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: EFFECTS OF HANDLING AND SHORT-TERM CAPTIVITY: A MULTI-BEHAVIOUR APPROACH USING RED SEA URCHINS, MESOCENTROTUS FRANCISCANUS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The article is well-written and polished. It was easy to read and flowed well. I did not see that the raw data was shared, however.
Experimental design: Research questions were well-defined and the methods were well-designed and clearly described.
Validity of the findings: Data appear to be robust, statistically sound, and controlled. Conclusions are clear and well-supported by their results.
Additional comments: Overall this study was well-designed, the paper was well-written and very easy to follow. This study is relevant and acceptance and publication is recommended with just a few minor revisions.

Specific comments:
Line 325 I might suggest that this sentence be revised since it is impossible to test all possible post-release behaviors.

Line 333 I believe the common name for D. antillarum is long-spined sea urchin. In addition this sentence appears to be an incomplete sentence.

The results for the distance travelled by sea urchins in 30 s after being induced to flee by contact with a predatory sea star (Fig. 3) were unexpected to me. The authors say that the urchins held in captivity appeared to recover from the handling event and “improved gradually after two days in captivity” (lines 302-304). Could this particular upward trend in the distance traveled be tested statistically? Is the slope different from zero? This was a very interesting result that could be highlighted further perhaps with a regression.

Then they suggest that there is a “possible reversal of the stress state”, however have other studies investigated this predator escape response of urchins held during longer periods. Do the authors think that this particular behavior needs to be investigated further? Can they suggest other methodologies in which researchers could test fleeing when in contact with a predator? They could include a few sentences on future research ideas, such as flight response from other predators—sheephead for instance may be more challenging to test in the field but perhaps this could be done at least for lobsters in the field.

Could urchins forget how to respond to a predator if held for a very long time? Further, could the captivity itself be a stressful experience if longer than the length of time examined in this study? At the end of this paragraph there are a few references pertaining to bird species, but could the authors include relevant references of studies of marine invertebrates. I know there has been some research on anti-predator responses of snails from water-borne stimuli or cues (for example: Alexander and Covich 1991 https://doi.org/10.2307/1542339). Perhaps a marine invertebrate example may be a bit more relevant than bird references—since the predator cues and captive environment are in the same medium as urchins (water rather than air). If you include the bird references, you may want to say these are birds, in case there are any readers unfamiliar with the common names saddlebacks and chukars.

What other behavioral responses could be tested in future studies?


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: TRANSCRIPTOME SEQUENCING OF OLFACTORY-RELATED GENES IN OLFACTORY TRANSDUCTION OF LARGE YELLOW CROAKER (LARIMICHTHY CROCEA) IN RESPONSE TO BILE SALTS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Clear and unambiguous, professional English used throughout.
Must be improved (see report below)

Literature references, sufficient field background/context provided.
'no comment'

Professional article structure, figs, tables. Raw data shared.
'no comment'

Self-contained with relevant results to hypotheses.
Must be improved (see report below)
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The present paper by Jiabao et al. investigated the expression of olfactory-related genes stimulated by bile salts in olfactory transduction in the fish Larimichthys crocea. The experimental level of study as well formal aspects are very good. The object of study has attracted larger research interest and present submitted study deepens the knowledge of this interesting model. As such, MS could be good contribution; however, the text itself suffers from many problems and unfortunately in present version must be rejected. This MS is a classical example of nice data lost in poorly, superficially formulated text. Both the English writing style (see comments below) and the general organization from Abstract/Introduction and particularly the discussion, which in the present form contains a lot of introduction material and looks like an expansion of the results section, more than in fact, discuss hypothesis and present a solid home message for the readers. I listed some (not necessary all) examples of incongruences in the text and suggest the authors to complete reformulate Abstract/Intro/discussion whole structure before any new attempt of submission.

Fish x fishes
Please keep in mind that Fish can refer to multiple fish individuals, especially when they are all the same species of fish. Fishes, however, usually refers to multiple species, especially in scientific contexts. Therefore, most of the places in the text the correct plural form should be Fishes.

Keywords should not repeat words from the Title. Please select a different set of.

Lines 48-57: The word “bile salts” appeared 8 times just in these sentences. Please find a different way of referring to it in order to avoid constant repetition of words (E.g: “Bile salts are biliary constituents derived from cholesterol that are synthesized in the liver and stored in the gall bladder (Haslewood, 1967). Regulated by different transport proteins, they are released into the intestinal”
Lines 56-57: This can be deleted or moved to final part of introduction, since its content is the same present in lines 69-70.
Line 85: Please provide the number of such protocols.
Lines 199-205: This paragraph can be moved to the introduction since it does not represent any discussion of your data.
In the same way, Lines 216-224 can be moved to the introduction since it does not represent any discussion of your data.
Line 232: Besides, we found that 17 TAAR genes were all up-regulated and were  …..
Lines 246- 254. Again can be moved to the introduction since it does not represent any discussion of your 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: TRANSCRIPTOME SEQUENCING OF OLFACTORY-RELATED GENES IN OLFACTORY TRANSDUCTION OF LARGE YELLOW CROAKER (LARIMICHTHY CROCEA) IN RESPONSE TO BILE SALTS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The text of the manuscript by Hu and colaborators is clear and the English is appropriate. There are some small mistakes that need correction: e.g. 1 "L. Crocea" needs to be changed to "L. crocea" in many parts of the manuscript; e.g. 2 "adaption" change to "adaptation". I think that the specially in the introduction some additional changes should be introduced: its somewhat ambiguous the central aim of the work - the dataset is valuable and the analysis look correct, but I would ask to provide in the introduction a better focus. For example, comparative analysis with similar studies; olfactory systems in fish etc, and the reference list is in some cases old (not that its bad but for sure other more recent studies exist). The figures and tables look appropriate (except figure 4 which looks a bit pixellated). Some sections should be more self contained: lines 231-236; lines 274-284 (not clear at all).
Experimental design: Most of the experimental design is appropriate. Yet, the authors should define better defined; what is the gap? I failed to see how the RNA seq was conducted - how many samples were sequenced? Individual sequencing or pulls? Why 5 minutes to observe the behavior?
Validity of the findings: I find the results valide but see comments above.
Additional comments: NA


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: LOW GENETIC DIVERSITY IN CAPTIVE POPULATIONS OF THE CRITICALLY ENDANGERED BLUE-CROWNED LAUGHINGTHRUSH (GARRULAX COURTOISI) REVEALED BY A PANEL OF NOVEL MICROSATELLITES
Review round: 1
Reviewer: 1

Output:
Basic reporting: Apart from some minor language issues (see below), language is appropriate.
Cited literature is appropriate.
The first figure is superfluous, one table needs additional information.
There are no clear hypotheses formulated, but the authors present what they announce.
Experimental design: Appropriate methods were used and are sufficiently described in the MS apart from minor issues (see below).
Validity of the findings: Some conclusions tend to reach a bit too far (see below), otherwise presentation and interpretation of results are good.
Additional comments: Lines 120 and 122 give different years.
Line 145 makes no sense: One cannot (directly) sequence a draft genome. What did you sequence? Was the outcome an assembly worth being called a draft genome? Which sample was used for the genome or were all samples pooled?
Line 175: why arbitrarily?
Lines 181 ...: consistently use l OR L for liter(s)
Lines 214, 226, 255: consistently use subscripts
Lines 342-4: Why are you so confident that your markers work in other or even all Garrulax (s.l. or s.s.?) species?
Line 430: Are museum specimens available? Why were they not used? Are they too old to provide DNA for MS studies?
Do you really need a photo = Figure 1?
Figure 4 is not a haplotype network.
Table 1: What is the orientation of the primer sequences?


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: LOW GENETIC DIVERSITY IN CAPTIVE POPULATIONS OF THE CRITICALLY ENDANGERED BLUE-CROWNED LAUGHINGTHRUSH (GARRULAX COURTOISI) REVEALED BY A PANEL OF NOVEL MICROSATELLITES
Review round: 1
Reviewer: 2

Output:
Basic reporting: The english is acceptable for publishing.

The reference list is sufficient but you will see that I recommend shortening the manuscript substantially which may result in removing references (see general comments).

The article structure is professional. Again, I recommend shortening the manuscript o the basic findings and this may allow fusing results and discussion (iff this is covered by the editorial rules).

As outlines in my general comments, I feel that the manuscript has the structure of a primer Note which is a technical paper. Thus, hypothesis stating and testing is limited and the paper is rather descriptive than hypothesis driven.
Experimental design: The motivation of the paper is justified.

The rather descriptive and technical character of the work allows just limited hypothesis building.

There are analytical flaws:

Number of samples:
Studies on conservation genetics and endangered species usually deal with low samples sizes and one cannot overcome this problem (this paper: OPHK = 14, NCZ = 9)! However, one has to be aware that a low sample size affects the accuracy of parameter estimates and the statistics behind it (as a rule of thump: 20 spec. minimum to get reliable values). This counts for the diversity and divergence analyses presented in tables 1 and 2. Thus, the authors should indicate this problem throughout the manuscript and scale down their interpretations, e.g. related to deviations from Hardy-Weinberg equilibrium or indirect conclusions about inbreeding (tables 1, 2 and text related to theses tables).

