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: MENTAL HEALTH STATUS AND QUALITY OF LIFE IN ELDERLY PATIENTS WITH CORONARY HEART DISEASE
Review round: 2
Reviewer: 1

Output:
Basic reporting: No further comments in this second review in addition to general comments to the authors
Experimental design: No further comments in this second review in addition to general comments to the authors
Validity of the findings: No further comments in this second review in addition to general comments to the authors
Additional comments: There are substantial improvements, but I still have some issues. First, the description of the construction of the scale scores for SCL-90 (line 101-113) is as far as I can see unchanged. I have loaded the data fila into SPSS from Excel and checked the ranges of the scale scores for Hostility, Phobicanxiety, Paranoidideation and Psychoticism, both in original and in mean form, and the minimum values of these are greater than zero, in fact the minimum values of the mean scores are always 1. From “Each item was scored using a 5-point scale (0=none, 1=slight, 2=mild, 3=moderate, 4=severe). This questionnaire indicated a positive item was with a score of more than 2, which may suggest potential mental health issues for that aspect. The number of positive items as per the score of each factor and a higher score indicated a more severe psychological problem”, I would have expected that at least some of these mean scores had a minimum value below 1. I still recommend a more detailed and precise description of how these scores are computed. In particular, even if data for individual items cannot be included in the data set supplementing the article for privacy reasons, a decision I support, the authors could still include syntax for the construction of SCL-90 scale scores from individual items (that are stated to range from 0 to 4, line 110) in the syntax and raw output supplement peerj-52224-Data_S2.docx. Concerning WHOQOL-BREF the computation of scale scores is precise now. Still, while it is stated, lines 121-122, that the mean scores are transformed to  0-100 to make interpretation easier, I support this, the scale scores for WHOQOL-BREF in the attached Excel data file range between 4 and 20. It is easy to construct 0-100 scores from this, but these transformed scores are not included in attached data. No reported analyses seem to use the scores transformed to 0-100.
Concerning the regression analyses the authors have expanded regression diagnostics as recommended, if I have understood it correctly, by the other reviewer. I will not recommend reduction of these diagnostics, with one exception (Durbin-Watson as detailed below). But I still recommend re-running at least some of the regression analyses with estimation of standard errors that is not dependent of the standard assumptions underlying least squares estimation. All the common possible deviations from these assumptions, including normality of error terms and homoskedasticity (or homoscedasticity, the spelling is disputed), have at least three issues in common; they do not bias the estimated regression coefficients, they may bias standard errors, usually downwards, and bootstrap estimation procedures do not assume that these assumptions are satisfied and give valid inference even if some of these assumptions are violated. For the convenience of the authors I attach syntax for the analyses (without some diagnostics for simplicity), including a bootstrap setup for the first regression analysis. The authors may consider running the regression analyses with bootstrapping, and report confidence intervals and p-values based on these if there are substantial deviations.
Concerning the Durbin-Watson test this is a procedure mainly to check for autocorrelation in time series. It may be possible to extend the procedure to observations with some other meaningful ordering, such as spatial data, but a Durbin-Watson test for observations with no statement of an interpretable ordering does not make sense. If the order of the respondents (rows in the data file) is changed, the Durbin-Watson statistics will change.
I have run the analyses reported in Tables 1-3 based on the supplementary Excel file imported into SPSS, and detected some minor differences in Table 2. And in table 3 most upper confidence bounds are lacking. I have also checked the numbers reported in Results.
Details on regression analyses, Table 2. To get anything close to Table 2 the mean versions of scores have to be used. Somatization, primary hypertension, p-value 0.854 in Table 2 vs 0.855 by running from syntax. Type 2 DM, coefficient 0.355 vs 0.354. Stroke coefficient 0.371 vs 0.370. Interpersonal sensitivity, primary hypertension, p-value 0.108 vs 0.107. Type 2 DM, p-value 0.042 vs 0.043. Stroke lower confidence bound 0.090 vs 0.089. Depression, primary hypertension coefficient 0.131 vs 0.132, lower bound -0.045 vs -0.044, upper bound 0.307 vs 0.308, p-value 0.143 vs 0.141. Type 2 DM, upper bound 0.354 vs 0.353. , Hostility, , primary hypertension coefficient 0.062 vs 0.061, Type 2 DM, coefficient 0.111 vs 0.112, Stroke lower bound 0.016 vs 0.015, upper bound 0.291 vs 0.290. Phobic anxiety, primary hypertension, lower bound 0.030 vs 0.031, upper bound 0.371 vs 0.372, Type 2 DM, lower bound 0.016 vs 0.015, stroke coefficient 0.280 vs 0.279, lower bound 0.117 vs 0.116. Paranoid ideation, lower bound -0.075 vs 0.076, stroke upper bound 0.339 vs 0.338. For Table 3 I have not found any discrepancies, but as commented above most upper confidence bounds have been deleted.

Details
Line 21, “was associated”, change to “were associated”, diabetes mellitus and stroke together constitute grammatical plural.
Line 27, “had no association”, as detailed in my first review there is no such thing as “no association”. If this statement is based on a pa-value > 0.05 state that, if the estimated association and both its confidence bounds support an interpretation as no substantial association state that.
Line 122, consider changing to e. g. “to ease the interpretation of the four domains”. In any case, “easily” with the ending “-ly” is not grammatically correct since this characterizes “interpretation”, a noun.
Line 176, “The results of univariate analyses are shown in Supplementary Tables 1, 2, and 3.” Table 1 is descriptive and Tables 2-3 report from regression analyses with more than one independent variable, none of these analyses are univariate.


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: MENTAL HEALTH STATUS AND QUALITY OF LIFE IN ELDERLY PATIENTS WITH CORONARY HEART DISEASE
Review round: 2
Reviewer: 2

Output:
Basic reporting: In the abstract and particularly in the explanation of the models, it is necessary to include that it was adjusted for BMI and hypertension and for all the variables in the model. The negative association also has to be explained as an adjusted association and adding the coefficient values, and p-value. Confidence interval not necessary.
In the statistical section I suggest you use STEPWISE to generate significant fit models. Add a paragraph on how each of the models will be evaluated.
In table 1, 2 and 3, I suggest to delete column for statists t, since it is not necessary.
In the paragraph of lines 177 to 183. It is not correct to interpret that there is an association of variables when the value of p is not significant. I think the association models are the main part of the manuscript and it should explain each of the models, including the coefficient values and p-values, and mention all the adjustment variables that have remained in the model, for example if in the model remain the variable hypertension and diabetes, then mention that each of them was adjusted by each other. It should include a main table where all the models with the coefficients, confidence intervals and p value are included. As a footer put the adjustment variables.
Experimental design: No comment
Validity of the findings: Review my comment
Additional comments: In the abstract and particularly in the explanation of the models, it is necessary to include that it was adjusted for BMI and hypertension and for all the variables in the model. The negative association also has to be explained as an adjusted association and adding the coefficient values, and p-value. Confidence interval not necessary.
In the statistical section I suggest you use STEPWISE to generate significant fit models. Add a paragraph on how each of the models will be evaluated.
In table 1, 2 and 3, I suggest to delete column for statists t, since it is not necessary.
In the paragraph of lines 177 to 183. It is not correct to interpret that there is an association of variables when the value of p is not significant. I think the association models are the main part of the manuscript and it should explain each of the models, including the coefficient values and p-values, and mention all the adjustment variables that have remained in the model, for example if in the model remain the variable hypertension and diabetes, then mention that each of them was adjusted by each other. It should include a main table where all the models with the coefficients, confidence intervals and p value are included. As a footer put the adjustment variables.


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: MENTAL HEALTH STATUS AND QUALITY OF LIFE IN ELDERLY PATIENTS WITH CORONARY HEART DISEASE
Review round: 3
Reviewer: 1

Output:
Basic reporting: No further comments in this third review in addition to general comments to the authors.
Experimental design: No further comments in this third review in addition to general comments to the authors.
Validity of the findings: I have one important comment regarding the use of stepwise regression, details are given in general comments for the authors below.
Additional comments: I have only one important issue now, the use of stepwise regression, due to a recommendation from the other reviewer in the second review round. While it is understandable that the authors followed this recommendation I have strong reservations against stepwise regression and therefore cannot recommend publication as long as this procedure is used. Since this is a case of fundamental disagreement between two reviewers I think the issue has to be resolved by the Editor. In any case this recommentation is as far as I can see not related to any recommentations from the other reviewer in the first round. In contrast, my recommentations in the second round are mostly related to changes from the first to the second versions of the article, or questions for further clarifications.
Concerning stepwise regression, I follow the recommendations of Frank Harrell’s book on regression modeling strategies, https://link.springer.com/book/10.1007/978-3-319-19425-7 , in particular the section on variable selection in chapter 4, probably section 4.3. Due to the pandemic I have not at present access to my office where I have this book, but I think the information above is correct. Briefly, independent variables that are selected by a stepwise procedure have p-values , standard errors and lengths of confidence intervals biased downwards. Also as shown in a large number of simulation studies the set of independent variables surviving a stepwise procedure is unstable, particularly for small and medium size data. Open  access sources making the same point are numerous, to give a few, https://towardsdatascience.com/stopping-stepwise-why-stepwise-selection-is-bad-and-what-you-should-use-instead-90818b3f52df , https://journalofbigdata.springeropen.com/articles/10.1186/s40537-018-0143-6 , http://www.danielezrajohnson.com/stepwise.pdf , http://assodiaf.org/en/how-bad-is-stepwise-regression/ .


