, which primary tip? The stroke planes do not seem to be completely parallel to global horizontal.  In general, what is the stroke plane angle relative to horizontal, and what is the estimated error involved in using only one camera to calculate amplitude given this average stroke-plane angle?

A previous reviewer suggested reporting downstroke ratio (percent time spent in downstroke within a wingbeat), but the authors replied that the measure is not possible given their frame rates.  250 Hz sampling seems sufficient for this purpose, and it is not clear how the authors can obtain wingbeat amplitude without being able to quantitatively discriminate between downstroke and upstroke.
Experimental design: No comment
Validity of the findings: Since wingbeat kinematics are reported to not correlate significantly with performance, it is vital to improve the description of how wingbeat amplitude was measured.
Additional comments: You report interesting and worthwhile data that explore issues related to your 2016 study.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: LIMITS TO LOAD-LIFTING PERFORMANCE IN A PASSERINE BIRD: THE EFFECTS OF INTRASPECIFIC VARIATION IN MORPHOLOGICAL AND KINEMATIC PARAMETERS
Review round: 3
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: No additional comments


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: LIMITS TO LOAD-LIFTING PERFORMANCE IN A PASSERINE BIRD: THE EFFECTS OF INTRASPECIFIC VARIATION IN MORPHOLOGICAL AND KINEMATIC PARAMETERS
Review round: 3
Reviewer: 2

Output:
Basic reporting: This is satisfactory.
Experimental design: This is satisfactory
Validity of the findings: This is satisfactory.
Additional comments: I appreciate your clarification of your methods for measuring wingbeat amplitude.  It is still unclear to me why downstroke (%) is not obtainable, but I don't view this variable as being critical to your experimental design and interpreting your results.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: IDENTIFICATION OF GENES AND FUNCTIONAL COEXPRESSION MODULES CLOSELY RELATED TO ULCERATIVE COLITIS BY GENE DATASETS ANALYSIS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The introduction text should be broken into multiple paragraphs.

The article does not cite previous gene expression studies of ulcerative colitis such as:

* Lawrence, Fiocchi, and Chakravarti. Human Molecular Genetics. 2001.
* Wu, et al. Inflammatory Bowel Diseases. 2007.    (This paper is referenced in a table but not cited in the bibliography.)
* Noble, et al. Gut. 2008.

McGovern, et al. Nature Genetics. 2010.  discusses a genome-wide association study for UC.  Although not gene expression, the part of the introduction mentioning GWAS studies for UC should be expanded, with more details (especially of results) and citations.

The word "biopsy" is misspelled in the methods. The R package name is truncated ("Robust Rank Aggreg" instead of "Robust Rank Aggregation").

Figure 1: Meaning of horizontal axis is not explained.

Results:
Statements such as "Among them, genes such as MMP1, REG1A and AQP8 had been confirmed to abnormally express in UC" should be followed by citations to the original studies.

I think it would be helpful to the reader to explain what the purpose of each method (e.g., RRA, WGCNA) is.  It might even be helpful to rename the results subsections to focus on the results rather than the analyses.  For example, "150 Genes are Differentially Expressed Between UC and Non-UC Patients", etc.

The language is not always as clear as possible.  For example, I had to reread the sentences "By setting p <0.05 and |logFC| >0.14, the number of differential genes result from RRA analysis would increase to 5,344. The expression matrix of these genes in GPL570 samples was performed for WGCNA." a few times to understand that (1) you used less stringent criteria to identify a large set of differentially expressed genes and (2) used the larger set of genes as input for the WGCNA analysis.  It might help to avoid phrases like "these genes" and instead by more specific about which genes.

In the methods, you explain why only the GPL570 samples were used for the WGCNA analysis.  It would be helpful to restate this in the results.
Experimental design: Your research goal seems to be to identify differentially-expressed genes that are associated with UC and identify their biological function.  Your analysis rests on the assumption that integrating multiple data sets will increase accuracy or statistical power versus analysis of a single data set.

The methods say that you used data sets that provided raw data or gene expression matrices.  It's not clear to me what the form of the input data was.  For example, did you have to compute the log-fold change yourself using something like DESeq?  Or, did you use lists of genes with statistically-significant changes in expression as determined by the authors of the original studies?  This is particularly pertinent as you are integrating data from multiple studies, which may have been analyzed differently.
Validity of the findings: The manuscript would benefit from more comparison to results from other studies.  What do you find that is new compared with the studies done on individual data sets?  What do you find is missing from your analysis versus the individual data sets?  How much agreement is there between what you find and others find?  How much evidence is there that what you find is of biological relevance?

A few times, comments are made on individual genes (e.g., MMP1, REG1A and AQP8) being studied previously but the other studies are not always cited in the same sentence.  It would be helpful to compare lists of genes found by the other studies and your studies and compare them.  Can you summarize the agreement quantitatively?  If possible, put this information in a table.