Bayes' analyses (STRUCTURE):
Please revise the presentation of these results both in the text (e.g. results: lines 306-311) and figure 2. Figure 2 shows in panel (a) the STRUCTURE bar plot (Please indicate clearly in the caption that the three plots refer to K2, K3, K4 and the two populations under consideration). These bar plots (particularly the upper one) indicate admixture (F1 like) between two genetic clusters. However, I miss any interpretation of this. I also think that the additional bar plots putatively representing results for K3 and K4 are meaningless and should be removed simply to keep the manuscript as concise as possible. Presentation of bar plots for K other than this considered to be the most likely one (e.g. as determined by the Evanno method) is only meaningful if they indicate additional and biologically interpretable substructures. Please also double check panels (b) and (c). Panel (b) is labeled with DeltaK... but the respective caption says Posterior probability (Ln P(D)). Panel (c) is labeled with L(K) but the respective caption says Delta K. I ask the authors to explicitly present a graph showing the mean Ln P(D) and to consider and discuss the possibility of K=1 which is not detectable by the Evanno-method but is a possible analysis outcome.
Validity of the findings: The manuscript has the character of a Primer Note rather than a research paper although microsatellites result from whole draft genome sequencing. This feeling is also due to the lack of data from free living populations which makes the results difficult to interpret.
Additional comments: Chen et al. develop a new set of microsatellites for the critically endangered Blue-crowned Laughingthrush. The motivation of the study is to provide a conservation genetics tool for monitoring genetic diversity and divergence that was exemplarily applied to two captive populations. The authors discuss their results in the context of current conservation strategies for the bird. The study is more than justified and gets is significance from the fact that genetics can substantially contribute to monitoring activities in combination with other disciplines, e. g. field work or ecology. This makes the content of the manuscript publishable and interesting for the scientific community particularly for people working in the field of ornithology and/or conservation.

However, I cannot support the publication of this manuscript in its current form. This primarily arises from what I said in respect to Experimental design and Validity of the findings. Furthermore, the paper has a strong focus and does not provide information for a broad scientific community in biosciences. Apart from this strong focus, the conclusions drawn about the population origin and the genetic status (e.g., inbreeding) of the two captive populations are limited (see discussion and conclusions part). I did not lose the impression that the content of the paper should be presented in the style of a Primer Note with an extended discussion covering the specific aspects of conservation. Consequently, I recommend substantial shortening the text just focussing on the main aspects, i.e. the need for this molecular tool, the basic properties of the loci, the main results and interpretations of the study of these two captive populations. I also think that the manuscript might get more attention in a journal with particular focus on ornithology.

I recommend addressing the aspects raised particularly in the paragraphs on "Experimental Design" and "Validity of the findings" and give some additional comments below. Once all these aspects are addressed I may be able to guaranty correctness of the study and may support publication.

Additional comments:

Figure 4: Please double check Figure 4. The document that I downloaded for review shows a bar chart rather than a network and the caption appears to not fit with what is shown there.

Comparison between diversity and divergence between different species and marker sets (Table 3):
Comparing between independent estimates of mitochondrial and microsatellite diversity between different species and different marker sets is dificult and under many circumstances even impossible and misleading. This is because different species have different life cycles and histories and one has to argue why a comparison is meaningful. Further, the comparison refers to non-orthologous loci which further limits the validity of the comparison. Genetic drift and/or selection acts differentially on the freely recombining nuclear loci and, therefore, affects locus specific estimates.

Discussion: This part of the manuscript provides a lot of general statements not least about the importance of conservation genetics (there is no doubt about). This makes the discussion overly long and partly unfocussed. The authors should rather concentrate on how their marker set could be incorporated in monitoring activities of free living populations and/or how their results on captive populations can be used for the management. Is it feasible to use the captive animals for reintroduction in the wild? If yes, I suggest an extensive discussion about putative problems arising from releases of captive animals in the wild, e.g. outbreeding depression. I argue that the scientific value of this manuscript greatly depends on the potential use of the data in a management plan (that could be developed or outlined here).


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: LOW GENETIC DIVERSITY IN CAPTIVE POPULATIONS OF THE CRITICALLY ENDANGERED BLUE-CROWNED LAUGHINGTHRUSH (GARRULAX COURTOISI) REVEALED BY A PANEL OF NOVEL MICROSATELLITES
Review round: 2
Reviewer: 1

Output:
Basic reporting: -
Experimental design: -
Validity of the findings: -
Additional comments: The authors intensively revised the MS, following our recommendations.

Only language needs some smoothing, e.g.:
Line 373: two populations
Figure 1: "Each line represents one individual, and the possibility of individual that assigned to the given genetic cluster is represent by the length of each line." Represented? Is possibility the right English term?


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: LOW GENETIC DIVERSITY IN CAPTIVE POPULATIONS OF THE CRITICALLY ENDANGERED BLUE-CROWNED LAUGHINGTHRUSH (GARRULAX COURTOISI) REVEALED BY A PANEL OF NOVEL MICROSATELLITES
Review round: 2
Reviewer: 2

Output:
Basic reporting: The first revision of this article is again written in an acceptable English for publication with an appropriate reference list. I appreciate that the authors followed my suggestion to substantially shorten the manuscript; particularly the discussion. I feel that the length of the manuscript is more appropriate with respect to results than the original submission.
Experimental design: I am disappointed about the way how the authors addressed my previous comments on several aspects of the genetic analyses.

First, I asked the authors to explicitly comment on the low sample size in respect to Hardy-Weinberg-Equilibrium (HWE) tests and interpretations about signs of inbreeding. Instead of doing so, they refer to the POWSIM test in respect to divergence (FST). Note, this was part of the original manuscript and my comment did NOT refer to this well chosen analytical tool. Furthermore, the authors discuss the impact of low sample size in assignment tests. I renew my comment and kindly ask the authors to interpret their results on HWE and inbreeding, e.g., in results under the headline genetic diversity.

Second, I explained in detail my concerns on the interpretation of the STRUCTURE results. Apart from correcting and improving the label in Figure 1 (Figure 2 of the original submission), the authors did not biologically interpret the clear signs of admixture but added methodological information. This was not meant by my comments. I renew my concern and state that STRUCTURE results could be consistent with K=1 given the high mean lnPD value (with minor SD) as shown in Figure 1 B of the revised version. I again ask to provide a biological interpretation for admixture which puts the statement about divergence between OPHK and NCZ in a slightly different context. I suggest that divergence is extremely low (FST 0.065; line 294 of the revised manuscript) and this low level of divergence is not appropriately reflected by STRUCTURE analyses but only (slightly) by PCA. I repeat again that the Evanno method cannot detect K=1 but K=1 is a possible outcome of Bayesian clustering that has to be considered. In addition, I asked to provide an explanation why bar plots for K=3 and K=4 are presented. I still argue that they do not add to the topic.
Validity of the findings: The validity of the findings are presented more focussed in the new (shortened) manuscript and become clearer.
Additional comments: If my concerns listed under experimental design are seriously and explicitly addressed based on scientific arguments, I will support publication of this manuscript. I currently feel that the authors did not provide convincing arguments to reject my suggestions from the first round of review.


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: LOW GENETIC DIVERSITY IN CAPTIVE POPULATIONS OF THE CRITICALLY ENDANGERED BLUE-CROWNED LAUGHINGTHRUSH (GARRULAX COURTOISI) REVEALED BY A PANEL OF NOVEL MICROSATELLITES
Review round: 3
Reviewer: 1

Output:
Basic reporting: This manuscript is in the second round of revision which required only few additional changes in respect to some scientific issues. The incorporated changes come in a clear and professional English and do not destruct the professional article structure. The new sentences explain results and hypotheses in in competent way.
Experimental design: The changes required in this seconds round of review did not refer to the experimental design or any methods used in the original paper/first review.
Validity of the findings: The new interpretations now correctly reflect the validity of the findings.
Additional comments: Thanks for the competent revision. I consider this version of the manuscript as a nice contribution to conservation.


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: LARGE CARNIVORE HABITAT SUITABILITY MODELLING FOR ROMANIA AND ASSOCIATED PREDICTIONS FOR PROTECTED AREAS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Basic reporting is solid
Experimental design: I do have several comments related to the fairly sophisticated modeling in this study.