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: MENTAL HEALTH STATUS AND QUALITY OF LIFE IN ELDERLY PATIENTS WITH CORONARY HEART DISEASE
Review round: 3
Reviewer: 2

Output:
Basic reporting: See comments in General comments for the author
Experimental design: No comments
Validity of the findings: View comments in General comments for the author
Additional comments: In general, improvements are observed in the manuscript, although there are still some observations that could improve the manuscript even more. These observations are described below.

In the paragraph between rows 178-189, in the explanation of the models, it does NOT refer to the fact that each of the coefficients is adjusted by a series of variables. In some models, diabetes was also adjusted for stroke and vice versa, in addition to the rest of the variables in the model. The adjustment variables were not common in all the models, so it is not possible to summarize the adjustment variables for all the models; it must be individualized for each model or groups of models. On the other hand, it is important that in the interpretation of the coefficients it refers to the type of positive or negative association (take into account the feasibility of this association), in addition to statistical significance. Apply the same for the corresponding section in the summary. I think that in this same paragraph (178-189) all the models and their results should be referred to or, where appropriate, eliminate them.

In discussion edit line 198
I think that the results of all the models should be discussed, if not eliminate them from the objectives

In the regression related tables, I suggest that the information corresponding to the constant be eliminated. For table 13, put in the footer that no variable was selected in the model and add a hyphen as a value. The latter applies to model 14 and 15 as well.
Table 2. Model 1 does not have age or BMI and in the abstract it is mentioned that models were adjusted by age and, BMI etc.

Variable age in model 7 it is necessary to put the units (years for instance).

In Table 3 is missing the confidence intervals data for model 11. Nor is information presented for model 13.


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: PHYLOGENETICALLY-CONTROLLED CORRELATES OF PRIMATE BLINKING BEHAVIOUR
Review round: 1
Reviewer: 1

Output:
Basic reporting: This paper reexamined factors influencing blinking behavior across primates. Using a previously published dataset, they used phylogenetically-controlled methods to examine the relationship between blinking behavior and locomotion, mass, group size, and trophic guild. Given that the previously published paper did not control for the phylogenetic relationships among species, this reanalysis is an important step in that direction. My main certain involves the selection of factors to consider. This paper uses some of the variables from the original dataset (body size, mass, activity) but not all (habitat type). It replaces the habitat type variable with locomotion data from a recently published database (Galan-Acedo) even though this database does include a habitat type variable. It would be helpful if more details could be provided on selection of variables.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Ln 27
“Performed” might be better than “conducted”

Ln 44
And peafowl (Yorzinski 2016)

Ln 53
What is meant by “other movement behaviors”?

Ln 72
“Account for” rather than “accommodate”

Ln 104
While Tada et al. termed the variable “Habitat Type,” they did group it into the same categories as you: terrestrial, arboreal, or a mix. How many species did you classify in the same way as Tada et. al? If you performed your phylogenetically-controlled analysis using their “Habitat Type” variable rather than your “Locomotion” variable, were the results qualitatively the same?

Galan-Acedo (2019) also present data on home range size and habitat type. Is there a reason you didn’t examine these variables? And, do their data for the other categories that you used from the Tada paper (i.e.., body mass and group size and activity) match?

Ln 106
Can you expand on this trophic guild more? What were the categories?

Ln 133
Why does locomotion appear twice in the model?

Ln 153
Galan-Acedo (2019) also present data on habitat type that seems like it could be used to directly examine habitat type to compare to Tada et al.

Ln 177
And chickens (Beauchamp 2017)


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: PHYLOGENETICALLY-CONTROLLED CORRELATES OF PRIMATE BLINKING BEHAVIOUR
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript is well written and well organized.
Experimental design: I have some major concerns that need to be addressed prior to publication:

1) The introduction leads to no hypotheses. The authors investigated three variables, namely the blink duration and rate, and tested the effect of several parameters. So, the study really needs clearly stated hypotheses, as some ecological conclusions are drawn in the discussion.

2) Because no hypothesis is stated, the authors don’t discuss crucial points highlighted by their results: namely the differences found between blink duration and blink rate.

3) The main aim of the study was to check the results of the original study of Tada et al. (2013) by taking into account phylogenetic similarity. However, the authors also changed the “habitat”/” locomotion mode” data. So, how to be sure that the differences found with the results of the original study are only explained by phylogeny?   It is not clear what is the difference between the two datasets, and what it may involve.
[They also added a “trophic guild” parameter, but it was removed from the model because of non-significance].

4) In the introduction, the authors clearly show that blinking can be affected by the situation (i.e., risky, stressful…), the social and the environmental conditions. However, we don’t know in which condition the animals have been filmed. These are useful elements to mention, as it may cause a serious bias when recording blinking behavior from these videos. Moreover, it seems that the group size data have been harvested from the literature; they may not reflect the living conditions of the captive subjects at all…
Before comparing all these species with an ecological framework, it would have been relevant to check whether the investigated variables (i.e., blink duration and rate) are affected by the animal direct environment and activity (i.e., locomotion, rest). This would have helped to develop a standardized measurement of blinking behavior reducing all the potential biases.
That being said, I have really appreciated that the authors addressed these different issues in their discussion. And they provide good tracks to follow for further research!

5) I have been worried about the sample size / species: 1 to 6 individuals. It is quite small, especially if the blinking behavior is situation-dependent… and that people who recorded the data did not really control for it.

6) Finally, I would like to remind the authors of the broad readership of the PeerJ journal. Some important points/notions are not well defined, while some more details are required to allow the reader a good understanding. (see the General comments section)

Please, see the General Comments section for specific points to address.
Validity of the findings: As I previously said, my first concern is that the authors did not just take into account phylogenetic similarity, they also used another dataset regarding the locomotion mode… As it is not clear what is the difference between the original “habitat” dataset and the new “locomotion” dataset, and what it may involve, we can ask the following question:  how to be sure that the differences found with the results of the original study are only explained by phylogeny?

Furthermore, regarding the effects of locomotion and group size: the conclusions have strong limitations due to the protocol used in the original study. A very small sample size per species, which is more concerning that if the blinking behavior is situation dependent, as it is suggested in the introduction, a great care should have been given to the experimental design. However, it is very vague in which condition animals have been filmed.
Also, group size data have been harvested from the literature. So, they may not reflect the living conditions of the captive subjects.

In addition, if the authors say that both blink rate and blink duration are related to locomotion mode, in fact, these results don’t go in the same direction.
“Blink duration was similar in solely terrestrial and solely arboreal species” L. 143
“Blink rate was lowest in arboreal, and highest in those that were solely terrestrial…” L.140
The authors need to provide precise hypothesizes in the introduction, and further discuss the differences they found between these two variables.

The variable having a powerful meaning, the trophic guild, had no effect at all…

For all these reasons, the results should be interpreted very cautiously… And I would not keep the present title. I would rather center on the need it highlights to consider phylogenetically-controlled correlations.
Additional comments: The main topic of the study is very interesting. The authors focus on the blinking behavior, which is a quite understudied aspect of animal behavior, while it may provide new insights into different aspect of the species’ ecology. They re-analyzed a previous study (Tada et al., 2013) by taking into account phylogenetic similarity. In fact, it changed the results, compared to the original study. They namely found the blinking rate and duration to be correlated with the locomotion mode.

So, this study deserves to be published, as it highlights a relevant point for further research: the real need to consider phylogenetically-controlled correlations when working with related species. However, I have some major concerns that need to be addressed prior to publication (please, see the Experimental Design section)

Below, the authors can find some  points that are meant to help them improve this manuscript:

Abstract:

L9. The use of the term “terrestrial” may be confusing here. I suppose you use this word in opposition to marine species. But shortly after, you use it again but in opposition to arboreal (Cf. L.18).

L.13 « recorded and analyzed a comparative study »: the wording doesn’t make sense to me, could you rephrase it? Maybe: just “realized”. In fact, you analyzed their study.

L.14 “a large sample of captive primates”. As you talk later about phylogeny, you should specify that this sample includes many different primate species (it could be a large sample of individuals of a same species…).

L.22-24: What about the relation with body mass and group size? Could you add a few words about how these results could be interpreted?

Introduction:

L.37: What do you mean by “this blackout is not perceived”? Could you explain a little more ?

L.42 You should explain why it is relevant to investigate blinking behavior in some sleeping animals. It is not intuitive: as we close our eyes when sleeping, “blinking when sleeping” deserves some explanations. The PeerJ journal has a broad readership, which requires to provide more details.

L.53 “blinks are timed to occur alongside other movement behaviors”: What kind of movements?  You should tell us more about it: are they movements related to social interactions, or locomotion? As in your study you test the effects of group size, and locomotion style, it would be interesting for the reader to know.

L.56 “which in turn will have been shaped by the selective processes that a species experienced during its evolution.” I am not sure of what you mean. Does “which” refer to “environmental and social conditions”?  If this is the case, could you explain? Or please, rephrase this sentence.

L.58 I am not an English native speaker, but the wording sounds curious to me. « will be tied» … You should rather say: “…is likely tied to its ecology”, or “many evidences suggest that … is tied to its ecology”.