Some statements do not seem to be supported with evidence or are vague. For example, "blue module was the most important functional module involved in UC."  Important is a vague term.  What makes it important?  Your results seem to suggest that it had the largest number of genes with statistically-significant biological functions.  But that does not imply importance.
Additional comments: This is an important study, and I enjoyed reading about your work.  I think that the paper would benefit from adding more detail and clearer analysis and comparison to other work.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: IDENTIFICATION OF GENES AND FUNCTIONAL COEXPRESSION MODULES CLOSELY RELATED TO ULCERATIVE COLITIS BY GENE DATASETS ANALYSIS
Review round: 1
Reviewer: 2

Output:
Basic reporting: -. The manuscript explored public genome-wide expression datasets annotated with ‘ulcerative colitis biopsy’ downloaded from ‘Gene Expression Omnibus database’. Authors integrated them to identify genes and pathways differentially enriched between healthy tissue and ulcerative colitis tissue trying to link their finding to pathogenic events that may contribute to new and valuable therapeutic targets.
-. The manuscript has spelling, grammar issues, and unambiguous English used throughout without clear evidence/ground/reference. For example, “in line 106: the absolute value of fold change was used for sequencing”. I don’t understand what this sentence meant. “in line 145: among them, genes such as MMP1, REG1A and AQP8 had been confirmed to abnormally express in UC”. This argument was made without reference. “in line 146: however, the possible value of HMGCS2, PCK1” in the pathogenesis of UC is still the first discovery. Authors pointed out only several genes among many differentially enriched genes. Then, how about others?
-. Authors used symbols without full name of symbols in many places, for example MMP1 and AQP8 in the Abstract.
-. Table 2 should have horizontal lines to distinguish different modules and explanation of table header (for example, count %, log10(p), log10(q)).
-. Figure 1 doesn’t contain legend of heatmap color and explanation of how fold change was calculated for each platform.
-. Figure 2 doesn’t contain legend of heatmap color.
-. Figure 3(B) doesn’t contain explanation of Dynamic Tree Cut and Merged Dynamic.
-. Figure 3(C) doesn’t contain explanation of heatmap color.
Experimental design: -. Authors have clear criteria to generate gene expression datasets comparable for downstream analysis to answer research question.
-. For downstream analysis, authors employed standard tools implemented in R to remove the batched effects from different datasets of the same platform, to identify differentially expressed genes of each platform, to select most valuable differentially expressed genes, and to group important genes into biological functional modules which were annotated by Gene Ontology. However, authors should describe on functions/methods and parameter values being used in detail in each step for reproducibility and comparison with future datasets instead of just mentioning which R packages being used.
Validity of the findings: -. The explanation/rationale of cutoffs on parameters involved in statistical analysis is lacking so that it is difficult to determine how rigorous the results reported would be.
-. Authors provided which public gene expression datasets included for downstream analysis in Table 1. However, authors didn’t provide a final dataset which was quality controlled
considering batch effects and different platforms.
-. Majority of discussion is based on Table 2 which contains functional enrichment analysis. Authors pointed out several pathways as important pathways out of many enriched pathways without the ground (is it based on count/p-value/q-value?) for each functional module.
-. Authors claims that blue module is the most important functional module without the ground.
-. Authors discussed only 4 modules out of 7 functional modules. In conclusion, I don’t see how authors chose modules as important modules, and pathways as important pathways within the modules.
Additional comments: No additional comments


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: IDENTIFICATION OF GENES AND FUNCTIONAL COEXPRESSION MODULES CLOSELY RELATED TO ULCERATIVE COLITIS BY GENE DATASETS ANALYSIS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The paragraphs are not well separated.  You should either add a blank line between paragraphs or indent paragraphs with a tab.

The English could still use some improvements.  For example, the phrase "of which obtained many biological processes through pathway enrichment analysis" from the abstract is not correct.  Could this be changed to "which pathway enrichment analysis indicated were associated with many biological processes"?

Re-define terms like DEGs and RRA upon first use in the results and methods.  (Don't assume that the reader has read the entire paper.) This will help the reader interpret a given section without needing to refer back to a previous section.
Experimental design: The Limma package supports a large number of different analyses on expression data.  More details on the particular analyses and the methods used by Limma are needed.

Would it be appropriate to use a correction for multiple hypothesis testing?  (e.g., Bonferroni correction or Benjamini-Hochberg correction). You don't mention this in your Methods section.  That said, given the small p-values you are seeing (e.g., 10^-7), I don't suspect that your results will change substantially.
Validity of the findings: The text still has some ambiguities and a few claims that are not well justified.  Specifically, what is a "key gene"?  I assume that you are looking for causative genes -- genes for which significant up or down regulation leads to the onset of symptoms associated with UC.  You should be clearer as to what makes a gene "key."

Secondly, the methods (e.g., rank aggregation of genes based on expression, pathway enrichment analysis) used are based on association testing.  Association testing cannot infer causation.  Consequently, your analysis may identify genes that are up or down regulated due to side effects (e.g., inflammation) of UC rather than causing the UC symptoms.  Be careful about drawing conclusions about causation.