First, it would be interesting to take a look at the raw data (simple maps of occurrence / non-occurrence used in this analysis). While investigating predictors of occurrence of large carnivores is an interesting exercise, I wonder how useful it is for the main question of the manuscript: overlap with different types of protected areas. It would really be even more interesting to run the analyses of overlap with protected areas using these raw data first. Would the authors get different results? I strongly suggest that the authors do this analysis. In case the results are not different, then the whole framework and thrust (and title) of the paper should be changed to something like ‘modeling habitat suitability for large carnivores in Romania’.
Second, more explanations are needed on the use of the Huber/White/ sandwich estimator. The authors claim that is was used to handle “heteroscedasticity and/or autocorrelation”. However, this technique does not handle spatial autocorrelation. By definition, the data used here are highly correlated spatially, especially since the land planning units used in this work can be smaller than the annual home range size of all three species. The Newey-West variance estimator is the adequate technique for handling autocorrelation (by specifying a given lag). I suggest the authors re-run their analysis using the Newey-West variance estimator and re-evaluate the findings. Moreover, the authors are running a logistic regression, so the distribution is not mis-specified; importantly, unlike Ordinary Least Square regression, heteroscedasticity is to be expected in logistic regression (you are using a binary predicted variable), and homoscedasticity of independent variables is NOT a prerequisite of implementing logistic regression.
Third, please see Hosmer, Lemeshow and Sturdivant (2013) “Applied Logistic Regression” 3rd Edition for variants that accommodate spatial autocorrelation, applicable to this analysis.

More on statistical analyses:
- were the data standardized prior to analysis? Based on Table 3, it is difficult to evaluate which of the many variables used in this analysis were most predictive (among those ). Standardization is common practice, which allows the evaluation of the relative predictive power of different variables by bringing them to the same scale. Also, the parameter estimates in Table 3 are almost useless; the authors implemented logistic regression, from which very useful information, such as odds ratios, can be extracted. Please replace these uninformative betas with odds ratios and their confidence intervals, and interpret the variables accordingly throughout the manuscript (e.g., X increase in cover type Y leads to an increase/decrease in occupancy of Z).
- For all variables for which a non-linear relation was expected, please provide hypotheses or other evidence for support for making this choice
Validity of the findings: I found that the conclusions of this study to be a bit misleading. Foremost is the assertion that different types of protected areas are “conducive” to the occurrence of large carnivore species. This is a rather vague statement and the authors imply causation, when in fact this is pure correlation. This needs to be reframed and presented for what it is: an analysis of overlap between modeled distributions of large carnivores (their habitat, actually) and protected areas. While large carnivores were likely on the species lists used to declare the new Natura 2000 sites protected areas, these new protected areas were not designed specifically to accommodate large carnivores (take for example the situation in the Western Carpathians). Few, if any, of these protected areas are actively implementing strategies for large carnivore conservation, including National Parks. Not exactly sure how these protected areas are “conducive”.
I encourage the authors to expand the ideas outlined here, and consider the limitations of their conclusions, and the potential uses of this research for management or conservation. In particular, the assertion in the Conclusions (378-383) seems far-fetched and not grounded in reality. What is the trend in declaring new protected areas in Romania? Are they still doing it? Do the authors think it is feasible to declare new PAs of sufficient size to accommodate carnivore ecology? Please bring this discussion down to the real-world situation, and make an attempt to place the findings of this work in this light.
Additional comments: This is an interesting manuscript evaluating relation between the occurrence of European large carnivores and different types of protected areas in Romania. The authors used logistic regression to model the probability of occurrence of the three carnivore species, and concluded that Natura 2000 sites overlap the most with brown bear and lynx, while National Parks overlap most with wolf distribution. Given the paucity of data on Romanian large carnivores (despite accounting for the bulk of the European populations of brown bear, wolf and lynx), the information in this manuscript has the potential to advance the conservation and management of these species in the EU.
I appreciate the modeling effort put in by the authors, and the general comments and concerns regarding the overall conclusions and the statistical analyses are aimed at improving the message and strengthen the conclusions.

A couple other comments:
 - Results section: this section really needs some numbers! The authors speak in terms of greater/lower/ medium etc., but no specifics are given to what those qualitative measures mean. Interpreting odds ratios would help greatly in honing message and strengths of this modeling.
- Discussions: need to be more "down-to-earth" in terms of real-world recommendations (see details above). Calculating  the odds ratios for variables in your best models should help with interpreting and discussing the findings on predictors of carnivore occurrence
- Figure 2: Are these predictions based on single-variable models, or on the full model with all the other variables at mean value? I assume is the former, given the strength of the predictions (they all reach 1!). Some of those relations are non-intuitive (national road for bear and wolf), while some of them seem insanely strong (conifer forest for wolf).

Thank you for the opportunity to review this 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: LARGE CARNIVORE HABITAT SUITABILITY MODELLING FOR ROMANIA AND ASSOCIATED PREDICTIONS FOR PROTECTED AREAS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript was relatively well written but there are several English usage errors that should be corrected. Examples are “have” on line 35 and “conducive of occurrence” on line 264, but I was not carefully checking so there are likely others that should be sought out and addressed.

I thought the study objective as framed by the title and abstract is very interesting, but needed a better treatment in the main text. I have suggested some ways forward in the author comments below.
Experimental design: The study design seems to be appropriate for the questions asked, but there are clarifications that are necessary for a reader to be clearly able to assess this. In particular, I was unable to discern some of the statistical model formulation details. See the comments to author below for specific comments.
Validity of the findings: The model seems appropriate for the task, and the conclusions justified. However, I would like to see some more clarification of how the models were specified and how the predictions relate to the headline objectives.
Additional comments: This study develops a predictive model of carnivore occurrence across Romania. It relates habitat and landscape features to presence absence data (footprint data) and then uses the best models as identified by AIC competition to predict where the carnivores could be present. It then aims to explore the relationship between protected area categorization and expected carnivore presence but it needs some work to better achieve this part of the study.

In general I appreciate the intent of the study and the methods that were used but the framing of the study, the details of the methods, and the descriptions of the results need to be clarified.

Major comments:
1.	The study appears to be framed as looking at the distribution of potential habitat for different carnivores across the different types of protected area categories. The framing misses on two fronts.

a.	First, the desired framing is not clearly laid out. For instance lines 75-77 seems to attempt to justify the framing as something to do with Natura 2000 sites but does not manage to convey to me exactly what the study is aiming to achieve. Objective 2 lays out a slightly different objective as merely coverage of carnivore habitat by the protected areas in Romania, but this doesn’t require all the background information about EU protected area types. Please carefully think about what framing is most appropriate for your study intent and then revise throughout to clarify.

b.	Second, the methods and results do not convey how the predicted values were used to explore the importance of the protected area categories. Depending on which framing you choose, these sections need to be revised to clearly lay out the analysis which achieves objective 2 and explain what is found.

2.	The statistical model that relates habitat and human density features to carnivore occurrence data needs to be explained more clearly.

a.	From the text I could not figure out how the footprint data were converted into the ones and zeros that were present in the data table and also could not determine what the independent variables associated with each FID represented. My best guess based on the supplementary figures is that presence or absence within each WMU is used as the independent variable but that needs to be much clearer in the text.

b.	I found the description of the predictor variables jumbled and confusing (Lines 158-201). This section would be improved if it was arranged according to the broad variable categories in Table 1 (Land cover, Terrain, Rivers, Roads) with each section describing the reason the predictor was included in the model, where the data came from, and what the units, scale etc of each predictor was.

c.	Please explain why you thought it necessary to re-rank supported models using a measure of deviance explained? I would have thought that the AICc value would have been adequate for determining the best parsimonious models because the sample size in the competing models was the same.

d.	The rectangular moving window component of the analysis (lines 195-201) needs to be explained better because based on the current text it is not apparent what was done, why, or how it relates to the predictive models.

e.	Similarly, the Huber-White sandwich estimator needs to be explained and justified better. In particular, why did you choose to use this estimator as opposed to fitting a basic GLM. I went to look at the Stata code in the supplemental for clues but I could not find an explanation there either.

f.	Finally, the process for deciding upon which models to specify for later AIC competition needs to be better explained. I can appreciate that you would not want to try every combination of variable because of the large number of predictors, but it is not clear to me why you chose the collection that you did, and in particular why did you choose to include non-linear combinations (any predictor squared) of predictors in the candidate model set?

Minor comments:

Line 75 and elsewhere. The term representation/ representativeness is not being used as is common in the literature. E.g., Chauvenet ALM, Kuempel CD, McGowan J, Beger M, Possingham HP (2017) Methods for calculating Protection Equality for conservation planning. PLoS ONE12(2): e0171591. https://doi.org/10.1371/journal.pone.0171591

Lines 91-94: These predictions do not match the framing of the paper or the objectives well.

Lines 143-144: This comment about individual footprints being missed implies a misunderstanding of the sampling approach. Of course sampling is going to miss some individuals!

Lines 145-155: The justification for data appropriateness needs clarification. In particular, the comment about concerns about the robustness of density estimates seems misplaced and the justification of the dataset as being appropriate because it is the only one.

Line 162: why did you exclude Natura 2000 sites with no known carnivores?

Line 217: I would like an example of when a squared term would be ecologically relevant. The provided example only includes linear terms.

Lines 230 -232: why did you calculate the VIF only for the top models? I would have calculated it for the predictors in general (in a full model) at the step where you assessed correlation of predictor variables.

Lines 243-244: this is the first mention of the different protected area groups. The reframing should make it clear earlier that this analysis will be occurring and what question it is designed to answer.

Lines 284-285: please explain why the 1% deviance rule was applied for this species.