L.65 “suggested that diurnal species blinked more than nocturnal ones”: as you did not introduce this duality (i.e., diurnal vs. nocturnal) earlier, you should provide a suggestion about what could explain this difference.

L71-73: “Species that are closely related are likely to show similar behaviors and traits due to this recent history, and therefore any analyses that compare species need to accommodate this nonindependence of the datapoints”
Again, PeerJ is a generalist journal with a broad readership. I think you need to give the reader more keys to understand this notion. It is the crucial point of your paper, and why you have done this re-analyze. So, you definitely need to explain more and provide some examples.

Methods:

L.83 You use three different measures of blinking. You should clearly explain to the reader why it is relevant to take these three measurements of this same behavior: what is the difference between them, and what do you expect from that?  Is there one standard measure used in previous studies, or always these three measures?
Maybe just say, for example: blinking/blackout can be reduced by 1) decreasing the rate, or 2) decreasing the duration, 3) decreasing both: so, you need to investigate these two parameters.
And you need to explain what do you expect from the third one.

L.89 “As well as providing these data describing blinking, Tada et al. (2013) also provided descriptive measures of the activity, habitat type, mean species group size and mean individual body mass, harvested from the literature.”
“Harvested from the literature”: do you mean that they did not take the group size and the body mass of the individuals they measured for blinking behavior? If this is the case, it should be clearly stated, as it is fundamental to interpret the results.
L.91 “In the current analysis, I use the original collected data for body mass (in kg) and group size”: are these data collected from the subjects, or the one harvested in the literature? It is not clear.

L.97-104: You should reread this paragraph:
“This classification of habitat [of Nada et al.] considered whether a species was primarily arboreal, semi-arboreal or terrestrial”
Vs.
“I instead collected data from Galan-Acedo et al. (2020) that described whether a species was primarily terrestrial, arboreal, or moved between both”
 With the elements that you provide, it is really hard for the reader to understand the difference between the two classifications… Please, rephrase it, or provide further explanations.

L.106 You should quickly define “Trophic Guild” for the broad readership of the journal. It is important because you integrated this variable in your model (Cf. L133). Also, did Tada et al. (2013) consider this variable?

L.133: “model “locomotion + mass + group size + trophic guild + locomotion”,
Why did you put « locomotion” twice?

L.134. “the least significant terms were then removed sequentially until the minimal adequate model was found.” Did you try the model selection approach using AIC?

Results:

Table1: In the caption, you should briefly provide the three modalities of the locomotion variable (i.e., terrestrial, arboreal, or moved between both, L.105), so the reader can understand what “both” means.

Discussion:

L.150 + L.166: The two variables blink rate and blink duration are not explained by the same parameters. There is a difference between both, could you provide a suggestion to explain it?
You found blink duration to be more sensitive to group size. It is interesting, as in the introduction, the result of the reference you provide about the effect of group size on blinking, is about blink rate (Rowe et al., 2020).  What about olive baboons and chickens? (L.50-51) you just talk about a “reduction in blinking” but you don’t specify if it involves blink duration or blink rate …

L.149: Also, you say that “that both blink rate and blink duration are related to locomotion mode”, but, in fact, both results don’t go in the same direction.
“Blink duration was similar in solely terrestrial and solely arboreal species” L. 143
“Blink rate was lowest in arboreal, and highest in those that were solely terrestrial…” L.140
You definitely need to discuss this difference.

L.147 « the isolated blink ratio is not correlated with any of the descriptive measures considered”
Again, what did you expect from this variable, and why?

L. 150 “in species that spend at some of the time”

L. 170-172: “Standard theory about how vigilance levels are influenced by size of group that an individual is associated with, where individual vigilance will decrease as the number of co-vigilant group members increases”
The syntax of this sentence is not correct. Please rephrase it

L.184: How did you test for it (diurnal vs. nocturnal)?
In the model you provided “locomotion + mass + group size + trophic guild + locomotion” (L.133), I don’t see this parameter.

L.189 « If the major cost of not blinking is a reduction in eye maintenance »
I think the « not » is not correct, and leads to a contradiction.


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: PHYLOGENETICALLY-CONTROLLED CORRELATES OF PRIMATE BLINKING BEHAVIOUR
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: This resubmission provided additional details on the analysis. While it is still an interesting study, further interpretation of the results are still needed.

Specific Comments:

Line 17:
This implies that the only change in the analysis was in controlling for phylogeny.

Line 27:
Not all animals blink using an upper and lower lid (e.g., many birds blink side-to-side)

Line 40
This is only known to be the case in humans. There has not been tested in nonhumans.

Line 198
Can you create the violin plot for the blink duration vs. movement?

Line 204-207
This sentence is not completely logical. If the riskiest type of locomotion is arboreal then why would animals that are arboreal+terrestrial have the lowest blink durations? Your results seemingly provide conflicting information (less blinks in arboreal species but longer blinks). Does this mean that the total amount of time species spend blinking is similar, regardless of movement type? More discussion on this result and its possible interpretations are necessary.

Line 207
Contradict rather than “do not confirm”

Line 216
A recent study within a species provides support for the importance of blinking in locomotion: Yorzinski 2020, A songbird inhibits blinking behaviour in flight.

Line 222
What frame rate was used in the Tada study? Was it above the minimum blink duration?

Can you provide the raw data as a supplement?


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: PHYLOGENETICALLY-CONTROLLED CORRELATES OF PRIMATE BLINKING BEHAVIOUR
Review round: 2
Reviewer: 2

Output:
Basic reporting: The author has very kindly and carefully provided thorough responses to all my comments. The paper now is really fluid, very enjoyable to read. I would like to thank the author because it has been really easy to track the changes.
The additional explanations in the introduction much better lead to the analyzes that have been done in this study. Also, results are more clearly discussed, and limitations are well flagged in the discussion. I appreciate the author has chosen to focus on blink rate and blink duration (as the third blinking parameter was problematic). I am very glad the author has provided a new title, that really better announces and depicts the paper.

I have no outstanding concerns regarding the publication of this manuscript. This paper will be very helpful for future studies, on the specific blinking behaviour, but not only.

I am pleased that the authors have found my comments useful, and I have just a few more minor suggestions (Cf. Comments for the author).
Experimental design: No additional comments.
The author has provided thorough responses to my previous review.
Validity of the findings: No additional comments.
The author has provided thorough responses to my previous review.
Additional comments: I have just a few more minor suggestions:

(Line numbers refer to the annotated document).

Abstract
L.20: “Described by the locomotion style of a species, where species moving through arboreal environments blink least, ground-living species blink most, and species that use both environments show intermediate rates. The duration of a blink was also related to locomotion mode.”
—> Locomotion “style” / Locomotion “mode”. Also, in the Methods, you talk about Locomotion “environment”.
I think it would be better to use the same term. Using too different terms for the same variable can be confusing.

Intro
Maybe, it could be nice to add a final sentence at the end of the introduction:
Something like: The results will help to assess (1) the importance of controlling for phylogenetic similarity when considering the evolution of behavior, (2) whether blinking behavior could bring new insights into the ecology of species.

Methods
L. 157: isn’t -->  is not
L. 158 : definition “of about” --> Definition of the

L.159 vs 161:
You are talking about one dataset but you refer to two papers Galán-Acedo et al 2020, 2019. That’s a little bit confusing, maybe you could integrate the two references differently.


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: COMPARATIVE TRANSCRIPTOME ANALYSIS OF DIFFERENTIALLY EXPRESSED GENES RELATED TO THE PHYSIOLOGICAL CHANGES OF YELLOW-GREEN LEAF MUTANT OF MAIZE
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The manuscript “Comparative transcriptome analysis of differentially expressed genes related to the physiological changes of yellow-green leaf mutant of maize” identified a yellow-green leaf mutant of maize and analyzed the differentially expressed genes related to the physiological changes by transcriptome. The methodologies used are appropriated and results are well discussed. However, there are some points that could be improved:
1.	In line 333, Fig.S3 should be revised as Fig.S2, and in line 335, Fig.S4 should be revised as Fig.S3
2.	Supplementary Table 2, 3 and 4 didn’t mention in the MS.
3.	Did the authors upload the transcriptome raw data? The accession number of the transcriptome raw data should be added in the MS.
4.	Gene name should be italic, for example, in line 228, “Zm00001d013013” should be “Zm00001d013013”.
5.	Formats of references should be in accordance with the requirements specified in peerj.