Your analysis produced hypotheses (e.g., relationship between UC and herpes virus) that are interesting and worthwhile for follow up.  However, I would be careful about assuming that your analysis conclusively proves anything.
Additional comments: Thank you for your efforts in responding to previous reviewer comments and implementing the request changes.  The changes you made a greatly improved the readability and depth of the paper.  Your analysis is interesting and a valuable contribution.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: IDENTIFICATION OF GENES AND FUNCTIONAL COEXPRESSION MODULES CLOSELY RELATED TO ULCERATIVE COLITIS BY GENE DATASETS ANALYSIS
Review round: 2
Reviewer: 2

Output:
Basic reporting: -. The manuscript explored public genome-wide expression datasets annotated with ‘ulcerative colitis biopsy’ downloaded from ‘Gene Expression Omnibus database’. Authors integrated them to identify genes and pathways differentially enriched between healthy tissue and ulcerative colitis tissue trying to link their finding to pathogenic events that may contribute to new and valuable therapeutic targets.
-. The author improved the manuscript a lot. I noticed the following sentences which need to be revised: Line 161, Line 168, Line 288, Figure 2 legend, Table 2 legend.
-. Table 1 has a column for Authors. I think that publication ID (for example, PMID) would fit better.
-. Table 2 has legend above and below the table two times. Please remove one.
-. Table 2 has three columns for %, log10(p), log10(q). Please control decimal floating-point numbers.
-. Figure 1 doesn’t contain legend of heatmap color. Please put ‘log10(FC)’ on top of color legend.
-. Figure 2 doesn’t contain legend of heatmap color. Please put legend on top of color legend.
Experimental design: -. Authors have clear criteria to generate gene expression datasets comparable for downstream analysis to answer research question. For downstream analysis, authors employed standard tools implemented in R to remove the batched effects from different datasets of the same platform, to identify differentially expressed genes of each platform, to select most valuable differentially expressed genes, and to group important genes into biological functional modules which were annotated by Gene Ontology.
-. Authors improved the manuscript a lot in explaining methods and parameter values. I have two questions. In line 164, authors said that they used 0.05 of adjusted p-value cutoff and |logFC| > 0.14 because these cutoffs have been used in Yan et al, 2018. The following is the sentence from Yan et al, 2018: “To include enough number of genes into WGCNA analysis, genes with Bonferroni’s adjusted p < 0.05 and logarithmic fold changes (logFCs) >0.26 were selected from the final ranked gene list.” It seems that Yan et al used different cutoffs. Please justify the reason of your cutoffs. In Line 182, authors said that p <0.05 was used for all hypothesis testing. Is this correct? It seems that adjusted q-value was used.
-. In line 169, authors mentioned ‘the ideal size of sampletree’. What do you mean by this?
-. In line 170, authors mentioned ‘scale-free topology index’. Please explain why they tried to generate clusters with certain scale-free topology index.
Validity of the findings: -. Authors diligently validated their findings comparing with results from literatures related to ulcerative colitis.
Additional comments: No additional comments


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: INCONSISTENT RESPONSE OF SOIL BACTERIAL AND FUNGAL COMMUNITIES IN AGGREGATES TO LITTER DECOMPOSITION DURING SHORT-TERM INCUBATION
Review round: 1
Reviewer: 1

Output:
Basic reporting: In this manuscript, Li and Yang describe a report in regards to the inconsistent response of soil bacterial and fungal communities in aggregates to litter decomposition. However, not only the designed experimental approaches raised many concerns regarding its scientific sounds, scarce data obtained from this study support the conclusions in this manuscript. Below, I have some comments in regards to additional experiments and modified approach that would help authors improve the quality of their manuscript in order to meet a general standard for publishing as an original research article.

While the authors invested their effort in bioinformatics analyses, which obtained from DNA sequencing of specific regions (16S rRNA and ITS1-2),  all of the data showing in this manuscript were observed in only one time point at the end of the incubation period (56-day after incubation) without a statement of specific or logical reasons. In my opinion, these data are insufficient to reflect a robust, convincible, and reliable interpretation. Rather, the author should perform a comprehensive comparison with at least three-time point: 0-, 28- (sometime in the mid-process), 56-day after incubation. This designed strategy would hopefully provide insights into dynamic responses of soil bacterial and fungal communities in aggregates to litter decomposition.

There are many concerns with regards to material collection and preparation. While the collection of top layer (0–15 cm) of the soil was stated, there were lacking detailed description regarding the representative of sample, including how many samples were collected, any specific season, ect.

For the preparation of materials, how was the contamination of un-indigenous microorganisms controlled during transportation and sample preparation? Importantly, the 1000 mL jars were utilized for incubation, how was the aeration managed, given that this is normally a major factor regulating the balance of fungal and bacterial growth in the soil.