Lines 301-334: This discussion describes lots of associations between predictor variables and the probability of occurrence. The model specification, specifically the addition of many squared terms that have unclear hypotheses as to their expected relationship with the response variable, makes this study design appropriate for identifying the best model for explaining the most variation in the variable of interest (presence or absence of carnivore) but it is not appropriate for commenting on hypotheses to do with the relationship between individual predictors and the response. This is because the observed strength of the relationship is modulated by the other predictors in the model and some of them are the squared terms with unclear meaning. Please rewrite to limit the discussion to inference that is appropriate to the model design.

Lines 335 onward: The reframed discussion needs to be focused around what the predictive models say about carnivore presence across the different types of parks if that is the framing that is chosen for the paper.

Lines 339-341: this statement cannot be made based on this model. Here you are using occurrence data to predict to protected areas. This statement could only be based on the predictions being from protected areas (i.e., protection type being included in the predicted model)

Table 1:
-	Not clear what the units of habitat type are. % cover? Present in pixel?
-	The units column should explain what the numbers in the data spreadsheet represent, not say whether the data is continuous or not.
-	What does the linearity column mean? A non-linear predictor value would be something that was squared. This does not seem to be what you are using it to mean.

Table 2:
I don’t think the null model comparison adds anything useful

Table 3:
I would prefer to see the coefficient presented as \Beta +/- the width of the confidence interval/2

Figure 2:
Please fix the scale the x axis of these plots from 0 to 1 so they can be interpreted easily.


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: LARGE CARNIVORE HABITAT SUITABILITY MODELLING FOR ROMANIA AND ASSOCIATED PREDICTIONS FOR PROTECTED AREAS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The article is much improved in terms of flow and narrative. The authors provide R code for all analyses. The literature cited and background information is in great shape and up to date. I have no concerns whatsoever on the Basic Reporting. Great job!
Experimental design: This work is original research and the authors performed new analyses as suggested by the previous review. The methods are rigorous and have a lot of detail (including R code and raw data). The work represents a great piece of the puzzle that is carnivore ecology in Romania. The conclusions about habitat suitability and representation of these species in Romanian protected areas are well supported.
I do not have any concerns about the validity of the analyses, but I do have a couple quick questions for the authors about the reporting of the logistic regression results. Table 3 presents coefficient estimates, yet the Results talk about odds ratios and the interpretation of carnivore-habitat relationships is also done based on odds ratios (increase in 1 unit of a habitat variable results in X% increase or decrease in occurrence). Odds rations are exp(beta). Table 3 should therefore present odds ratios, not betas. Also, sometimes the authors talk about standard deviation as the unit increase or decrease, sometimes about actual values (proportions of cover types) as the unit. What are they?  I am not 100% convinced that the explanations are correct, and that the authors interpret the coefficient estimates as odds ratios. For example, at line 293 the odds are increasing by a factor of 0.82 (82%). If this was an odds ratio, then the correct explanation is that the odds are decreasing by 18% for a unit increase in cover. Another source of confusion may relate to the Intercept in those models. Depending on parameterization, the intercept could be one of the levels in a categorical variables, and the rest of the coefficients are differences from that level. As such, interpreting the odds ratio for a given variable based on that coefficient is wrong, as it is relative to the Intercept, not as a standalone value. The outcome can be a huge increase or decrease in odds ratio values bc they represent changes relative to some variable (the intercept). Maybe try to run the models w/o intercept and then obtain and interpret the odds ratios that way? This should remove the issue of comparisons relative to the intercept and showcase the real odds ratios associated with each variable.
Validity of the findings: I have no concerns about the validity of the findings. Everything is well interpreted and make sense from an ecological standpoint. The only issues (if indeed they are issues and not just my misunderstanding of the presentation of results) are with the odds ratios described above.
Additional comments: I commend the authors for a great revised draft and solid and much needed contribution to the literature on large carnivores in Romania. My comments above are meant to clarify the presentation of results, not to raise concerns about the rigor or validity of your work.
Hopefully enough science is being published on this topic that will eventually make the wildlife authorities pay attention and incorporate it into management (and move their thinking from the stone age to current times). Fingers crossed...
Thank you for the opportunity to review this work!


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

Input:
Title: LARGE CARNIVORE HABITAT SUITABILITY MODELLING FOR ROMANIA AND ASSOCIATED PREDICTIONS FOR PROTECTED AREAS
Review round: 2
Reviewer: 2

Output:
Basic reporting: This is the revised version of a paper modeling habitat suitability for large carnivores in Romania, and then examining the variation in predicted suitability across different protected area types.

The authors have done a good job incorporating many of the previously requested changes and the paper reads much more clearly and coherently. However, there are still style and clarity improvements that should be made before the paper is ready for publication.  Please see the detailed comments below.
Experimental design: There are a few places where better explanation of methods would improve the manuscript. Please see the detailed comments below.
Validity of the findings: The manuscript still needs careful editing to remove statements of effect or causality that are outside the scope of inference provided by the analysis. See detailed comments below.
Additional comments: Major comments:
1.	The framing of the justification for analysis of differences in suitability across different protected area types is improved, but I think it still needs work.
a.	This paper essentially has two steps. 1) develop three valid predictive models that can be confidently used to say something about the expected occupancy of different regions of the landscape by the three large carnivores. 2) Use these predictions to say something about the possible variation in habitat suitability across the different protection types.  In this second step it is still not clear what the “something” is that you want to explore with the predictions. I would like to see a much clearer justification for why differences in predicted suitability across protection type might be relevant for conservation or management of large carnivores in Romania in the introduction. I would then like to see a clearer discussion of how managers should interpret the trends in the results and what they could practically do with this information.
b.	The inclusion of the summary of raw footprint data across the different protection types is useful, but it needs to be more carefully justified, introduced, and explained. There is a difference between where individuals of rare species currently are (potentially range restricted due to threats) and where they could potentially be (this is what your predictive model is aiming to estimate. These raw data represent the first and the predictions represent the second. They are not equivalent. But a discussion of what they do represent and the differences between them is worthwhile.

2.	The description of the study aims is muddied by trying to frame the objectives as hypotheses. For hypothesis 1, it is not particularly useful to describe what predictors you expect might be useful, when what you are trying to do is just find a set of relevant predictors that provide meaningful predictions. Hypothesis 2 is more reasonable to frame in this way, but it could also be framed more clearly as a question. This relates to my comment above about needing a better set up for why you are interested in the difference in predictions across protection types.

3.	I would like to see a clearer explanation about the grain of the predictor data and justification for how a predictive model that is developed using response variables that are aggregated to the WMU scale is reasonable for predicting to the 1km scale.

Minor comments:
The abstract needs work to better convey the objective and broad implications of the study and less focus on specific details that are difficult to interpret out of context. Also, the statement on lines 48-50 seems overly broad to take away from this study.

The paragraph about previous research into European carnivores (lines 73-83) currently does not add useful information to the introduction. It could be modified to explain how previous work can inform expected predictions, or why there might be expectations of variation in predicted occupancy across protection types

Line 85: I don’t think that model selection and multi-modal inference minimizes uncertainty

Lines 92-99: this text starts to justify why different protection types may have different predicted occupancy. It should be in a separate paragraph and moved to earlier in the introduction because it is informing half of your study objectives.

Lines 133-135: mention that detectability issues also would preclude an absolute census

Lines 230-234: I am unconvinced by this example why the included quadratic forms are ecologically relevant. Please clarify.

Lines 239-242: please add a citation for this statement

Line 293: This is the first mention of odds. It would be better to describe the logistic model more clearly in the methods and describe there how the odds is calculated and what it represents.

Lines 306-308: This is the first mention of assessing the raw data across the protection types. Add a section to the methods justifying and describing this.

Lines 308-310 and elsewhere: be very careful about not making strong statements such as this. You can talk about mean differences across the protection types, but there is no obvious absolute difference across the protection types because the SD bars overlap for all of them. The text at 331-332 is better.

Lines 403-405 and elsewhere. Also be careful to avoid causal language in describing possible implications of observed results. Here you could say that the patterns you are seeing could be because “human land use could displace lynx” but a low probability of occurrence says nothing about the specifics of why they might not be there.