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: COMPARATIVE TRANSCRIPTOME ANALYSIS OF DIFFERENTIALLY EXPRESSED GENES RELATED TO THE PHYSIOLOGICAL CHANGES OF YELLOW-GREEN LEAF MUTANT OF MAIZE
Review round: 1
Reviewer: 2

Output:
Basic reporting: This manuscript described the difference in physiological and transcriptomic characterization between the yellow-green leaf mutant and the normal green leaf inbred line of maize. Physiological experiments on photosynthesis parameters revealed that the yellow-green leaf mutant of maize contained fewer active PSII centers with lowered net photosynthesis rate, but have the similar efficiency of the light absorption and energy transfer of the light-harvest complex with that of the wild-type plants. And, Comparative transcriptomic analysis of the mutant plants and wild-type plants led to the identification a lot of differentially expressed genes response to their physiological change. These results not only provided valuable genetic resources for further studies of chlorophyll-deficient mutants in maize, but also contribute to our understanding of the molecular basis of morphological and physiological characteristics of the yellow-green leaf mutant.
In general, this is a well written and well organized manuscript, but some corrections and observations indicated as follows must be attended.
Experimental design: There are many physiological and biochemical indexes were determined, including chlorophyll content, chlorophyll a fluorescence parameters, photosynthesis parameters, and so on. The author should clearly state if there are biological repeats were created for each experiment. It is the necessarily case the scientific experiments.
Validity of the findings: All underlying data have been provided. They are robust, statistically sound and controlled. Conclusions are well stated.
Additional comments: Authors should prepare the corrected manuscript following very carefully the Instructions to Authors Guide. There are quite some evident grammatical errors or typos, which need to be intensively examined and corrected.
Line 12, it seems that the author provided the wrong email address for the corresponding author.
Line 61, the “YS83 (LOC_Os02g05890)” should be written in italic. The same mistake were found in the following line 62, the cpSRP should be in italic too.
Line 100, line 98-105, there should be a space between the number and the unit. For example, 250*4.6mm, 5um, 40℃, the author should be more careful to avoid similar mistakes
Line 172-173, the authors should briefly state the details of the method to map the target genes to metabolic pathways using the MapMan software.
Line 210 the unit “photonsm-2s-1” was wrote in the wrong way.
Line 182 there is should be an indentation at the beginning of the second graph.
Line 208, 222, 223, and 236, there should be a space between Fig. and numbers.
Line 223-225, the numbers stated in the text are inaccurate, the authors should clearly state and make the result more easier to be understood.
Line 253-262, there are many typos and some inaccuracies about write of the genes that should be corrected.
Line 333 and 335, there are mistakes when the authors cited the supplementary figures in text, the authors needs to carefully check it thorough the whole text
Line 343-348, Authors should follow the Instructions to Authors Guide, the full name of authors is needed.
The incited references in the text should be written in italic. The author should double check the format of the references (Line 367-479). The full name of magazine is required. The author needs to make the revision though all cited references.
Legend of figure 1 should be clearly state using letters instead of left or right.


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: COMPARATIVE TRANSCRIPTOME ANALYSIS OF DIFFERENTIALLY EXPRESSED GENES RELATED TO THE PHYSIOLOGICAL CHANGES OF YELLOW-GREEN LEAF MUTANT OF MAIZE
Review round: 1
Reviewer: 3

Output:
Basic reporting: Literature references, sufficient field background/context provided.
Experimental design: Research question well defined, relevant & meaningful.
Validity of the findings: Although the authors claimed comparative transcriptomic analysis was carried out, however, accessibility to raw data is not availiable.
Additional comments: The manuscript by Tingchun Li et al. titled “Comparative transcriptome analysis of differentially expressed genes related to the physiological changes of yellow-green leaf mutant of maize” isolated a new yellow-green leaf mutant. Subsequently, chlorophyll contents, chlorophyll fluorescence parameters, and photosynthesis characteristics were determined. In addition, comparative transcriptomic analysis was performed and 21 genes involved in chlorophyll metabolism, tricarboxylic acid cycle and sucrose to starch conversion expressed differentially between wild-type and the mutant. Based on the original and meaningful work, the manuscript is acceptable. However, the presentation should be improved since there were so many demerits as listed following:

1)Although the authors claimed comparative transcriptomic analysis was carried out, however, accessibility to raw data is not availiable. I suggest authors deposite their raw data to public database and provide the accession number in their manuscript to make their analysis repeatable.
2)Are biological repeats for both wild-type and mutant applied in RNA-Seq? Sampling is not described in details, thus I’m not convinced by their DGE analysis results.
3)At line 141, the title mentioned qRT-PCR, but qRT-PCR protocol was not described bellow.
4)Diffenretial genes were identified based merely on DGE anlysis and no experimental validation was performed. I suggest the authors quantify the expression of those genes by qRT-PCR.
5)The relationship between physiological changes and gene expression was not well discussed, thus the discussion section should be revised.


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: COMPARATIVE TRANSCRIPTOME ANALYSIS OF DIFFERENTIALLY EXPRESSED GENES RELATED TO THE PHYSIOLOGICAL CHANGES OF YELLOW-GREEN LEAF MUTANT OF MAIZE
Review round: 1
Reviewer: 4

Output:
Basic reporting: 1.There are no accession number of Genbank for transcriptome data in paper, if the data will be uploaded?
2.It is necessary to make clear for the notes of figures, eg. A, B and C should be locatted on the upper left corner. And suggesting that Fig. 2 and Fig. 3 should be merged.
Experimental design: 1.Chlorophyll is the main pigment in plant photosynthesis. And maize is an important food crop in the world, increasing the yield of maize is one of most important goals in breeding. Therefore, whether it can supplement the correlation analysis between the content of chlorophyll and yield? Whether the yield of yellow-green leaf is lower than green leaves? In order to achieve the purpose of increasing yield of maize, finding the yellow leaf genes and transfering the yellow leaf genes to green leaf by gene knockout or Crisper cas9 technology can be used in further study. It will be meaningful in molecular breeding for maize.
2.Specifically, the change of yellow green leaves occurred in which period of maize development? Could you show the specific spatiotemporal changes in phenotype of yellow-green leaf?
3.It is necessary to specify the period of leaf as the material. Why only choose one stage of leaf as the material? Suggesting that choosing more stages to analyze transcriptome, it will be better to find DEGs.
4.Why only choose two sets of datas for correlation analysis?
Validity of the findings: 1.Many differentially expressed genes in chlorophyll synthesis pathways were found by transcriptomic analysis. The differentially expressed genes were verified by RT-PCR, but further verification by methods such as transgene or gene knockout or Crisper cas9 were still needed to ensure the reliability of the detected differentially expressed genes.
2.How to contact the physiological data and DEGs to explain the difference of yellow-green leaf and normal green leaf ?
3.How to regulate and control the formation of leaf colour between DEGs of many metabolic pathways, conjointly?
4.There are no integrated conclusion by combining the every passway to analyze the mechanism of the formation for yellow-green leaf.
Additional comments: This study analyzed the differentially expressed genes related to the physiological changes of yellow-green leaf mutant of maize by comparative transcriptome analysis.  A number of genes in chlorophyll biosynthesis, photosynthesis, tricarboxylic acid cycle, and sucrose-to-starch conversion were found to be differentially expressed in the yellow-green leaf mutant and the normal green leaf inbred line. These findings provide potential explanations for observed morphological and physiological changes in the yellow-green leaf mutant.  I have the following comments or suggestion for the author's reference.


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: COMPARATIVE TRANSCRIPTOME ANALYSIS OF DIFFERENTIALLY EXPRESSED GENES RELATED TO THE PHYSIOLOGICAL CHANGES OF YELLOW-GREEN LEAF MUTANT OF MAIZE
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The manuscript is acceptable after revising.


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: COMPARATIVE TRANSCRIPTOME ANALYSIS OF DIFFERENTIALLY EXPRESSED GENES RELATED TO THE PHYSIOLOGICAL CHANGES OF YELLOW-GREEN LEAF MUTANT OF MAIZE
Review round: 2
Reviewer: 2

Output:
Basic reporting: NO
Experimental design: NO
Validity of the findings: NO
Additional comments: The authors have made careful corrections to their manuscript, and all my advices have been accepted in the revised manuscript. I recommend it to be 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: COMPARATIVE TRANSCRIPTOME ANALYSIS OF DIFFERENTIALLY EXPRESSED GENES RELATED TO THE PHYSIOLOGICAL CHANGES OF YELLOW-GREEN LEAF MUTANT OF MAIZE
Review round: 2
Reviewer: 3

Output:
Basic reporting: The manuscript was well written and well organized.
Experimental design: The research question well defined, relevant and meaningful.
Validity of the findings: All underlying data have been provided. The findings are reliable.
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: A COMPARISON OF FOURTEEN FULLY CHARACTERIZED MAMMALIAN-ASSOCIATED CAMPYLOBACTER FETUS ISOLATES SUGGESTS THAT LOSS OF DEFENSE MECHANISMS CONTRIBUTE TO HIGH GENOMIC PLASTICITY AND SUBSPECIES EVOLUTION
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The paper by Nadin-Davis et al describes the complete sequences of three C. fetus subsp fetus (CFF), two C. fetus subsp venerealis (CFV) and two C. fetus subsp venerealis biovar intermedius (CFVi) strains and the comparison between the species including seven other fully sequenced genomes of C. fetus spp. The paper is well written, methods are clear, sequences have been deposited. The availability of more completely sequenced genomes of C. fetus spp. is useful however most of the conclusions have been described before in various papers. I have some minor comments.

Minor comment
1. please add the 7 other C. fetus strains and their genomes to table 1 and table 2. remove table 3. I think their phenotypes are known or at least they can be determined making the comparisons in the text easier to understand while removing a superfluous table.

2. The title suggests that the mechanism for CFV genome plasticity has been found, however a mechanism has not been really described, merely an ineffective CRISPR/CAS system. I would not call that a mechanism. CRISPR/CAS is a mechanism to prevent introduction of foreign DNA, it's absence or ineffectiveness is not a mechanism in CFV to increase plasticity.