The authors should clarify why was the Leymus chinensis chose as plant litter (leaf and stem) in this study.
Experimental design: As above
Validity of the findings: No comment
Additional comments: No additional comments


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: INCONSISTENT RESPONSE OF SOIL BACTERIAL AND FUNGAL COMMUNITIES IN AGGREGATES TO LITTER DECOMPOSITION DURING SHORT-TERM INCUBATION
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript is well written. Clear English is used throughout for the easy understanding of readers. Sufficient background and citations are also provided.
Experimental design: The experimental design is well constructed.
Validity of the findings: Please see my review attached.
Additional comments: No additional comments


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: INCONSISTENT RESPONSE OF SOIL BACTERIAL AND FUNGAL COMMUNITIES IN AGGREGATES TO LITTER DECOMPOSITION DURING SHORT-TERM INCUBATION
Review round: 1
Reviewer: 3

Output:
Basic reporting: 1. Basic Reporting
The English is largely very good, but there are some minor corrections outlined below.
The raw data appear to all be present and correct. The only omission is that Sheet 2 of the .xlsx should include the sample names as present in the SRA, so that the reads can be matched to the correct sample.
Specific comments on abstract/ introduction section:
Line 8: please provide an academic/professional e-mail address if possible
Line 13: please remove the comma after ‘grassland’
Line 15: there were four aggregate classes, not three
Lines 17-23: the summary of the results makes no mention of the different aggregate classes, and whether the observed patterns differed across them. Please add in some information on this.
Line 28: avoid repetition of the word ‘factors’, e.g. “…three main drivers: climate factors…”
Line 42: I’m not sure what you mean by ‘converse’. Do you mean there was an inverse relationship?
Lines 46-51: the hypotheses section needs some work. The whole set up of the experiment is designed to test whether different aggregate sizes respond differently to litter addition, and whether the quality of the litter has any effect on the relationship. Neither of these is mentioned in the hypotheses. In addition, please justify hypothesis 2. The layout of the hypotheses, results section and discussion section should mirror one another in order to present a clear story.
Experimental design: 2. Experimental design
The stated hypotheses need some work (see above). In particular, the main research questions are implicit rather than being fully stated.
Specific comments on methods section:
Line 57: what area or weight of soil was removed?
Line 63: how was the soil stored between May and September?
Lines 66, 70: was the plant material dried, or did you obtain dry weight from a subsample?
Line 69: you mention in your previous paper (Yang, 2019) that air-drying soil can alter the microbial communities. If so, it would be good to a) discuss that in the present paper (where community analysis is central) and b) if this is experimentally demonstrated, back it up with a reference to the literature.
Line 71: why was the control designated CK?
Line 81: 2 ml NaOH from (not of) each trap
Lines 83-85: did you measure weight loss in the plant material as a indicator of decomposition? If so, this would be very interesting data to add.
Line 94: when citing primers, please refer to the paper that originally described them
Line 97: I assume you mean µL (microlitre) not mL (millilitre)! Please check carefully throughout the rest of the paper that you have referred to the correct unit.
Lines 99-100: this sentence doesn’t make sense. Was it sequenced on a HiSeq in the USA or a MiSeq in China?
Lines 104-108: Step i) why a 300 bp cutoff, when the target region was aprrox. 450 bp? Step ii) this suggests that exact barcode matches were removed - I assume you mean barcode errors were removed. Step ii) it’s not clear what this means.
Line 112: as for the primers, please cite the source of these equations or a reference work (eg Begon, Harper and Townsend) rather than a paper that simply used them.
Line 117: please give these abbreviations in full at the first mention. Also, do you mean a two-way ANOVA, seeing that you give two predictor variables?
Line 118: were the LSD P-values adjusted for multiple comparisons?
Line 118: please capitalise P (here and throughout)
Line 120: you use both Shannon and Chao1, which capture different aspects of diversity, but at no point do you discuss how they differ or what they tell you. If you are going to include more than one index, please include this information and discuss it in the discussion section.
Validity of the findings: 3. Validity of the Findings
The conclusions section makes no mention of aggregate size classes, which is strange given that this was the main component of the experiment. In general, statements in the discussion need to be better justified and checked carefully to see if they can really be derived from these results. For example, lines 216-217 “Soil fungi diversity plays a more important role in litter decomposition than soil bacterial diversity”. This is probably true, but I cannot see how it could be derived from the results of the current study.
Always keep in mind that you measured diversity, not abundance, of bacterial and fungal communities. This means that you only have a measure of change within each community, not between them. It is therefore not possible to make any statements about a shift between bacterial vs fungal dominance (e.g. line 189).
Specific comments on results and discussion:

Results:
The first section of the results comes across as rather confused. It needs reworking to take the reader through each ANOVA model in turn and tell a coherent story.
There is no need to report main effects where there is a significant interaction, as the two factors depend on each other. In the first paragraph, you mention leaf but not stem addition, and sometimes refer to litter addition without specifying whether this is leaf, stem or both treatments lumped together. Be very careful about statements suggesting causation, e.g. line 160-161 – you can’t actually tell whether the pH and SOC influenced the bacterial communities or vice versa, as the relationship is only correlative.
The section on SEM could do to have a bit more information to make it clear for those unfamiliar with the method (like me). In particular, the meaning of lines 166-167 is unclear.
Line 134: there is no need for the word “on”

Fig 1: if the data were analysed together, do treatments also separate from each other?
Fig. 4 would benefit from more information in the legend.