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

Input:
Title: IDENTIFICATION OF GENE-PHENOTYPE CONNECTIVITY ASSOCIATED WITH FLAVANONE NARINGENIN BY FUNCTIONAL NETWORK ANALYSIS
Review round: 1
Reviewer: 1

Output:
Basic reporting: 1.The English language should be improved and the manuscript needs professional care. For example, in Line 21 “consequences showed that naringenin...” should be “the results showed that...”
2. The legends for the figures should be better described. In figure 4, what’s the difference between the links with and without arrows?
Experimental design: 1.The PPI network construction of naringenin-mediated proteins is not sufficiently described. The STRING web server has provided the PPI network for the input protein list. But in line 77-78 and 109, the PPI network was constructed by Cytoscape. Did the authors used any special plugin or modules of Cytoscape to construct the PPI network? Or just visualize the network? Please make it more clear in methods part.
2.Why did the authors select the hub proteins by node degree larger than 25? It’s a relative high degree, please give more explanation.
Validity of the findings: Why just prostate cancer was chosen as the phenotype associated with naringenin-mediated proteins? Is it just because of hormone related? If so, why other types of cancer that hormone related were not considered, such as breast cancer.
Additional comments: No additional comments


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

Input:
Title: IDENTIFICATION OF GENE-PHENOTYPE CONNECTIVITY ASSOCIATED WITH FLAVANONE NARINGENIN BY FUNCTIONAL NETWORK ANALYSIS
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: Speculation is welcome, but should be identified as such.
Additional comments: This paper used a set of bioinformatics tools to integrate available data on naringenin, and find some prostate cancer related genes. The authors proposed that they find gene-phenotype connectivity. What is the genetype and phenotype here? PPI network and prostate cancer? I don’t think the relationship between PPI and prostate cancer is the most important finding here. So, I suggest using a more specified title. More comments are listed in the following:
1.	The first step is to put naringenin into the 8 targets returned by DrugBank without screening. Among them, the ttgR gene belongs to Pseudomonas putida species and the other seven genes belong to human. I was wondering if is more reasonable to not include ttgR gene in the further analysis, as prostate cancer is the human disease
2.	In the network analysis, degree > 25 was used to select key proteins. This analysis seems too simple, and the authors may use more network parameters to make their finding more convincing.
3.	What the network scale used for pathway analysis? STRING can give networks with different scales. Please clarify it
4.	There are various English problems. It it better to have a proofreading service.


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

Input:
Title: IDENTIFICATION OF GENE-PHENOTYPE CONNECTIVITY ASSOCIATED WITH FLAVANONE NARINGENIN BY FUNCTIONAL NETWORK ANALYSIS
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The authors have addressed all my comments and have accordingly improved the presentation of their manuscript.  But there are still some English problems, please make improvement.


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

Input:
Title: IDENTIFICATION OF GENE-PHENOTYPE CONNECTIVITY ASSOCIATED WITH FLAVANONE NARINGENIN BY FUNCTIONAL NETWORK ANALYSIS
Review round: 2
Reviewer: 2

Output:
Basic reporting: has been improved
Experimental design: has been improved
Validity of the findings: has been improved
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: INTEGRATED BIOINFORMATICS ANALYSIS OF AS, AU, CD, PB AND CU HEAVY METAL RESPONSIVE MARKER GENES THROUGH ARABIDOPSIS THALIANA GEO DATASETS
Review round: 1
Reviewer: 1

Output:
Basic reporting: 1 Line 109-110, the author thinks the key genes and pathways can be used as a biomarker to control heavy metal pollutants. I think that the word “control heavy metal pollutant” is not accurate.
Experimental design: 1 Authors obtained lots of genes to responsive to heavy metal through integrated bioinformatics analysis, such as 59 up-regulated genes and 109 down-regulated genes. But these theoretical results need more some experiments to verify. Therefore I suggest that authors should add some experimental verification to some critical gene expressions to responsible to heavy metals in their future work and try to screen the most sensitive genes as marker genes.

2 Line 118-120, the author has mentioned 5 kinds of heavy metal experiments, but I could not find the experiment details, so I think that the related experiment methods should be described in this paper.
Validity of the findings: no comment
Additional comments: The manuscript by Chao Niu et al. represents an interesting work regarding integrated bioinformatics analysis of As, Au, Cd, Pb and Cu heavy metal responsive marker genes through Arabidopsis thaliana GEO datasets. In this manuscript, the authors have obtained 168 differentially expressed genes (DEGs) which contained 59 up-regulated genes and 109 down-regulated genes through comparative bioinformatics analyses. The results are benefit to understand the potential molecular mechanisms or signaling pathways responsive to heavy metal stress in plants.

In my opinion, the manuscript can be accepted with minor revisions.


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: INTEGRATED BIOINFORMATICS ANALYSIS OF AS, AU, CD, PB AND CU HEAVY METAL RESPONSIVE MARKER GENES THROUGH ARABIDOPSIS THALIANA GEO DATASETS
Review round: 1
Reviewer: 2

Output:
Basic reporting: In the present manuscript submitted to PeerJ, Niu et al. analyze data from eleven microarrays to identify differentially expressed genes (DEGs) in Arabidopsis thaliana after different heavy metals exposures. So, the results and conclusions of this manuscript are of great interest because provides some clues for the detection of heavy metals pollutants in water and soil. This study is interesting, could be useful in the field and will be interesting for readers of PeerJ.
English is not my first language and I do not claim particular competence. However, I think that the paper is clearly written and logically structured. Overall, the references present are appropriate, well discuss and related with the results obtained in this work.
Experimental design: In my opinion, although my laboratory´s work does not focus on the field of microarrays, the experimental approach is adequate to analyze the DEGs that are common after exposure to several heavy metals. Until now, differentially expressed genes had only been analyzed individually after exposure to a single heavy metal.
Validity of the findings: In my opinion, the discussion seems to be well written. However, this section is weak and I think the authors should discuss, apart from the DEGs in common, the DEGs that are not common among all heavy metals. The discussion would benefit from a minor revision.
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: INTEGRATED BIOINFORMATICS ANALYSIS OF AS, AU, CD, PB AND CU HEAVY METAL RESPONSIVE MARKER GENES THROUGH ARABIDOPSIS THALIANA GEO DATASETS
Review round: 1
Reviewer: 3

Output:
Basic reporting: Summary:
Authors in this study analyzed several microarray expression datasets towards developing a biomarker for detecting contaminations of a group of chemicals in Arabidopsis thaliana plant. The study applies differential gene expression analysis using mainly traditional approaches and summarizes finding of several genes. Regarding the approach, the work is based on a very typical bioinformatics analysis and no novelty is realized. The work lacks any systematic evaluation of the accuracy and stability. Also, there is no comparison with any existing method or previously developed notable signatures like S1500 and L1000.

Positives:
1- Applying bioinformatics for biomarker detection of environmental chemicals
2- Extending DEGs analysis with PPI networks and exploring more significant genes.

Negatives:
1- Approach is oversimplified for biomarker detection and analysis.
2- Lacking systematic evaluation using accuracy and stability measures needed to judge the validity of the results.
3- Lacking comparison with any other existing related signature such as S1500.
Experimental design: - The approach for finding differentially expressed genes is based on limma which is one of the standard statistical approaches used for ranking genes. While this is one of the well-known solutions, biomarker development generally goes beyond in an effort to tackle limiting assumption like linearity and independence of genes. Related and notable works of signature development are S1500 (toxic exposures related) and L1000 (drug treatment related). Both signatures and other works, propose more steps such as clustering gene space, dimensionality reduction, and expression space coverage. Authors are highly advised to:
--Study these developed solutions closely
--Compare with the signatures at the conceptual or performance level

- Another point regarding the approach, is the limited elaboration on common genes between the different subsets. DEGs were generated for each dataset but how much these groups share. This is very useful to understand stability and relevance (or importance) of the selected group of genes. Related to this point, is providing details on methods like RRA and possible variants of selecting a final group of genes.
Validity of the findings: - A strong limitation of this study is lacking any systematic evaluation of the accuracy and stability of results. Authors resorted mainly to enrichment analysis and related literature to highlight the significance of selected genes. However, established accuracy measures like the sensitivity of selected genes in defining toxic samples should be used. If samples are not labeled (i.e. phenotype is not known) to perform such evaluation, then, showing gene expression coverage using the selected genes will be useful (see L1000 evaluation). Other types of evaluations can be found in the literature as well. As an example, see the following study:
Wang, Pingping, et al. "A reduced transcriptome approach to assess environmental toxicants using
zebrafish embryo test." Environmental science & technology 52.2 (2018): 821-830.
For stability evaluation, were the 9 cores genes found using network construction step among the top 168 ranked earlier? PPI network was introduced as another way to choose genes and while this is true, it was not clear how results were related to the previous analysis.

- It was not clear from the result section, how specific the developed set of genes are for the Arabidopsis thaliana plant.
Additional comments: Minor points:
- For figure 3, what about a heatmap for all experiments together. For figure 4, it should be annotated with control vs. treatment (or exposure) and then, maybe the name of experiments.

- "the cluster heatmap of the top 200 DEGs identified was displayed by" --> How the top 200 were defined? Was p-value score average from different experiments or what?

- Not sure about a minor issue as in the compiled PDF file of the manuscript, two abstracts exist where the first one has some typos."detecting their expression in plants growing in those environments. anisms or signaling
... through detecting their expression in plants growing in those environments."!!

- "afterwards, it enters the animal" --> "Afterwards,"
"Then these datasets" --> "Then"," these datasets"
Review and fix other related typos.

- "However, these solutions are not convenient nor efficient" --> Elaborate briefly on this.

- In "Retrieval of gene expression profile data" sub-section please provide brief details
about the platforms processed and using which approach. As in the supplementary table 1,
RMA and MAS5.0 were applied to most of the datasets. How did you address combining
different types of normalizations??

- "in 20 raw were.." --> What "raw" refers to here?

As a side note, it was interesting to see that MAPK signaling pathway is among the top enriched ones. This complies with previous findings.