3. The start of the discussion mentions that CFF evolved in a human population (Iraola et al 2017). That paper uses a discrete trait analysis using human and animal strains, suggesting that the genomes of ancestral strains of strains infecting humans looked like strains infecting humans now. This is correct. This however does not mean carriage by humans, or "human strains", it means that the strains infecting humans look like other strains having infected humans, but these strains most likely still originated from fecal contaminated bovine or ovine products (e.g. unpasteurised milk). In livestock they did live their life as commensals or potentially even pathogens. There is little to no evidence that these strains evolved in humans as the assumption was flawed to begin with (the flawed assumption being: strains isolated from humans are carried and transmitted by humans to other humans). Clades of CFF are simply extremely undersampled in livestock (carriage) and extremely oversampled in humans (invasive disease). Not surprisingly as CFF is not notifiable in livestock unlike CFV while at the same time CFF in humans is associated with invasive disease, which usually does draw the attention of clinicians and researchers. Perhaps some nuances on the statement "originated in humans" is needed in the start of the discussion as the evidence for that statement is quite thin.

4. Header of table 3 has a typo, but if the strains in table 3 are added to table 1 and 2, this comment can be ignored.


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: A COMPARISON OF FOURTEEN FULLY CHARACTERIZED MAMMALIAN-ASSOCIATED CAMPYLOBACTER FETUS ISOLATES SUGGESTS THAT LOSS OF DEFENSE MECHANISMS CONTRIBUTE TO HIGH GENOMIC PLASTICITY AND SUBSPECIES EVOLUTION
Review round: 1
Reviewer: 2

Output:
Basic reporting: The paper doesn’t emphazise the relevance of subspecies differentiation at the genomic level in the introduction. I think this is very relevant to understand the logics behid the whole experimental design.
L111: inconsistencies in the genetic and phenotypic characteristics of the strains are found. Several papers support it. Some examples are:
Van der Graaf-van Bloois et al. 2014. J Clin Microbiol 52(12): 4183-8; Calleros et al. 2017 J Microbiol Meth 132: 86–94.
This is important to the discussion of the results.
L260: there is an extra parenthesis
L413: please replace “ruminants” for “mammals”
Fig S1, Fig 1 to 4 and TablesS1 and S2: legends for these are missing.
Fig. S1: the quality of the figure is not good. One cannot read the names of the strains
Experimental design: Experimental design is sound and takes into account several aspects of the issue. Technical aspects are well performed. Methods are well described and detailed.
Validity of the findings: Results of the paper are very important and it gives useful information to the understanding of genetic variation and pathogenesis of the species. However, improvement on the discussion is necessary.
The discussion is centered in CRISPR variation, but it fails at citing previous work where some of the hypotheses were already presented (Calleros et al. 2017).
Some of the results are not discussed. Some examples of interesting results that could be discussed:
L224: results from ELISA on serotype are contradictory with previous foundings.
L318-326: the relationship between virulence genes and phenotipic characteristics could be discussed. Some previous work (for example Van Bergen et al. 2005 J Clin Microbiol 43(12): 5888–98; Wang et al. 2013 Vet Microbiol 164(1): 67-76; Kienesbergeret al. 2014 PloS one 9(1): e85491) raises some hypothses about.
Also, subspecies differentiation at the genetic level is a matter of discussion in this species. I think it would be interesting to take this into account.
L393: parA is one of the genes used as a target for diagnostic methods
Several of these results could shed light on the discussion (for example, referred to the genomic evidences of a differentiation between Cff and Cfv) if taken into account.
Additional comments: CGB is a major concern to cattle productive systems. The species C. fetus is increasing its importance as a human pathogen. Accordingly, the analysis of its genetic variation is of paramount significance. Also, it is a fastidious microorganism with very strict growing requirements, so obtaining strains to sequence the genomes with high accuracy and circularized is difficult. This emphasizes the importance of the work presented in the paper.
Results of the paper are very important and it gives useful information to the understanding of genetic variation and pathogenesis of the species. However, improvement on the discussion is necessary.


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: A COMPARISON OF FOURTEEN FULLY CHARACTERIZED MAMMALIAN-ASSOCIATED CAMPYLOBACTER FETUS ISOLATES SUGGESTS THAT LOSS OF DEFENSE MECHANISMS CONTRIBUTE TO HIGH GENOMIC PLASTICITY AND SUBSPECIES EVOLUTION
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript has professional english used throughout.
Lines 102-111 -
-The authors should cite McMillen et al 2006 as the original parA real time assay which was bunked by Spence et al. See - https://jcm.asm.org/content/44/3/938.short. Chaban et al 2012 tried to adapt the parA assay to a SYBR green assay however this target has been shown to not be specific.
The authors should also cite the current OIE - Animal Health standards for CFV diagnostics - this is up to date and summarises the inaccuracies of all currently potential CFV molecular tests. See link https://www.oie.int/fileadmin/Home/eng/Health_standards/tahc/current/chapitre_bovine_genital_campylobacteriosis.pdf
-Also the authors mention biotypes - to this reviewer's knowledge they are 'biovars' - CFV biovar venerealis and CFV biovar intermedius - the authors refer to CFV and CFVi only? please confirm the use of this nomenclature in this manuscript - this reviewer differs.
Line 331 - bound not 'bounded'
Line 387 - within 'most' isolates - quantify exactly rather than generalising
Lines 390-391 - quantify instead of the use of the term 'some'
Experimental design: The experimental design is good -the reporting could do with some additional organisation.
The results section entitled 'genome organisation' needs structure and could include sub-headings. It is otherwise an un-ordered section with many ideas.
The methods are well described.
Validity of the findings: The authors have made several assumptions from their data but lack in interpretation. They have made lists of differences but have not entertained the reason why CFVs are so different to CFFs.
It is not usual to refer to tables and figures in the discussion section.
Table 1 should include the new accession numbers for the Canadian isolates in this study - this is very important and should be included in the manuscript.
The publication cannot be accepted without accession numbers for the new genomes - this is very important. Raw data and annotations need to be reported in the manuscript with accession numbers.
Additional comments: The authors have not really contemplated in the discussion as to why the differences in CFV genomes have evolved? the discussion in the current form is not very intuitive or suggestive.

The publication is worthy of publication - there are several groups who have dominated this field - however the authors have not set themselves apart and the manuscript could be better organised.

Lack of accession numbers for the new genomes is a big issue - this is important for relevance. It is difficult to evaluate without this raw data.


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: A COMPARISON OF FOURTEEN FULLY CHARACTERIZED MAMMALIAN-ASSOCIATED CAMPYLOBACTER FETUS ISOLATES SUGGESTS THAT LOSS OF DEFENSE MECHANISMS CONTRIBUTE TO HIGH GENOMIC PLASTICITY AND SUBSPECIES EVOLUTION
Review round: 2
Reviewer: 1

Output:
Basic reporting: Issues have been adressed.
Experimental design: Issues have been adressed.
Validity of the findings: Results of the paper are very important and it gives useful information to the understanding of genetic variation and pathogenesis of the species.
Additional comments: The paper has improved substantially. The discussion section is now more complete and ordered.

However, some major concerns remain:
•	In the light of the results, the authors suggest in the conclusion (and I agree with this), that genetic variability of the specie is a continuum and may be subspecies classification is no longer adequate to explain its clinical and ecological behavior. But the problem is this conclusion is not reflect along the discussion, because the treatment of the subspecies separation and the adaptations of the strains to bovines is treated to loosely, confusing some concepts. For example: “While the CFFs contained just one copy of this element, bovine-adapted strains tended to harbour greater copy numbers of these elements and very notably these prophage sequences were often located at the boundaries of sequence rearrangements and inversions supporting the importance of this element type to genome plasticity.” Cff/Cfv classification and adaptations to hosts are not interchangeable concepts. It has been demonstrated in several previous studies. I do recommend to the authors to thoroughly think about this issue and change some statements in the discussion accordingly.
•	Some conclusions about bovine adaptation cannot be made because of the absence of comparison with strains isolated from other hosts, so these statements are not supported by the results. There are a human and an ovine strain included in the study, but discussion on this comparison is lacking. Also, all Cfv and Cff strains were collected from the genital tract or aborted fetuses, so it is difficult to extract differences in adaptation from this data.

There are also some minor issues that must be corrected:
•	CRISPR-cas system is written in three different ways along the paper, please correct this.
•	“Consistent with our observations, screening of a collection of 102 C. fetus isolates for the presence of cas1 failed to detect this gene in all 62 CFV samples (Kienesberger et al. 2014) thus supporting prior suggestions that disruption to the CRISPR-cas system could be a significant factor contributing to the emergence of bovine-adapted strains of C. fetus (Calleros et al. 2017).” Please rewrite to avoid saying that 2017 is prior to 2014.


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: A COMPARISON OF FOURTEEN FULLY CHARACTERIZED MAMMALIAN-ASSOCIATED CAMPYLOBACTER FETUS ISOLATES SUGGESTS THAT LOSS OF DEFENSE MECHANISMS CONTRIBUTE TO HIGH GENOMIC PLASTICITY AND SUBSPECIES EVOLUTION
Review round: 2
Reviewer: 2

Output:
Basic reporting: the article meets the standards
Experimental design: the article meets the standards
Validity of the findings: the article meets the standards
Additional comments: Thank you for undertaking the necessary edtis


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: HISTORICAL AND CURRENT DISTRIBUTION RANGES AND LOSS OF MEGA-HERBIVORES AND CARNIVORES OF ASIA
Review round: 1
Reviewer: 1

Output:
Basic reporting: The papers is well organised and clearly written. There are some minor lapses in English expression that I note in comments to the authors.
Experimental design: This is solid. The study is primarily descriptive, reporting summary data as maps. This is fine, other than for one point concerning screening for data quality that I mention in comments to authors.
Validity of the findings: The findings are sound, and I have no doubt significant enough to justify publication.
Additional comments: This is a valuable analysis, which provides strong and useful results. The paper is well organised and clearly written. I have only three substantial comments, and some minor corrections to the text.