Discussion:
I’m puzzled by the appearance of Aphelidiomycota in your results. This is not a decomposer taxon, but aquatic parasites associated with algae (Karpov et al. 2014. Front Microbiol 5: 112). I think you should look carefully into the identification of these OTUs and how come they are in your data. Also, the Cercozoa are not fungi but protists. They presumably were co-amplified by the fungal primers, and really should be excluded from the analysis as these primers do not deliberately target protists.
The model shown in Fig 5 seems to be one of the most important parts of your manuscript, yet it isn’t really mentioned in the discussion.
Lines 177-178: what do you mean by the proportion of bacteria? The bacteria-fungus ratio? I feel that the first few lines of the discussion would be better placed in the introduction, as they don’t directly relate to interpreting your findings.
Lines 183-184: I don’t see how you can imply competition from these results.
Line 197: bacterial phyla
Lines 198-199: be careful! You didn’t measure abundance, so in what way do Sun’s findings corroborate yours?
Line 204: again, the fungal communities didn’t increase, but alter in composition
Additional comments: 4. General comments
This is a well-designed study, which explores an often-overlooked aspect of soil diversity: the physical heterogeneity of soil, and the different microbial communities associated with different aggregate size classes. However, the presentation and interpretation of the results needs considerable work in order to be clear and robust.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: INCONSISTENT RESPONSE OF SOIL BACTERIAL AND FUNGAL COMMUNITIES IN AGGREGATES TO LITTER DECOMPOSITION DURING SHORT-TERM INCUBATION
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have revised and added some additional information as the request in the review report. However, the revised manuscript did not address major concerns that would improve its quality and meet a scientific sound. Again, all the experimental procedures from sample harvesting to sample preparation and experiment design were insufficient to obtain robust, reliable, and repeatable results. Importantly, there were no alternative approaches to validate the achieved data represented in the manuscript. For publishing as an original research article, this study should be thoroughly re-conducted with additional sets of well-designed experiments, thereby hopefully obtaining scientific-sounded evidence for the questions the author asked. Below are comments based on the authors' responses:

1. While the authors invested their effort in bioinformatics analyses, which obtained from DNA sequencing of specific regions (16S rRNA and ITS1-2), all of the data showing in this manuscript were observed in only one time point at the end of the incubation period (56-day after incubation) without a statement of specific or logical reasons. In my opinion, these data are insufficient to reflect a robust, convincible, and reliable interpretation. Rather, the author should perform a comprehensive comparison with at least three-time point: 0-, 28- (sometime in the mid-process), 56-day after incubation. This designed strategy would hopefully provide insights into dynamic responses of soil bacterial and fungal communities in aggregates to litter decomposition.

Response: Thank you for the kind suggestion. We are sorry that we only measured soil microbial structure at the end of the incubation period (56-day after incubation). We have supplemented the data of litter mass loss after 28 and 56 days of incubation in Table 2, which helps us to understand the temporal dynamics of litter decomposition, and to make the paper more logical, we mention that this experiment is a short-term incubation experiment.

Comment: I am not convincing by this response. In this case, a comprehensive comparison with at least three-time point: 0-, 28- (sometime in the mid-process), and 56-day after incubation should be performed for all of the CO2 release rate, SOC storage and soil microbial communities. Otherwise, it seems to be inadequate to prove the role of soil bacterial and fungal communities in aggregates to litter decomposition. Additionally, I do not agree with the statement “during” in line 17, because it was only at the end of incubation process.

2. There are many concerns with regards to material collection and preparation. While the collection of top layer (0–15 cm) of the soil was stated, there were lacking detailed description regarding the representative of sample, including how many samples were collected, any specific season, ect.

Response: Thank you for the kind suggestion. The soil samples were collected from a natural grassland located in Guyuan, Hebei Province, China (41°46′ N, 115°41′ E, elevation 1380 m) in May of 2018, the initial stage of growing season. The site has a calcic-orthic Aridisol soil with a loamy-sand texture.

In brief, the top layer (0–15 cm) of the soil (~200 kg) was collected in plastic bags with a shovel, and was quickly transported to the laboratory by car, upon which the plant roots and leaves were carefully removed by hand and the soil was air-dried.

Comment: This response did not answer exactly how many samples were collected. Additionally, based on this response, ~200 kg of soil were collected at only one location? Any specific proposal for this soil collection? Why designed square-sample model and representative approaches were not applied in this field work?

3. For the preparation of materials, how was the contamination of un-indigenous microorganisms controlled during transportation and sample preparation? Importantly, the 1000 mL jars were utilized for incubation, how was the aeration managed, given that this is normally a major factor regulating the balance of fungal and bacterial growth in the soil.

Response: Soil aggregates were stored hermetically at room temperature after until collecting the litter samples. The 1000 mL jars are new and unused, so that the contamination of un-indigenous microorganisms can be avoided as much as possible. Also, each jar was covered with perforated cling film, which can not only keep the air permeability but also avoid the contamination of un-indigenous microorganisms.