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: INTEGRATED BIOINFORMATICS ANALYSIS OF AS, AU, CD, PB AND CU HEAVY METAL RESPONSIVE MARKER GENES THROUGH ARABIDOPSIS THALIANA GEO DATASETS
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: All the questions have been solved.


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: INTEGRATED BIOINFORMATICS ANALYSIS OF AS, AU, CD, PB AND CU HEAVY METAL RESPONSIVE MARKER GENES THROUGH ARABIDOPSIS THALIANA GEO DATASETS
Review round: 2
Reviewer: 2

Output:
Basic reporting: Authors in this review have addressed most of the major comments.
Experimental design: It is appreciated that they have performed an experimental validation to support the previous findings. Experimental validation using qPCR is certainly a valuable inclusion and it supports the conclusion.
Validity of the findings: The findings were supported by several statistical analysis tools and experimental validation.
Additional comments: - For "mentioned above (Cd, Pb and Cu) or no mentioned above", change "mentioned above" and "no mentioned above" in all text to "training compounds" and "testing compounds" or "study compounds" and "external testing compounds" or other more clear expression. In addition, in Abstract and Experimental validation sections, list the chemicals  "mentioned above (Cd, Pb and Cu) or no mentioned above (Zn and Cr)"

- In Introduction "..Library of Integrated Network-based Cellular Signatures (LINCS ) L1000 data", add citation to L1000:
Subramanian, Aravind, et al. "A next generation connectivity map: L1000 platform and the first 1,000,000 profiles." Cell 171.6 (2017): 1437-1452.
Please make sure to cite L1000 in text.

- Fix statement:
"The real time quantitative PCR (RT-qPCR) experiments on RNA collected "-->
"... RNA 'were' collected "

- Experimental validation: add "quantitative PCR (RT-qPCR) validation" to abstract.


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: ESTIMATING PROBABILISTIC CONTEXT-FREE GRAMMARS FOR PROTEINS USING CONTACT MAP CONSTRAINTS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Major points:
1. It’s not immediately clear to me who the audience of this paper is. Though I’m interested and fairly well read in protein evolution, sequence-structure constraints, etc. there are a number of terms and concepts presented in this manuscript that are new to me and writing assumes that readers have a pretty far ranging prior background. While this of course may (and surely does to some extent) reflect limitations of this humble reviewer’s knowledge, the authors are nevertheless missing an opportunity to more clearly explain the value of this research more broadly to people who may be interested in these concepts (i.e. me). “Grammar” in general will of course be familiar to most but care should be taken to explain the usefulness of this concept to protein sequences/evolution. “Context-free grammar” on top of that is a term that many/most will likely have not heard of or will be only vaguely aware of. Other concepts such as “syntactic trees” and “parse trees” are just kind of thrown out there and I’m doubtful that most readers will immediately know what the authors are talking about. A toy example presented in a figure or two would really work wonders to teach the importance of these concepts and make the authors advances more clear and more widely applicable.

2. Of all the writing, the abstract in particular is dense and difficult to parse. This is going to be the “sales pitch” for most potential readers and I strongly encourage the authors to clean this up and simplify it with clear problem statements and proposed solutions.

3. There were a few typos/grammar issues throughout but it wasn’t anything too bad. That said, the abstract (in addition to being dense) had much worse English than the remainder of the manuscript and really starts out on the wrong note and should be thoroughly edited in addition to being simplified. A few examples throughout that I came across:

a.	An overall poor start in terms of English/grammar is that the first sentence of the abstract is a bit unwieldy and I believe grammatically incorrect. “Learning language of protein…” == “Learning the language of protein…”.
b.	Still in that first sentence of the abstract… having 2 “which” clauses in a sentence is clunky. This could be changed by giving a full sentence to defining context free grammars as I believe few readers will know/understand this term. It’s all to say that a sentence like this could slide by more freely in the middle of a manuscript but this reviewer is of the opinion that the abstract is the portion of the manuscript that should have the most careful editing / precision of language to hook potential readers
c.	Also in the abstract “Within the framework…” == “Within this framework…”. “The” is a bit ambiguous as if there were one and only one whereas “this” specifies that we are discussing the authors contribution.
d.	I’d avoid any mention of heavy concepts like parse trees in the abstract entirely if possible because these will need to be defined.
e.	Page 2 lines 57/58 “which do not outperform significantly HMMs” == “which do not significantly outperform HMMs”
f.	Page 12 line 346: “amino acid sequence of” == “amino acid sequences of”
g.	Page 17 line 467: “Complex character…” == “The complex character…”
h.	Page 17 line 497: “…may even more benefit…” == “…may benefit even more…”

4. Code should be made publicly available

Minor comments:
1. Figure 2: actual structures here would be nice in addition to the simplified diagrams

2. The mathematical abbreviations in Tables 2 and 3 headings could be simplified to increase readability. I had to keep flipping back to where these terms were defined to understand the differences in the columns. A more straightforward textual description might be easier to grasp for those who won’t be diving fully into the methods

3. Page 1 Line 28: “a near infinite number of sequences”. Perhaps it’s a mis-understanding on my behalf but I don’t see that an infinite number of sequences can exist, unless I suppose they are of infinite length but for a finite alphabet and bounded size it’s an of course astronomical and near infinite number but not infinite.

4. The introduction is very well written and seems to have been edited far more closely than the abstract. Though an orienting “summary” paragraph at the end of the introduction might be helpful to add to again focus the readers on the structure of the paper, the problem, the solution, etc.

5. The methods are extraordinarily dense (Pages 4-11). Which may suit some readers fine and I don’t think should necessarily be cut down. But I note it here only that this reviewer was unable to properly evaluate them and more generally would encourage the authors to make sure that their results are written predicated on the fact that few readers will have read / fully grasped their methods.

6. Conclusions/Discussion could probably be combined into a single section. Or more typically if both occur the conclusion is typically a shorter summary paragraph rather than the longer of the two. I thought that the conclusion section was well written, fairly addressed the limitations, and hypothesized paths forward for this research and this should probably be put in the discussion.
Experimental design: Major points:
1. I really struggled after two read-throughs to pin-point the problem that the authors were addressing. Learning the language of protein sequences seems to be the goal, but this is of course a very abstract goal and hard for the reader to grasp in practical terms. While working towards this goal, the authors do present results that are concrete and I was kind of surprised when they came given that this information is not clearly laid out in the abstract. A clear practical goal/application seems to be to use contact information to supplement sequence information in order to better discriminate protein family members but nowhere is this made entirely clear. Being able to better define protein families using these methods could for instance have a large practical benefit of increasing the accuracy of protein homology search (a very valuable application). Page 2 lines 37-53 are the clearest articulation of this vision in the context of broader research but this definition of the problem appears nowhere in the abstract and really seems limited in the overall arc of the paper. If I’m correct that this seems to be the main practical benefit/goal of such research, this really needs to be made more explicit and scattered throughout the abstract/intro/results/discussion. If this is one of the main practical goals, well then it would seem apt to compare the methods developed here to the more limited profile-HMMs in the results (or are profile-HMMs fully equivalent to the grammars estimated without contact constraints? Entirely possible that this is the case, but if so, I think that’s another point of confusing terminology as I believe the term profile-HMM will be far more widespread to the likely readers of this paper than context-free/-sensitive grammars. The solution would simply to be to reiterate the fact that those methods are equivalent, but I’m still not sure that they are).

2. I still don’t fully get the “Descriptive power” section. Which again just relates to some lack of clarity in the writing or assumption of prior knowledge that I simply do not have. What precisely is even being classified/described here with the precision scores? True positive contacts from the structure? It’s not clear how their method converts probabilities into binary predicted contacts. Which is to say, it might be described there but again in a very abstract way that I have difficulty extracting using their terminology. It seems that this information comes directly from parse trees but if the link is kind of clear and obvious well then perhaps a diagram or figure again could show this link to someone who is unfamiliar with some of these concepts (i.e. has never heard of a parse tree, as I suspect some/many readers may be in a similar situation to myself). Additionally the value of that “descriptive power” problem seems a bit weak. Which is to say training a model with contact information helps better predict contacts? I’m not sure if I’m getting all of that properly, but it seems a bit tautological and not entirely surprising. The authors should clarify/expand why I’m being unfair in that regard.

Minor comments:
1.Table 1: It’s unclear to me why the number of nneg varies for the different protein fragment targets. Is this a consequence of the different lengths? 829 negative sequences were used, and it is said that these are cut into matching lengths for the positive set (are these cuts overlapping? i.e. is a 100amino acid sequence with target length 20 cut into 5 sequences or 80?) so this would make sense but could be made a bit more methodologically explicit.
Validity of the findings: No comment.
Additional comments: Dyrka et al. approach an important and topical issue, the relationship between structural constraints and protein sequences, using the concepts of context-free grammars. Notably, they show how to incorporate protein contact constraints into context-free grammars to develop methods that can better discriminate protein family members and re-capitulate the structural properties of the protein families in question. Overall, I find that the research is solid and can think of few/no actual analytical objections to any of the research presented that would require any re-analysis or further experiments. However, the biggest qualms I have with the paper are that it presents a very high bar of assumed prior knowledge and a lack of clear goals that severely limits comprehension of their results and advances.