1. at Lines 127-129 the authors comment that primary data on species locations were screened for data quality. I think some more explanation is needed on criteria that were used to wither accept or exclude records on the basis of their strength. The point is an important one as a matter of scientific rigour, and also because is is clear from comments in the Results that significant numbers of records were excluded.

2. The database the authors have collected seems very valuable. It is not clear from the review copy opf the paper whether the database will be made available in supplementary material or in some accessible data archive. In my view, it should be made available.

3. In Figure 1, it is difficult to see the current distributions. Perhaps this should be shown on separate maps without the overlays that make them difficult to see on the current maps.

Some minor corrections to the text:

Line 55: change ‘on’ to ‘in’.

Line 56: ‘OWEN-SMITH’ should not be in capitals. Same for SOULE at line 57, and in a few other cases.

Line 64: a more up-to-date reference might be valuable here. I would suggest Smith et al (2018) Science 360, 310.

Lines 183-189: there is some repetition here, that should be removed.

Line 300: the word ‘less’ should be inside the brackets.

Line 334-335: can you give a reference and brief explanation for this term? I’m not sure what it means.

Line 352: should be ‘persecution’ rather than ‘prosecution’.

Line 353: ‘got vanished’ should be changed to something like ‘disappeared from’.

Line 360: should be ‘retreating’ rather than ‘retrieving’, I think.


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: HISTORICAL AND CURRENT DISTRIBUTION RANGES AND LOSS OF MEGA-HERBIVORES AND CARNIVORES OF ASIA
Review round: 1
Reviewer: 2

Output:
Basic reporting: The MS is general well written, although there are some minor technical problems that need addressing, such as the inconsistent use of capitals when citing references. Avoid the word “got” and see lines 421-422 for a confusing sentence.

Title: This currently too broad and ambitious as it suggests that all megafauna are covered in all areas of Asia, whereas this study is restricted to a subset of ten species and not all of Asia is covered. Interestingly, the offered short title is more evocative, although it again needs refining and the gardening term is not valid based on data provided here.

The section on other studies of range contractions (lines 77-94) does not cover the important studies in Asia by Turvey and his collaborators, nor the comprehensive historical mapping of large mammals in China by Wen and his collaborators. A useful recent paper is Qian et al (2020. Changes in the Historical and Current Habitat Ranges of Rare Wild Mammals in China: A Case Study of Six Taxa of Small- to Large-Sized Mammals. Sustainability 12, 2744; doi:10.3390/su12072744), which covers some of the species addressed here for the area of China, and also highlights considerable literature not used by Mahmood et al.
Experimental design: Mahmood et al collate information on earlier distributions of a suite of ten large mammal species and compare this to their occurrence in a set of protected areas, this in tropical/warm temperate Asia. Given the decline of large mammals globally, this is clearly a relevant and important topic, as we need to better understand the patterns, causes and consequences of these declines.

The study focuses on a set of ten large mammal species, but it is not clear what the basis for selecting these species may be. So the authors should justify this choice, this based on their hypotheses or research objectives, or some other justification around data quality, etc.

A key aspect of this study is the assessment of the representation of these taxa in a set of the larger protected areas  - this is an important focus as it talks to the efficacy of conservation areas and should possibly be highlighted in the title.

How robust are the earlier distribution records compiled here? Monsarrat et al (2018. Ecography 41:1-12. https://doi.org/10.1111/ecog.03944) recently demonstrated spatial biases in reporting of historical distributions of large mammals. In addition, Monsarrat & Kerley (2018. Biological Conservation 223:68-75. https://doi.org/10.1016/j.biocon.2018.04.036) demonstrated taxonomic biases in the historical reporting of large mammals. The authors need to recognise the risks inherent in such biases and how these may influence their study. This is especially the case given the absolute paucity of data for some of their species across such a huge area, as well as the varying socio-political histories (and hence reporting prospects) across their study area.

The delimitation of the study period as “a period of several thousand years” (line 96) is rather vague and also needs to be justified. Later this is given as ~10 000 years (line 110) – which is more specific but also not equivalent to “several”.

The method of developing historical occurrence maps is extremely crude, given that it is based on interpolation between location records, as well as some apparently arbitrary (as seen in the maps provided) extrapolation beyond these records. Hence, this approach does not reflect any assessment of habitat suitability in these ranges.

The classification of occurrence in the various PAs (lines 158-159) assumes that the habitat within the PA was, and still is, currently suitable for the species in question. This needs to be validated.

The estimation of proportional loss of megafuanal species from PAs (lines 163-169) will provide a misleading answer, given that the analyses focusses only on a subset of the megafauna that may have occurred in these PAs. Hence this section of the study is not valid and should be removed.

The section on the literature used (lines 173- 180) adds no value to this study as this is background information and does not provide information on the actual data used. The authors should provide these datasets of literature used and their validity in a supplementary information file or accessible database, and delete this section. This should be replaced with a description of the number of actual records and their representation through time for each species. These data should also be made available in a data repository - see submission system.

Lines 297-303 are to some extent a repetition of the section in the Methods. Delete to focus on Results.
Validity of the findings: The historical data analysed for each species ranges from 29 to 458 points (no data provided for leopard), and this for a period of up to 10 000 years. There is clearly a need to justify the extrapolation of these sparse data over millions of km2 and thousands of years. In addition, there is a need to validate the application of the range estimation approaches using such sparse data. Clearly, simply filling the gaps between points (as done here) has considerable risk of overestimating areas occupied, particularly when there is no assessment of habitat suitability in the intervening areas. This is glossed over in the Discussion, but is a foundational issue for this paper, and must be addressed in a robust fashion.

The findings for the 10 species reported on here cannot be used to refute (lines 340-343) a much larger earlier study based on 173 species, as the present study uses a non-random sample.
The Discussion tends to focus on detailed patterns of loss (often without supporting literature) that is at odds with the large scale of this study. This focus needs to be kept to the larger scale to be valid.
The generalization around megafuanal biogeographic patterns (starting line 404) are not justified, as only 10 taxa were considered, and this is clearly a biased sample.
The section on the loss of the ecological function of these species (lines 433-443) could be replaced by two sentences, an also needs to relate to the predators.
Additional comments: Missing from the Discussion is the concept of relaxation – and a key question would be whether these species have been lost in these PAs prior to or subsequently to the establishment of the PAs.


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: HISTORICAL AND CURRENT DISTRIBUTION RANGES AND LOSS OF MEGA-HERBIVORES AND CARNIVORES OF ASIA
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: I am happy with the revised version of the manuscript. My one further comment relates to availability of primary data and sources as supplementary material. In their response the authors say that these data and sources can be made available if the journal editor requests it. It is my strong view that the data should be provided in this way.


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: DETERMINING NOVEL CANDIDATE ANTI-HEPATOCELLULAR CARCINOMA DRUGS USING INTERACTION NETWORKS AND MOLECULAR DOCKING BETWEEN DRUG TARGETS AND NATURAL COMPOUNDS OF SINISAN
Review round: 1
Reviewer: 1

Output:
Basic reporting: Zhang et al. investigated the main functional molecules in SiNiSan and their potential targets by a network-based polypharmacology strategy. The authors recommended some targets in the p53 pathway, however, there is no in vitro and in vivo data to support their hypotheses.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Some concerns should be addressed before its publication in this journal.

1.	Figure 3: The figure legend needs to be revised because there is no “Black cube” in the network.
2.	Figure 4: What do the yellow nodes represent?
3.	Figure 7:
The figures are very ugly, and should better be re-rendered.
The figure legend needs to be revised because the P53 pathway-related targets listed are not consistent with ones studied by molecular docking.
In the main text, the binding modes of quercetin to the selected targets should better be discussed with more details. Intermolecular interactions apart from the hydrogen bonding interactions also need to be discussed. Besides, if there are common binding patterns of quercetin to the three selected targets?
The docking energies to the three selected targets are too small, which suggests that quercetin is unlikely to bind. The authors should better add the unit for the docking energy.
4.	Quercetin is a well-known multi-target drug, and the reported targets of quercetin need to be discussed.
5.	Tables 1 and 2: Why the table titles are duplicate?
6.	There are lots of spelling and grammar errors. For example, the presentation of the name of prof. Feng in Acknowledgements is wrong.