Comment: In a typical microbial investigation, “The 1000 mL jars are new and unused” is inadequate to support the idea “the contamination of un-indigenous microorganisms can be avoided”. Therefore, a sterile condition should be applied. Importantly, how can sterile condition be assured for the section “Experimental design and incubation study” in line 79-93?
Experimental design: As above
Validity of the findings: As above
Additional comments: No additional comments


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: INCONSISTENT RESPONSE OF SOIL BACTERIAL AND FUNGAL COMMUNITIES IN AGGREGATES TO LITTER DECOMPOSITION DURING SHORT-TERM INCUBATION
Review round: 2
Reviewer: 2

Output:
Basic reporting: See below
Experimental design: See below
Validity of the findings: See below
Additional comments: I would like to commend the authors for their improvements to the manuscript, which have greatly improved it. There are still a few points I would like addressed prior to publication, which are listed below:

Line 17: “control” not CK as the abbreviation hasn’t yet been defined
74: laboratory, and dried
76: cited, not sited
120-122: still doesn’t full make sense. How about this? “(ii) exact barcode matching, less than two nucleotide mismatches in the primer, and no ambiguous characters in the read”
125: which database?
178 &179: significantly correlated with
202: still implies competitive dominance, which can’t be assessed from the data. I would suggest “in this aggregate, bacterial diversity is favoured”
216-217: This is still misleading – it currently tells the reader that both you and Sun et al. found that litter addition did not change the bacterial abundance, ie the total number of individual bacterial cells. Actually, what you both found was that litter addition did not change the bacterial relative abundance, ie community composition. This says nothing about total abundance, because if one taxon increases another will decrease relative to it. Conversely, the number of cells could double between samples, but if the taxonomic composition was unchanged you would detect no difference.
221-22: see previous point – you can’t say that fungal relative abundances increased, because what did they increase in relation to? You can say that fungal relative abundances changed, and that the relative abundance of specific taxa increased.
223: I concur that Aphelidiomycota are fungi, but based on their typical ecology their appearance is very surprising. I would therefore like to see some discussion of this point, at least to explain to the reader that they are not typical soil fungi.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: ADAPTATION, PHYLOGENY, AND COVARIANCE IN MILK MACRONUTRIENT COMPOSITION
Review round: 1
Reviewer: 1

Output:
Basic reporting: This is a well done study though some more details are needed in the methods section.

Please see the attached PDF for minor questions/edits related to grammar/sentence structure.
Experimental design: Line 69: It would be helpful to include the total number of species in the analysis (written in the text and not just the supplement)..
Line 101: It would be helpful to include the R code in the supplement so the reader could better evaluate the models.
102-103: Please explicitly list the predictor variables and why they may be important for milk composition.
Line 107: Please explain this analysis in more detail? Also, if this is not a phylogenetic test, then why use it?
Line 110: Explaining this method a bit more would be good.
Validity of the findings: Line 139: You mention alternative models are similar but it is not clear how this was judged? Is it based on AIC?

Line 169:170: So doesn't this support the idea that using more traditional or recently developed phylogenetic methods have little influence on the results? i.e. recently developed PCMs are not advantageous (at least with this dataset)

Line 191: Though it may also be possible that diet has a stronger association with milk composition due to biological factors
Additional comments: Some additional comments about the figures and tables:
Fig2A is too small. Fig2b&C is also too small and should be full page width.
Fig3 is quite small. The tree is not labeled so it's difficult to discern each clade's identity
Table 3: can you highlight predictors with p values <0.1 and <0.05


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: ADAPTATION, PHYLOGENY, AND COVARIANCE IN MILK MACRONUTRIENT COMPOSITION
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript is well written.  The discussion needs greater development. Several of the figures are hard to read and need additional labels for clarity.
Experimental design: This paper is original and appropriate for PeerJ.  The questions are well defined and while the results have some overlap with prior work, I think it is critically important that we revisit taxon-wide studies evaluating important adaptations, such as milk composition, as new methods become available. The methods appear to be in sufficient detail, however, I am not an expert on multivariate phylogenetic comparative methods.
Validity of the findings: The authors did not note if the data are available.  However, the data from Skibiel et al are available at J Anim Ecol.  The data are sound and appropriate measures have been taken to ensure that the data are valid.  As described below, the discussion focused more on the value of the method that the findings themselves. I would have like to hear a lot more about the significance of the data.  The author explicitly presents the result as selection regimes - this will be exciting to several readers. Thus, the patterns observed warrant further discussion.
Additional comments: I have reviewed Blomquist’s manuscript titled ‘Adaptation, phylogeny, and covariance in milk macronutrient composition (#34219). This study updates the findings of Skibiel et al. (2013) by applying multivariate phylogenetic comparative methods to the data. The results of this analysis confirm many of the findings of the original study and add the findings that primates are unique and suggest that distinct selection regimes underlie the evolution of milk in pinnipeds, primates, perissodactyls, and marsupials. While the results of this study have some overlap with Skibiel et al., I think it is critically important that we revisit taxon-wide studies evaluating important adaptations, such as milk composition, as new methods become available.

Major comments. Overall, I think the author did a nice job with this reevaluation. I particularly appreciated the nutritional geometry and the clade shift plots (but see comments below). A lot of the data presented will be unclear to a reader who is not familiar with multivariate phylogenetic comparative methods. Further, the phylogenies are small and not labeled, and thus, lactation biologist interested in these patterns will have a hard time gleaning patterns upon inspection. The discussion focused more on the value of the method that the findings themselves. I would have like to hear a lot more about the significance of the data.  The author explicitly presents the result as selection regimes - this will be exciting to several readers. Thus, the patterns observed warrant further discussion.