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: ESTIMATING PROBABILISTIC CONTEXT-FREE GRAMMARS FOR PROTEINS USING CONTACT MAP CONSTRAINTS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The authors present the description of sequences of protein fragments using probabilistic context-free grammars (PCFG) using contact map constraints. This approach is successful in statistical models of RNA since the contacts in RNA secondary structure naturally fit the framework of PCFG (nested but no crossing contacts). These methods have not been applied much to proteins, which are more frequently described via profile HMM. The latter cannot include non-local interactions, and thus structural constraints for contacts bringing together residues distant in the primary structure. The authors propose PCFG to overcome these limitations.

While the generalization beyond HMM is an important question, I have a number of basic remarks concerning the manuscript. In general, it is written quite clearly, but also in a very formal language accessible mostly to computer scientists. It seems more written for the proceedings of a computer science conference than for a life-science journal. An effort to give motivations behind formal constructions should be made.
Experimental design: se below under "Validity of findings"
Validity of the findings: 1) The authors propose PCFG as structurally informed probabilistic models for protein sequences. I do not understand the motivations for this choice, since contact maps of proteins have many characteristics, which do not seem compatible with the construction rule of CFG: Crossing contacts like in alpha helices (e.g. (i,i+4) and (i+1,i+5)) or parallel beta sheets (e.g. (i,j), (i+1,j+1),…, (i+l),(j+l)), residues with multiple contacts two distinct regions of the protein instead of one-to-one contacts. So the target might be small fragments like the ones shown in Fig. 2 and analyzed in the paper (by the way, Fig. 2 is basically unreadable, a little graphical effort might be helpful there). But wouldn’t the effort of developing PCFG be large for fragments of very specific structure? I miss an overall motivation and justification of the model setup, and a serious discussion of the limitations of application. More general graphical models or Markov random fields (MRF) seem more adapted to the complex structure of protein contact maps.

2) In the definition of te PCFG used, it remains unclear why V_T is not directly identified with \Sigma? Probably the use of three symbols for V_T ans four for V_N makes the model more flexible (more parameters) than PCFG used for RNA, but why values 3 and 4 for these cardinalities? The justification seems to be the computational complexity of the problem, but is there any argument that less symbols would be worse in performance? Also profile HMM do not have this kind of multiplicity, since each match state has a single amino-acid emission matrix. The precise model settings remain thus somehow unmotivated.

3) Even if the number of elements of V_T, V_N remains restricted, the PCFG framework currently does not seem to be applicable to sequences longer than 20-30 amino acids, and alignments have to be gapless. This seems quite a restriction. MRF are currently used for hundreds of amino acids in multiple-sequence alignments with gaps.

4) In the tests on protein fragments, very few positive sequences (24-160) are used. Why so few? The fact that performance decreases with npos is counter-intuitive: One would expect that model parameters are more precisely estimated when samples are larger. Larger samples would also, in principle, make average-precision results less noisy.

5) The entire protein space is approximated by 829 single sequences of length 300-500, constructed from old data in 2006. Uniprot contains more than 100 million sequences, Pfam lists more than 16,000 protein families, the majority of them containing structurally resolved examples in the PDB. So I would guess that the negative set is an extremely rough approximation of protein sequence space, but the resulting sample is already three orders of magnitude larger than the corresponding npos values.
Additional comments: I am afraid that I miss some basic point, but even after reading the manuscript several times I continue to miss it. I would therefore suggest the authors to substantially revise their manuscript to make their work more accessible and the motivations and limitations – in particular for biological applications – more clear.


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: ESTIMATING PROBABILISTIC CONTEXT-FREE GRAMMARS FOR PROTEINS USING CONTACT MAP CONSTRAINTS
Review round: 2
Reviewer: 1

Output:
Basic reporting: In my initial review I raised a number of concerns about the writing style and presentation of the results, finding it to initially be a very dense paper for a biological audience. In this revision, the authors have completely overhauled the manuscript. In the current version, the writing is clear, the problem is introduced well, the conclusions are clear, software is made available (with a working link), and the figures (particularly the addition of a toy example) are much improved. I see no other major concerns to note at this point
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: Responses to my initial comments/concerns were both very thorough and courteous. I believe that the revision to be a much improved manuscript, and thank the authors for taking the time and care to address those initial concerns so clearly and comprehensively.


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: ESTIMATING PROBABILISTIC CONTEXT-FREE GRAMMARS FOR PROTEINS USING CONTACT MAP CONSTRAINTS
Review round: 2
Reviewer: 2

Output:
Basic reporting: In their revised version, the authors have made a major effort to improve the presentation of their paper. However, as pointed already out by both reviewers in the first round, the paper remains quite hard to access:

- It contains valid scientific work, even if the application to small protein fragments appears limiting as compared to the formal development beyond the PCFG-CM approach.
- The presentation is very formal. Much of the notations is far beyond what is needed to follow the manuscript, but they make the article inaccessible to a large fraction of the potentially interested readership (e.g. researchers interested in protein sequence modeling and annotation).
- The discussion of the sample applications in terms of, e.g., discriminative power and average precision remains quite superficial, concentrating on the comparision of a few numbers characterizing global performance.
- Some of my questions have been answered in the rebuttal letter, but these answers have not necessarily found a clear way into the manuscript (e.g. the selection of data sets, scanning e.g. SwissProt or PDB with the Prosite motifs gives much more positive hits; the selection of some model details like the cardinalities of VN and VT etc). The motivation and generality behind these choices remains thus unclear in the paper.

So the impression is that the almost the entire interest of the authors went into the formal development of the PCFG-CM framework, and little into the biological problem. From my point of view (but I might be the wrong reviewer for this kind of manuscript, not having a background in computer science), this is a pitty. Early papers suggesting very similar mathematical structures - PCFG with structural constraints – written by authors like Eddy, Durbin, Haussler and others in the 1990s, are much more accessible to a broader audience. These papers have, without doubt, changed our way to bioinformatically look at RNA.
Experimental design: -
Validity of the findings: -
Additional comments: I sincerely think that a less formal presentation of the material would facilitate the access to this work. However, the style of presentation should be a choice of authors.


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

Input:
Title: GENOME-WIDE CHARACTERIZATION OF ENDOGENOUS RETROVIRUSES IN SNUB-NOSED MONKEYS
Review round: 1
Reviewer: 1

Output:
Basic reporting: 1) The English language should be improved throughout the manuscript, with particular attention to typing errors, plural vs singular nouns (especially when using possessive forms), and in the use of past tense. In addition, some sentences are unclear and should be rephrased. Some examples are given below:
“The vast majority (of) ERVs lack(s) infectious capacity due to the fact that they may accumulate as  (accumulated) nonsense mutations, insertions, and deletions“
“envelop(e)”
“However, ERV in colobinae has remained unclear and lack of research.”
“Moreover, based on the resulting (Figure 5), the evolutionary of ERVs tends to broadly follow the evolutionary history of their host”
“LTR is one of the main groups of retroelements. ERVs, which are members of the LTR elements, represent the most complex of retroelements”

2) At page 8 lines 51-53 the authors report the association of ERVs with various diseases, stating “Some ERVs, however, have retained infectious properties and can be associated with diseases such as diabetes, some neoplasms, chronic diseases of the nervous system and autoimmune diseases (Brodziak et al.2012)”. This sentence, in my opinion, includes various misleading concepts. In fact, i) it seems that an ERV should produce infectious particles to have an effect, while it is known that most of the ERVs exert their effects as individual proteins or, more often, as noncoding RNA and cis-regulating agent, while there are no known example of any infection-competent ERVs (at least in humans); ii) authors claim various associations between ERVs and diseases, but up to date - despite the efforts done – no causal association has been demonstrated for any HERV in any disease. In general, while many studies detected the general expression of a given HERV group in a given diseased condition, very few of them identified specific HERV loci involved and/or a defined model of pathogenesis, preventing any corroborated causal association. Hence, the Reviewer thinks that such current lack of definitive etiological link should also be presented in the introduction, and suggests some recent reviews on the topic: i) HERV Envelope Proteins: Physiological Role and Pathogenic Potential in Cancer and Autoimmunity. Grandi and Tramontano 2018; ii) Human endogenous retroviruses in the aetiology of MS. Christensen 2017; iii) The role of molecular mimicry and other factors in the association of Human Endogenous Retroviruses and autoimmunity. Trela et al. 2016”;

3) At page 8, lines 55-56 Authors state that: “There is growing evidence that ERVs have played an important role in the evolution of many mammalian lineages including nonhuman and humans by providing new functions and evolutionary stimuli” and the same concept is reported again at page 9 lines 83-84 “Growing evidence suggests that ERVs have significantly contributed to primate evolution, diseases and development”. This is surely true but, being growing evidences, I would suggest to cite also more recent references given that the ones indicated date back to 10-15 years ago. Here some suggestions: i) Human Endogenous Retroviruses Are Ancient Acquired Elements Still Shaping Innate Immune Responses. Grandi and Tramontano 2018; ii) Transposable elements are the primary source of novelty in primate gene regulation. Trizzino et al. 2017; iii) Co-option of endogenous viral sequences for host cell function. Feschotte et al. 2017; iv) Regulatory activities of transposable elements: from conflicts to benefits. Chuong et al. 2017. In addition, given that the impact of ERVs on primate evolution is indicated as a main input for the work done, I think that the Authors should discuss more in details the main contribution of ERV sequences to primate physiology and evolution, allowing the readers to have a comprehensive overview of the findings’ significance.