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: DETERMINING NOVEL CANDIDATE ANTI-HEPATOCELLULAR CARCINOMA DRUGS USING INTERACTION NETWORKS AND MOLECULAR DOCKING BETWEEN DRUG TARGETS AND NATURAL COMPOUNDS OF SINISAN
Review round: 1
Reviewer: 2

Output:
Basic reporting: (1) The language in this manuscript is ambiguous, and the conclusion is exaggerated. Please see Major Q1 and Q7.
(2) There are some problems in the figures. Please see Minor Q1 to Q3.
(3) Raw data are supplied.
Experimental design: (1) The experimental method has many problems and needs to be redesigned. Please see Major Q2 to Q7.
(2) There is no problem with the statistical method in this manuscript.
Validity of the findings: (1) The findings could not be verified. Please see Major Q1.
Additional comments: Hepatocellular carcinoma (HCC), the most common form of primary liver cancer, typically develops within the background of chronic liver disease. It is an aggressive disease with a dismal prognosis. Therefore, it is necessary to develop more effective therapies. Sinisan, a traditional Chinese medicine, is effective for the treatment of gastrointestinal disorders. In this study, Dr. Qin Zhang and colleagues investigated the potential mechanism of SNS for the therapy of HCC by constructing interaction networks and molecular docking. The study provides the conclusion, which showed that quercetin in SNS may treat HCC by targeting on SERPINE1, CCNB1 and CDK1 gene in p53 signaling pathway. There are some problems in this study, which require the author to revise the research design, and there are some major concerns that need to be addressed.

Major concerns:
1. “Validity of the findings & Introduction” Line 95-99. “It has been believed to be effective for treating liver stagnation and spleen deficiency, improving disorders of digestive system and alleviating mental depression (Jiang et al., 2003). While the unclear mechanism of TCM has hampered its clinical practice in the world, the therapeutically effect of SNS on Hepatocellular Carcinoma remains to be further elucidated.” The purpose of this study was to analyze and predict the potential active components and potential targets and mechanisms of SiNiSan against HCC by using the pharmacobioinformatics method. The significance of this study is based on the premise that SiNiSan has significant anti-liver cancer activity. However, according to the literature investigation described by the author and myself, there is no clear evidence that SiNiSan is clinically used in anti-liver cancer therapy, nor has it been found to have significant anti-liver cancer activity in animals. Therefore, the existing experimental data in this manuscript cannot support the conclusion of the study. The author must verify the anti-liver cancer activity of SiNiSan by adding in vivo and in vitro experiments. The first method can directly verify the anti-liver cancer activity of SiNiSan by using the transplanted tumor model of mice, or the second method can confirm the anti-liver cancer activity of the active components of SiNiSan screened in this study by using in vitro methods.
2. “Materials & Methods – Screening of Bioactive compounds and targets in SiNiSan” Line 124. “Set the filter criteria as OB≥30% and DL≥0.18 for potential bioactive compounds…” What is the rationale for selecting these two screening criteria? What are the references? The authors must elaborate in the text.
3. “Results – Bioactive compounds and targets in SiNiSan” Line 169-174. “In all, 113 compounds were identified as potential bioactive molecules for further study.” What is the content of these 113 compounds in SiNiSan? The effect of the drug depends on its activity and content, and it is hard to believe that all 113 compounds are effective. The authors should indicate the content of these 113 compounds in SiNiSan using experimental or database data.
4. “Result – Hepatocellular Carcinoma-associated targets” Line 176-178. Why choose the GSE101685 data set with few samples? The expression profile of HCC with different pathological types, clinical stages and carcinogenic factors is different. Therefore, the author should select a specific type or clinical stage of HCC. It is recommended that the authors use the TCGA data set, taking into account patient risk factors (HBV, HCV or alcohol) and clinical stages.
5. “Result – Analysis of Protein- protein interactions (PPI) network” Line 191-195 and Figure 4. In this part of the experimental results, the author established the interaction network of differentially expressed genes of HCC. However, the author did not elaborate on the relationship between this part of the experimental content and the theme of this study. This makes the description of the results in this section less logical.
6. “Result – GO and KEGG pathway enrichment analysis and KEGG network” Line 197-205 and Figure 5 and 6. In these results, the effect of SNS on HCC by GO and KEGG pathway enrichment analysis and KEGG network were analyzed, but the authors did not specify which genes were used for analysis.
7. “Result – Docking results of compound and core target” Line 207-216 and Figure 7. In this part, the author studied the potential direct effects of quercetin and SPRPINE1, CDK2, CCNB2, and IGFBP3 proteins using docking. But there are a lot of problems here. First of all, quercetin is an active ingredient widely distributed in Traditional Chinese Medicine and has been studied the most. Therefore, quercetin has the most identified targets, and using Quercetin as a key component of SiNiSan clearly lacks logic. Second, molecular docking is used to study the potential direct interactions between molecules, and only those molecular interactions that interact with the pharmacodynamics pocket may be biologically active. The authors do not explain the impression of molecular docking conformation on the biological conformation of proteins, so these data are meaningless. Third, AutoDock is a semi-flexible docking software, which requires the crystal structure of interaction between positive drugs and target proteins to be obtained first. Based on the drug action pocket, the screening docking is conducted by AutoDock. Obviously, the authors did not obtain the corresponding crystal structure, and did not use a reasonable docking method, but simply docking. Generally speaking, this part of data lacks verifiability and practical significance.

Minor comments:
1. Figure 4. What does the yellow node represent?
2. Figure 5. Please ensure the clarity of the text.
3. Figure 6. Please add the x-coordinate title.


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: DETERMINING NOVEL CANDIDATE ANTI-HEPATOCELLULAR CARCINOMA DRUGS USING INTERACTION NETWORKS AND MOLECULAR DOCKING BETWEEN DRUG TARGETS AND NATURAL COMPOUNDS OF SINISAN
Review round: 1
Reviewer: 3

Output:
Basic reporting: NA
Experimental design: NA
Validity of the findings: NA
Additional comments: In this manuscript "A strategy to find novel candidate anti-hepatocellular carcinoma drugs by constructing interaction networks and molecular docking between drug targets and natural compounds of SiNiSan formula" Zhang et al. have analyzed a traditional Chinese medicine -SiNiSan which has been used as a treatment strategy for liver and spleen related diseases. They analyzed the active compounds in SiNiSan and performed various bioinformatics analysis and commented on quercetin and its target genes. Although the question of understanding the application of Chinese medicine to hepatocellular cancer patients sounds interesting however manuscript in it’s current form is very poorly written and lacks novel insights gained from the bioinformatics analysis.

Specific comments that need to be addressed are listed below:

1.	TCGA has a huge dataset of hepatocellular cancer patients. It is not clear why authors have confined their analysis to very few patient sample data from GEO to identify differentially expressed genes in normal vs. cancer patients. What pathways these differentially expressed genes represents? A GSEA can help in understanding the core genes/pathways contributing to hepatocellular cancer in patients.  Figure2: Does distinct clusters shows different pathways? It is also not clear how the expression data was normalized.

2.	Figure3: what is the biological significance of this network analysis? What does different colors represent? Figure legends should clearly explain the figure. Why this analysis was restricted to only top differentially expressed genes? A detailed functional interpretation of such complex network can aid in understanding the biological significance of these differentially expressed and target genes.

3.	What was the criterion used to call a gene as differentially expressed? Did authors perform any normalization of gene expression data downloaded from GEO? It should be clearly mentioned in the methods section of the manuscript.

4.	It is not clear what authors meant by preliminary screening network (line 194)? What does DC>61 and BC>600 represents? What does yellow node represents in Figure 4? What are the biological insights gained from this protein-protein interaction data analysis?

5.	Please provide all the technical details (including parameters) of how autodock was performed for the docking analysis. What is the rationale for performing a docking experiment here? Does quercentin has significantly higher binding affinity to SPRPINE1, CDK2, CCNB2 as compared to any other protein? Line 212-217 what is protein A, protein E and protein C?

6.	What is the overlap of bioactive compounds and targets among various individual herbs in SNS? Do these targets belong to same distinct pathways?

7.	Figure 1: It is not clear what authors meant by OB+DL  screening. Abbreviations should be limited in the figures for better understanding. "Differential anglysis" -> Differential analysis  Abbreviation used in figure should be clearly described in figure legend.

8.	Figure quality is very poor and needs significant improvement.

9.	For the screening of bioactive compounds in Chinese herbal medicine database how filtering criteria as OB≥30% and DL≥0.18 were identified for potential bioactive compounds identification.

10.	Figure 5: what does colors indicates?

11.	Figure6: what does the x-axis represents?


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: DETERMINING NOVEL CANDIDATE ANTI-HEPATOCELLULAR CARCINOMA DRUGS USING INTERACTION NETWORKS AND MOLECULAR DOCKING BETWEEN DRUG TARGETS AND NATURAL COMPOUNDS OF SINISAN
Review round: 2
Reviewer: 1

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


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: DETERMINING NOVEL CANDIDATE ANTI-HEPATOCELLULAR CARCINOMA DRUGS USING INTERACTION NETWORKS AND MOLECULAR DOCKING BETWEEN DRUG TARGETS AND NATURAL COMPOUNDS OF SINISAN
Review round: 2
Reviewer: 2

Output:
Basic reporting: (1) The language in this manuscript has improved.
(2) Picture quality has improved.
(3) Raw data are supplied.
Experimental design: (1) The experimental method has some problems and needs to be redesigned. Please see Major Q2 to Q3.
(2) There is no problem with the statistical method in this manuscript.
Validity of the findings: (1) The findings of this manuscript should be verifiable.
Additional comments: Hepatocellular carcinoma (HCC), the most common form of primary liver cancer, typically develops within the background of chronic liver disease. It is an aggressive disease with a dismal prognosis. Therefore, it is necessary to develop more effective therapies. Sinisan, a traditional Chinese medicine, is effective for the treatment of gastrointestinal disorders. In this study, Dr. Qin Zhang and colleagues investigated the potential mechanism of SNS for the therapy of HCC by constructing interaction networks and molecular docking. The study provides the conclusion, which showed that the SNS component has a large number of stage III HCC targets. Among the targets, the sex hormone receptors, the AR and ESR1 genes, are the core targets of SNS component and the most active proteins in the PPI network. In addition, quercetin, which has the most targets, can act on the main targets (BAX, CDK1, CCNB1, SERPINE1, CHEK2, and IGFBP3) of the P53 pathway to treat HCC. Although the author answered all of the questions, I still have some questions about the responses, which need further answers from the authors.

