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: ECMPRIDE: PREDICTION OF HUMAN EXTRACELLULAR MATRIX PROTEINS BASED ON THE IDEAL DATASET USING HYBRID FEATURES WITH DOMAIN EVIDENCE
Review round: 1
Reviewer: 2

Output:
Basic reporting: In “ECMPride: Prediction of human extracellular matrix proteins based on the ideal dataset using hybrid features with domain evidence” the authors present a method for the in silico prediction of extracellular matrix proteins using a Random Forest classifier trained on features of vetted proteins contained in the Human ECM Atlas.  The approach shows a modest improvement over ECMPred and predicts ~600 human proteins not found in MatrisomeDB or other data sets.  The authors have validated via immunohistochemistry and immunofluorescence that a small number of putative ECM proteins are indeed present within biologically relevant contexts i.e. the extracellular space.  The authors propose that ECMPride will be useful in the prediction of ECM proteins from other genomes and that the generated list of human predictions may be a useful resource to expand our knowledge of ECM function and potential therapeutic targets.

The authors clearly explain their rationale in light of previous work on this dataset and have used acceptable professional English language throughout.  Figures are generally of acceptable quality though text and lines in the figures show some rasterization/non-transparency – are these the final figures?  It is evident that much more work went into writing technical aspects of the methods than into the biological context and interpretation of the results.  Some additional raw data (esp. venn overlaps) should be included as noted below.  Please address the following suggestions / questions.

General Concerns:

1.  Broadening the scope of the introduction a little, would provide the reader with a better overview of the field and strengthen the rationale.  There is tendency to over-rely on just two or three papers which are over-cited.  There has been other work in this field to globally define the set of ECM proteins using curation and network methods for example, Cain et al. (pubmed: 19755719) on elastic fibres, Cromar et al. (pubmed: 22275077) on ECM, and various work out of the Richard-Blum lab in France who host MatrixDB (pubmed:20852260).  In addition, the seminal paper on protein domain combinations was Apic et al. (pubmed: 14649290) whereas Bornberg-bauer et al. (pubmed: 23562500) covers theory of protein domain evolution.

2.  The results/discussion sections are entirely too brief.  Much of the text of the paper as a whole is devoted to methods and is largely impenetrable to non-machine learning experts.  Matrix biologists will require a logical, lay-explanation of the Random Forest approach, the rationale for choosing this method in light of the available data and commentary assisting their interpretation of the results rather than an impenetrable methods section and simple statement of results.

3.  The Discussion section, in its present form, is more of a summary paragraph than a critical consideration of the results and their implications.  As such, I suggest that the Results section could be re-titled Results/Discussion and expanded to address some of the concerns raised below.  Content currently in discussion could be merged with the concluding paragraph.  Otherwise, the discussion section would need to be re-envisioned as a more critical commentary addressing the same concerns.

4.  Due to the relatively large number of supplementary files for a paper of this size, I would recommend consolidating the supplementary text into one file of supplementary methods with subheadings.

Detailed comments:

Main text Line 45:  Rather than ‘disorder’, I suspect you mean ‘dis-regulation’, or ‘disruption’ via mutation, possibly contributing to ‘mis-assembly’.  Disorder is a topic unto itself and quite essential to the normal function of a number of ECM proteins – see e.g. Peysselon et al. (pmid:22009114) on intrinsic disorder in the ECM.

Main text Line 59:  The lack of overlap between the matrisome predictions and experimentally identified ECMs was explained in Naba et al. 2016 as largely due to the fact that tissues in which certain matrix proteins were known to be exclusively expressed (e.g. teeth, bones, etc.) were not represented in the experimental data at the time.  The difficulty in solubilizing and analyzing such tissues is a reasonable limitation the authors faced.    Rather than making a sweeping criticism about the lack of overlap (which was still >60% for the curated set and 50% overall for the predicted set) you should qualify your remarks by stating the facts (i.e. a low overlap ~ 50-60% which likely reflects the poor representation of insoluble matrix tissues in the experimental set used for comparison).  Still, this reinforces the point that there is an opportunity for in silico methods to circumvent these significant experimental limitations!

Main text Line 65 – 74:  The discussion of pre-existing methods is appreciated, especially the more detailed discussion in Article S1.  Could the authors include a comparison of the results of these tools in terms of the sensitivity and specificity or, total numbers of ECM proteins predicted at a particular confidence level?  Without this, it is difficult to appreciate the relative contribution of these advances in methodology or, the assertion that, (Line 73) “their lack of a connection with experimental biological features” is a significant drawback.

Main text Line 297-298:  Pertaining to Figure 3, please refer to your own subheadings when referring to your observations.  For example, in Figure 3a (second venn), your dataset does not cover nearly a quarter of the core matrisome which, are a highly curated set.  This deserves comment.  As a suggestion, it may be more succinct to label your dataset simply as “ECMPride” within these figures and, more correct to refer to ECM proteins you have predicted as “putative ECM proteins” since the predictions are unverified.  Also in Figure 3a (first venn), since the Matrisome comprises both curated and predicted data it would be nice to see how much of ECMPride overlaps these two categories within the Matrisome.  In supplementary material, please provide the raw data for these figures, namely the list of proteins in each section of each venn.