4) At page 8, lines 58-64, authors present the general classification and taxonomy of Retroviridae family. This section should be rephrased avoiding repetitions (e.g.: the presence of 7 genera is mentioned two times in a couple of text lines) and should include all relevant information (e.g.: class II retroviruses includes not only Betaretroviruses, but also lentiviruses, alpha- and deltaretroviruses). I also suggest to cite more recent references for HERV classification and nomenclature, such as i) Classification and characterization of human endogenous retroviruses; mosaic forms are common. Vargiu et al. 2016; and ii) On the classification and evolution of endogenous retrovirus: human endogenous retroviruses may not be ‘human’ after all. Escalera-Zamudio and Greenwood 2016.

5) After a quick check, it seems that the article structure does not always conform to PeerJ standards. I suggest the Authors to check again the journal guidelines and to conform to their indication. Some examples: i) abstract should be organized in the following section indicated in bold: Background, Methods (not considered in the present abstract), and Results. ii) References sometimes lack the DOI number and the journal names are sometimes abbreviated and sometimes not. iii) all figures have no caption describing the results shown.

6) Some supplementary material was provided, but it was never referenced in the main text. Regarding the two excel files, I can suppose that one lists the sequences identified in R.roxellana and the other in R. bieti, but it is not clear which one is which because there are no titles or legends. In addition, the files contain some information about the ERV sequences found (such as length and identified genes) but does not provide their genomic coordinates, making impossible to localize them in the considered species genome without making individual blast searches.
Experimental design: 1) In the Methods section, page 10 lines 110-116, Authors reported that to identify ERV sequences in the golden snub-nosed monkey genome (GCA_000769185.1) and the black and white snub-nosed monkey genome (GCA_001698545.1) they performed a TBLASTN using as queries the Gag protein and Env protein sequences of the gibbon ape leukemia virus (GenBank number: NP_056791.2; NP_056789.1). In my opinion, this constitute a relevant bias for the search outcome, because i) it excludes all ERV sequences devoid of Gag and Env coding portions, which could however have a physiological and/or pathological significance as well. In fact, it has been reported that also highly-defective ERVs and even solitary LTRs can exert biological functions; ii) it excludes all ERV lowly related to the gibbon ape leukemia virus in terms of structure, also considering that no cut-off threshold of identity and/or blast score is reported. In fact, authors only report that they downloaded 100 hits for each genome, that is the default number of results commonly displayed by the program.

2) In the result section, please use the proper nomenclature for viral genes (italics) and proteins (capitalized)

3) At page 13, lines 225 to 241, Authors found that 13 species showed an ERV sequence similar to a pair of proposed orthologs in the analysed monkey species (RR146/RB237). I think that this is quite expected, given the overall high similarity of primate ERVs from the same class, not necessarily indicating that they are orthologous loci. In addition, authors conclude that “results suggest that primates of a shared common ancestor may inherit the same ERV sequence from a common ancestor.” Also this concept is rather old and yet assessed, given that such a mechanism is at the bases of ERV spreading among primates. As an example, the ERV-W group has been exaustively characterized in the human genome (Contribution of type W human endogenous retrovirus to the human genome: characterization of HERV-W proviral insertions and processed pseudogenes. Grandi et al. 2016) and its individual members have been identified and compared one by one in 5 Catarrhini species, revealing that the great majority of them where shared among all these primates, being orthologous insertions (HERV-W group evolutionary history: characterization of the group in non-human primates and identification of highly related sequences in New World Monkeys. Grandi et al. 2018). Hence, I suggest the Authors to improve the conclusions taken from these results beside the general presence of shared loci.

4) At page 14 lines 244-255, Authors refer to “ERV-related genes around or within ERV sequences”. Related to what? The co-localization with ERV is not a relation and has not necessary a functional effect neither on the gene nor on the ERV sequence. Hence, the use of the expression “ERV-related genes” is misleading. Moreover, the fact that some monkey genes have the ERV insertions and the corresponding human genes do not does not provide any information, given that that specific ERV could have been deleted in humans, or have been integrated after the divergence between the two lineages. Hence, which conclusions can be taken from this result?
Validity of the findings: 1) At page 15 lines 257-274, Authors should discuss the results relative to the ERV identification in the two monkey genomes considered. However, there is no mention of the results but only a repetition of the general ERV state of the art, including the accumulation of mutations and the general ERV structure. The latter is also imprecise, reporting the viral genes in the wrong order and omitting some proteins and functions (e.g. protease). I suggest the authors to rephrase the information and move most of it in the introduction, discussing here the ERV sequences identified during the study.

2) At page 16 lines 299-300, authors state that “The genes we found out indicate that the fusion of ERV with genes expressed by the host is ubiquitous”. This sentence contains various misleading concepts and should be removed or rephrased: i) what does it means “the fusion of ERVs with genes expressed by the host”? Are the two sequence fused together? If yes, how did authors established this? Did they checked for chimeric transcripts? ii) what does it means that the fusion is ubiquitous? Are all expressed genes co-localized with ERVs? Can authors provide the proportions of the intergenic and intragenic ERVs found?

3) At page 16 line 311 Authors state that “Some of the ERV-related genes we have identified appear to play a role in disease expression.” Did the Authors performed genome-wide association studies that are not reported in the present paper? Otherwise, how can they support this statement? Of course, the sole co-localization with human genes, even if involved in diseases, does not support in any way an involvement of the ERVs in any disease. Authors should state this clearly when claiming for uncertain associations.

4) Page 17 lines 335-345: as mentioned above (basic reporting section, point n° 2), to date, no definitive causal association has been demonstrated for any HERV in any disease, and neither specific HERV insertions nor a demonstrated mechanism of pathogenesis has been confirmed. Hence, the sentence “human endogenous retroviruses(HERV) activity has been strongly associated with ovarian, colon, gastric and pancreatic cancers and HERVs have also implicated in autoimmune diseases including rheumatoid arthritis(RA), multiple sclerosis(MS) and systemic lupus erythematosus(SLE)” is misleading and should be rephrased and completed referencing the relevant literature in the field.
Additional comments: The manuscript by Wang et al. analyze the presence of ERV sequences in two species of colobine primates, namely Rhinopithecus roxellana and Rhinopithecus bieti, through a Blast search and a subsequent structural and phylogenetic characterization. Overall, the manuscript has some potential, investigating two species for which ERVs have not been characterized yet. However, the methodological design is not sufficiently detailed and present some major biases, and both the results contents and discussion need extensive revision prior to publication. In addition, the presentation of the state of the art is often misleading, and the english language needs to be revised by a native speaker.


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: GENOME-WIDE CHARACTERIZATION OF ENDOGENOUS RETROVIRUSES IN SNUB-NOSED MONKEYS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The authors performed comparative bioinformatic analysis of the endogenous retroviruses in golden snub-nosed monkey and the black and white snub-nosed monkey. The writing is clear except a few places, the background and discussion contains enough information, and the manuscript has standard sections. They also provided all the data and results within the main text and supplementary. The results also provide new insight into the evolutionary of endogenous retroviruses within primates.
Experimental design: The whole method is standard, and also provide sufficient information to repliacate.
Validity of the findings: The data and findings are robust, and they also provided the sequences of the endogenous retroviruses as supplementary files.
Additional comments: The authors performed comparative bioinformatic analysis of the endogenous retroviruses (ERV) in golden snub-nosed monkey and the black and white snub-nosed monkey. They found more than 200 ERV sequences in each monkey, and further identified the ERV related genes, which may play an important in the expression and also may be associated with diseases. I enjoyed reading the entire manuscript, and the science of this manuscript is sound for me, thus I think this work can be published with a few minor changes which are merely my personal perspectives.

1. The language should be improved. For example, there are many tense errors, e.g. Line 108, "discuss" should be discussed; Line 118, "download" should be downloaded. The whole writing is clear, but the authors should pay great effect to correct such errors.

2. The last section of the results. They found 48 and 63 ERV related genes in each monkey, and also discussed some of the genes in the Discussion. However, I could not see the whole list of these genes, please provide them as supplementary files.

3. Lines 311-333 in the Discussion. They described some important ERV related genes. This is not a problem, but I would like to see this part more than just simipliy telling us how important of those genes.


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: GENOME-WIDE CHARACTERIZATION OF ENDOGENOUS RETROVIRUSES IN SNUB-NOSED MONKEYS
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment.
Experimental design: no comment.
Validity of the findings: no comment.
Additional comments: The authors have done a good job revising, and the paper is much clearer with the details and organization. I would fully recommend accepting the manuscript.