Specific comments.
•	Perissodactyl is misspelled throughout the manuscript.
•	Line 18: replace ‘less sugary and more fatty milks’ with more from language ‘milk that this is lower in sugar and high in fat’
•	Line 120: replace ‘Unusual clades stand out both displays’ with ‘Unusual clades stand out in both displays’
•	Line 128-129: regarding the statement: ‘The top 5 clades are indicated but typically the distinctiveness drops of steeply from the highest-ranked clade’, please clarify where the ranking is coming from.
•	Figure 2: A - I can’t read the titles for the axis. Further, please give a clear explanation of what A represents in the figure description.  For B & C - it is unclear how the three trees differ. What are the numbers? They are very hard to read. Please explicitly give the color associated with each taxon.
•	Line 154: change ‘resulted a large significant increase’ to ‘resulted in a large significant increase’
•	Figure 3: please name the taxa associated with each color.
•	Table 3: I would be much easier to interpret this is if ‘regression coefficient, (credible interval, MCMC) p-value’ were placed above each column. I recommend making all significant p-values bold to help your reader pick them out.
•	Table 4: see Figure 3 – please make it more explicate what is in each column.
•	Line 172: see comment for line 18


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: ADAPTATION, PHYLOGENY, AND COVARIANCE IN MILK MACRONUTRIENT COMPOSITION
Review round: 2
Reviewer: 1

Output:
Basic reporting: The author has done a good job revising the figures and revising the text
Experimental design: I appreciate the added methodological details, including the additional code in the supplement.
Validity of the findings: This is a rigorously performed study
Additional comments: I appreciate the edits made to this latest version. I look forward to seeing the paper published.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: RETHINKING THE LAKE TROPHIC STATE INDEX
Review round: 1
Reviewer: 1

Output:
Basic reporting: The MS is written in a highly technical (statistical) language that the main potential audience for the this paper may find hard to understand. I would suggest expanding on the explanation of the approaches and methods you used so the information is clear to the non-statistics expert. In addition, the text seemed at times to be very brief creating unclear sentences and links between sentences (examples below). There are a number of places where clarification is required:
ln 88- why were the great lakes omitted? They would be a good test case.
ln 148- define cMeso/Eu
lns 155-156- not clear
ln156- discrepancy between what?
ln 158- The connection between "We used a hold-out..." to the previous sentences is not clear.
ln 161: which three?
ln 178: Not clear how figs. 7, 8 summarize uncertainty- please expand on this.
ln 185-189: a mixing of topics and issues and therefore not clear.
ln 195-197: again, text not sufficiently clear
ln 223-234: Please provide an explanation as to how this could be done
ln 280: please expand on this statement so it is clear to the reader "This phenomenon is also graphically explained in Fig. 7,8"

Given the extended use of trophic indexes around the world over the last several decades I found the literature references not to be entirely up to date and somewhat limited. I would for example recommend looking at the papers by A. Parparov and colleagues who have addressed the limitations of the TLI approach and proposed several different approaches. But others are also available.
Figures: The figures are not numbered in the order they are referred to in the text. In addition there are cases in which figure numbers are not given. For example, ln 173 "(Figure ??).
The figures in the supplement material are not mentioned in the supplementary text.
Experimental design: The research question is well defined and justified and can be extremely useful. As mentioned above I do have concerns as to the description of the methods as they not necessary clear to the study's main target audience since the MS is written in a highly technical manner.
My main reservation about the approach and design used in this study is that they develop an approach for determining a new trophic index that may vary spatially between lakes but not temporally over time for a given lake. And while they state that it can be used in that way and may assist lake reclamation the fact that 1 of the 4 variables is constant (elevation) reduces the sensitivity to change in the index even if change occurs in a lake.
In the extended application the situation is even more extreme as 2 out of the 3 variables are constant (latitude and ecoregion) thus changes in conditions in a lake over time will have very little impact on the overall index value.
Validity of the findings: The largest shortcoming and limitation of the approach is the use of spatially varying variables and not temporally varying variables. The consequence is that the approach can indeed be used to compare between lakes but can't be used to look at changes over time. Hence, it is not an effective tool for lake restoration, in contrast to the statement made by the authors. This is also in contrast to the statement on ln 253 as to the ability to "quantity altering nutrients can move a lake across the trophic continuum".
The development of the extended application is a very good idea as it can be used in tandem with remote sensing and thus can be used at a global scale. However, here too, the variables selected prevent using the application to see change over time as 2 of the 3 variables are constant for a given lake. Limiting the selection of the variables that can vary over time would greatly enhance the applicability of the application.
Another point of inaccuracy relates to the statement on lns 244-245: "...quantifies the uncertainty of lake trophic responses to changes in nutrients, as the response varies from lake to lake" however the response can vary within a lake and not only between lakes. Thus there are two types of uncertainty-associated with responses to nutrients- between lakes and within lakes. The within lakes uncertainty is not addressed in the MS.
Additional comments: No additional comments


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: RETHINKING THE LAKE TROPHIC STATE INDEX
Review round: 1
Reviewer: 2

Output:
Basic reporting: There are a few unformatted citations and figure references, but otherwise OK.
Experimental design: It was unclear to me what was the primary goal of the analysis being described. As the authors point out on the top of page 2, "the definition of trophic state should be differentiated from its predictors."  Yet, the analysis described seems to confound these issues. Are the authors trying to define a new (continuous) *indicator* of trophic state, or better predict the already-identified trophic state of a lake from other variables? The authors assert that the goal is the former, but then I don't agree that variables such as elevation, latitude, ecoregion, or %evergreen should be part of the indicator *definition*.  Rather they seem to me to be *predictors* of the indicator. This would then imply that the goal of the paper is to create a better model to predict trophic state, which is quite different and less novel. I suggest that the authors be clear about their goal and better justify why they take the approach they do in light of that goal. There is some discussion along these lines in the last paragraph, but it comes too late and does not adequately clarify things.