Major concerns:
1. “Introduction” The authors need to clearly explain why the differentially expressed genes for stage III HCC were analyzed.
2. “Prediction and Gene Set Enrichment Analysis (GSEA) enrichment analysis of stage III hepatocellular carcinoma targets” Line 134-147. In the GSEA analysis, how did the authors group the samples and then identify the target genes associated with stage III HCC? The author needs to explain in detail.
3. “Differential analysis of stage III hepatocellular carcinoma targets and the building of a compound-disease target network” Line 150-157. Same as major Q2, which two or more groups were compared to obtain differentially expressed genes?
4. “Response to Major Q1” The supplement of in vivo and in vitro experiments is my suggestion to the author, which is the most direct way to increase the significance of this study. I can understand that the authors do not want to add too much workload, but I do not suggest the authors to use the epidemic situation as a reason. As we all know, the COVID19 epidemic situation in mainland China has been well controlled in May.


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: DETERMINING NOVEL CANDIDATE ANTI-HEPATOCELLULAR CARCINOMA DRUGS USING INTERACTION NETWORKS AND MOLECULAR DOCKING BETWEEN DRUG TARGETS AND NATURAL COMPOUNDS OF SINISAN
Review round: 2
Reviewer: 3

Output:
Basic reporting: NA
Experimental design: NA
Validity of the findings: NA
Additional comments: Fig1: A bar plot representing the enriched termed at a fdr threshold will be helpful  as compared to Figure 1A. What does yellow region represent?

How much is the overlap between GSEA enriched terms in GEO and TCGA datasets?

Figure3: What are the top genes? Please mark them for clear understanding

Figure4: A statistical analysis of significance of intersection of targets of SNS component and HCC genes will be helpful as compared to non informative network representation. Similarly what is the significance of PPI analysis as shown in Figure 5


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: DETERMINING NOVEL CANDIDATE ANTI-HEPATOCELLULAR CARCINOMA DRUGS USING INTERACTION NETWORKS AND MOLECULAR DOCKING BETWEEN DRUG TARGETS AND NATURAL COMPOUNDS OF SINISAN
Review round: 3
Reviewer: 1

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: Hepatocellular carcinoma (HCC), the most common form of primary liver cancer, typically develops within the background of chronic liver disease. It is an aggressive disease with a dismal prognosis. Therefore, it is necessary to develop more effective therapies. Sinisan, a traditional Chinese medicine, is effective for the treatment of gastrointestinal disorders. In this study, Dr. Qin Zhang and colleagues investigated the potential mechanism of SNS for the therapy of HCC by constructing interaction networks and molecular docking. The study provides the conclusion, which showed that the SNS component has a large number of stage III HCC targets. Among the targets, the sex hormone receptors, the AR and ESR1 genes, are the core targets of SNS component and the most active proteins in the PPI network. In addition, quercetin, which has the most targets, can act on the main targets (BAX, CDK1, CCNB1, SERPINE1, CHEK2, and IGFBP3) of the P53 pathway to treat HCC. The paper is improved and most concerned raised by the reviewer have been addressed. I think it is might suitable for publication at this version of revised manuscript.


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: LOWER LOCUS COERULEUS MRI INTENSITY IN PATIENTS WITH LATE-LIFE MAJOR DEPRESSION
Review round: 1
Reviewer: 1

Output:
Basic reporting: Language is mostly clear but more descriptive terms should be used as detailed below. Some suggestions for additional references are given to provide a more exhaustive overview of recent additions to the literature. A few typos are still in the main text but those can be quickly resolved.

________________
[Major comments]:

-	The authors often use a language that suggests within-person (i.e., longitudinal) change in locus coeruleus ratios. For instance, they often use the words “decrease(d)”, “reductions”, “alterations” etc. (including in the title). Their data, however, merely suggests between-person differences. Please adapt your wording throughout the manuscript. That is, based on your cross-sectional data you cannot claim that there is a decrease or reduction.
-	Similarly, the contrast mechanism of current locus coeruleus-sensitive MRI sequences is not well understood (see e.g., the Betts et al., 2019 consensus statement in Brain). Thus, please refrain from using terms like “neuromelanin signal” etc. throughout the manuscript.

________________
[Minor comments]:

-	Results: Please always report statistics in full in the main text (not only p-values [e.g., “Due to the consecutive recruitment strategy, groups differed in age (p=0.001) …”]).

-	Table 1: Please specify which long-term memory test is reported (in the legend).
-	Methods: Did the FSE sequence include online or offline averaging? Please specify.

-	Results: Please report whether locus coeruleus ratios were related to age and sex (as corrected values are reported throughout).

-	A number of dedicated recent studies demonstrated age differences in locus coeruleus ratios and their topography along the rostrocaudal axis of the nucleus in large samples (https://pubmed.ncbi.nlm.nih.gov/30447418/ ; https://www.nature.com/articles/s41562-019-0715-2 ), please consider adding these to your discussion of (A) age differences (B) rostrocaudal differences to provide a more complete picture of the literature.

-	Methods: “However, due to the small size of the locus coeruleus and the anisotropic voxel size of the images, the neuromelanin sensitive sequence was not manipulated and used as reference” – as there is a particular “reference” used to calculate the locus coeruleus ratios, please consider exchanging the word reference here (e.g., use “fixed image”; “target image” instead) as it is misleading.

-	Please double-check your reference “German et al., 2988” :)
Experimental design: As detailed below, the methods (MRI-processing) need to be described more thoroughly.

________________

[Major comments]:

-	The description of the coregistration procedure needs more detail so potential readers can reproduce the pipeline. Please add in the main text (A) which software and version was used, (B) what type of coregistration type (linear, nonlinear etc.), (C) what type of interpolation. Related to this:

o	The authors appear to threshold (binarize) the transformed ROI images. This suggests that some type of interpolation other than nearest neighbors was used (which would maintain the binarized properties of the ROI image over the transformation). Please adapt/explain.
o	As brainstem coregistration is quite challenging for standard software (see e.g., Keren et al., 2009, NIMG or Betts et al., 2019, Brain for discussions), please provide some visualization (in the supplementary information) for coregistration accuracy in the brainstem.

-	Methods: Please justify why the mean over the 10 brightest voxels is a valid metric for the (integrity of the) locus coeruleus. Considering the native space voxels size (0.4 * 0.4 * 2.5), 10 voxels do not capture the whole locus coeruleus? Cf. Tona et al., 2017 in Brain Struct. Funct.
Related to this aspect: Please justify why you did not employ the suggested formula to calculate the locus coeruleus intensity ratio, cf. Liu et al. 2017, NBR (Eq. 1), as this limits comparability to other studies.
-	Methods: Unless you are expecting interhemispheric differences in the association of locus coeruleus ratios, please consider averaging ratios across hemispheres to (A) yield more reliable integrity estimates (B) reduce the overall number of comparisons (and the need to correct for multiple comparisons).
Validity of the findings: ________________
[Major comments]:
-	Methods: What other medication affecting the central noradrenergic systems did participants take (e.g., beta blockers)? Did these relate to locus coeruleus ratios as well?

-	Figure 1: Does this visualize a single participant in native space? – the voxel size does not appear to differ substantially between x (y) and z dimensions (should be 0.4 vs 2.5 mm).

________________
[Minor comments]:

-	Figure 1: Please show the same participant with the same sections in panel A and B so potential readers can evaluate the fit of the locus coeruleus and reference ROI to the hyperintensity. Also, please add the reference ROI to the image.

-	Table 1: Healthy controls show a MMSE range of 24—30, this indicates that some HCs fall below the often employed cut-off of 26 points? Please report and discuss. In addition, the range of MMSE values indicates that in the MDD group, at least one subject received a score of 13 points? – this seems particularly low?

-	Methods: Why was this specific age range (60—76 y) chose, please justify.
Additional comments: General:
In their manuscript titled „Locus coeruleus neuromelanin signal intensity is decreased in patients with late-life major depression“ Guinea-Izquierdo, Mónica Giménez et al. investigate MRI-indexed locus coeruleus integrity in later life. In particular, a group of healthy controls is compared to patients suffering from depression and/or MCI (in groups of n ~ 30). The authors report lower locus coeruleus ratios is depression which may be related to intake of SNRI.
The study is timely and of interest to a broader readership. Locus coeruleus imaging is a relatively new scientific field and the study may help to better understand previously reported age differences in locus coeruleus contrast. However, before publication a series of major concerns need to be addressed. As detailed below/above (depends on the order of these boxes):

-	The description and justification of employed methods is often insufficient
-	A more descriptive language should be used at several instances
________________