I also suggest that the authors justify their use of random forests for variable selection.  Random forests allow for highly nonlinear and non-monotonic relationships, but the final fitted model is linear. Therefore, there may not be a correspondence between the most important variables in the two stages. In fact this can be clearly seen by comparing the apparent importance of % Evergreen Forest in Figures 5 and 6 against the results in Table S1. I suggest a stepwise linear regression approach to variable selection instead.

The authors need to be clearer about the form of their predictor variables.  It only became apparent to me when I saw figures S4 and S5, that they were using some jagged function of latitude, rather than a linear or smooth relation.  As noted below, I don't think such a jagged function can be interpretable as a meaningful component of a trophic state index.
Validity of the findings: I agree with the authors that regression coefficients that are not statistically significant should not necessarily be ignored.  However, I am concerned that some conclusions are being drawn based on insignificant relations.  For example, conclusions are being drawn about the important role of watershed conditions, such as %Evergreen, but Table S1 shows that the estimated coefficients on %Evergreen are 0.00 and -0.00 for nitrogen and phosphorus, respectively, with standard errors of 0.01. This implies that %Evergreen does not have an interpretable influence.

I am also concerned about overfitting when I see Figures S4 and S5, indicating a highly jagged influence of latitude.  I don't think that such a jagged dependency plot should be used to draw conclusions about the role of latitude and I suggest that a much wider smoothing window should be used.
Additional comments: I suggest that sigma and tau values be reported in Table 1. I think Figure 2 would benefit from the mu and sigma nodes having "index" or "indicator" added to their subscripts, to distinguish from the square box of trophic state at the bottom.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: RETHINKING THE LAKE TROPHIC STATE INDEX
Review round: 2
Reviewer: 1

Output:
Basic reporting: no further comments
Experimental design: no further comments
Validity of the findings: no further comments
Additional comments: I am happy with the corrections and responses provided by the authors to my previous comments.
I do however have two minor comments:
1. lines 213-214: "This is extremely important as lake misclassification has management implications.": This is indeed correct but how does the manager know the the lake has been misclassified? In otherwords, how does the POLR approach assist the manager in this issue?
2. lines 224-225: "The developed TSI model’s balanced accuracies are 0.93, 0.83, 0.72, 0,73 for oligotrophic, mesotrophic, eutrophic, and hypereutrophic classes, respectively." How were these values calculated? I calculated different values of .875. 0.54, 0.67, 0.67. These were calculated by (number of correct cases in a category of actual state /total number of cases in category of the actual state).


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: RETHINKING THE LAKE TROPHIC STATE INDEX
Review round: 2
Reviewer: 2

Output:
Basic reporting: The article is clearly written and appropriately formatted and cited.
Experimental design: 1. My previous criticism continues to hold, as it has not been addressed by the authors. Moving the extended application to the SI does not fully address my concern. To paraphrase it here: It was unclear to me what was the primary goal of the analysis. The authors state that "the definition of trophic state should be differentiated from its predictors." Yet, the analysis seems to confound these issues. I am still not clear whether the authors trying to *define* a new (continuous) *indicator* of trophic state, or better *predict* the already-identified trophic state of a lake from other variables? The authors assert that the goal is the former, but then I don't see why a variable such as "elevation" should be part of the indicator *definition*. Is it reasonable to assert that a lake at a higher elevation with the same nutrient and secchi disk values should be *defined* as being of a lower trophic state?
It might be *predicted* to be, but I don't see why it should be *defined* to be. I continue  to suggest that the authors be clear about their goal and better justify why they take the approach they do in light of that goal.
2. I am not fully satisfied with the response to my comment regarding random forests.  There are ways to address multicollinearity with linear regression models, and having used the method in previous studies is not a meaningful justification. However, if the authors continue to feel strongly about this point, I won't insist.
3. My concerns regarding the form of the predictors have not been addressed, although I acknowledge that I did not state them clearly. I should have said:  As evidenced by the partial dependency plot, the predicted value of the response variable is changing in a very jagged way in response to changes in the predictor variable. I don't think that such a jagged dependency plot should be used to draw conclusions about the role of latitude and I suggest that a much wider smoothing window should be used. This is relevant whether this information is in the main body or the SI.
Validity of the findings: All of my previous concerns regarding the validity of findings continue to apply, even though the relevant material has been moved to the SI.  They are still part of the manuscript and should adhere to the same standards.
Additional comments: The authors addressed my additional comments regarding notation.