S2 Line 6-8:  It is difficult to tell whether the main reference here is intended to be Ulen et al., Thul et al. or both?
Experimental design: Given the limitations of the data, the authors are to be applauded on their ambition and creativity in seeing relevant dimensions on which to base their classifier.  Having said this, the following concerns need to be addressed:

General Concerns:

1.  It is not entirely clear what classes of genes are represented in the positive ECM set.  The authors state that they used genes derived from the Human ECM Atlas corresponding to 478 proteins defined by Swiss-Prot identifiers.  It would be helpful if the authors provided a characterization of the composition of the data similar to Fig 3 in Nada et al. 2016 but showing the number of genes in the positive data set comprising each matrisome category.

2.  The purpose of using the Atlas was to base the positive set on experimentally supported ECM assignments.  However, there are two notable problems with this.
a) First, the atlas is partly made up of ECM data derived from tumor tissues, reflecting abnormal proteins and ECM compositions.  Did the authors include or exclude proteins derived on the basis of only tumor data?
b) Second, we know that the Atlas does not include a significant number of bona fide ECM proteins that are known to be exclusively expressed in tissues like teeth, bones etc. that are resistant to solubilisation (a requirement for MS analysis).  This means that the positive data set is (unavoidably) biased.  This should be acknowledged.  It also means that any bona fide ECM proteins that are known not to be present in the Atlas need to be either added to the positive data set or, removed from consideration as novel predictions when evaluating ECMPride.

3)  Given the large structural/functional diversity of proteins comprising the ECM, I am surprised that a PSSM would provide much useful information.  Even among collagens there is enormous sequence/structure/function diversity.  Can the authors clearly state how much weight was given to the evolutionary sequence conservation vs. other factors?  It is difficult to see this having a large influence given that other factors like domain composition, presence of a signal peptide or transmembrane domain (all of which can be reliably predicted) are themselves quite diagnostic.  Have the authors considered breaking their ECM positive set into different categories, similar to Nada et al. to see whether predictions based on an ensemble of category-specific classifiers provides increased predictive power?

Detailed comments:

Main text Line 253:  Re: domains being introduced for the first time.  Are the domains not implicitly represented in the PSSM data already and therefore included in previous methods?  Arguably, they represent the least noisy and most diagnostic information in your sequence based data.  Could this be the reason that their inclusion adds little more improvement over the previous PSSM based methods?  i.e. Is it consistent with your results to suggest that all you have effectively done is boost the signal to noise ratio by spiking in a repeat of the most diagnostic, most conserved sequences?

Table S6:  Among ECMPride predictions that are not found in MatrisomeDB, there appears to be a very high enrichment for membrane proteins.  Perhaps this is due to the exclusion of membrane-associated genes from the negative training set.  Would it not be better to include membrane proteins in the negative set and screen them against Matrisome for identified ECM genes as you did for intracellular genes?  Please discuss your choice here.
Validity of the findings: The authors have applied a rigorous methodology and confronted important and challenging issues such as the imbalance in the size of the positive and negative training sets as well as construction and optimization of features used for prediction.  As with any predictive method, the quality of the predictions is necessarily limited by imperfections in the training data and, our evaluation of them, by the present state of annotations.  These limitations are not explicitly acknowledged by the authors, leading to some over-reach in their claims.  Nevertheless, the work herein remains a meritorious advancement on previous methods.  Please address the following comments / concerns:

General Concerns:

1.  The experimental immunohistochemistry and immunofluorescence results provide validation that a very select number of ECMPride predicted proteins are expressed as extracellular proteins.  They do not confirm that they are ECM proteins and here, the authors are appropriately careful not to over-reach in drawing their conclusions.  As a suggestion, the presentation of any experimental interaction data for these proteins might help bolster support for the functional associations that are suggested here, particularly if they establish a physical interaction with known ECM proteins.  For this, I would suggest looking at MatrixDB as a starting point.

2.  Due to the difficulty in assessing the potential validity of the predictions at a glance, I recommend providing additional links in Table S6 to aggregated functional information.  One solution would be to provide a hyperlink to the corresponding entrez gene summary for each gene.

3.  The suggestion that this method could be useful if applied to other species should include the qualification that its performance would depend on the degree to which similar features are conserved in that group.

Detailed comments:

Main text Line 81-82:  The conclusion that “ECMPride achieved relatively good performance in ECM protein prediction” is a generalization that unfortunately, is weakened by the lack of comparisons with previous tools.  At most the authors can claim a modest improvement in performance over ECMPred and refer to the specific measures of sensitivity and specificity that they achieved.  On the other hand, the authors may wish to emphasize other aspects of their software tool such as availability, ease of use, the robustness of the underlying training data (albeit with some biases)– as improvements over previously available tools.

Main text Line 257-258:  Figure S1 contains interesting inflection points.  Can the authors provide any insight into the nature of the feature sets (1-25) vs. (30-100) vs. (101 – 146) representing the corresponding inflections?  In addition, other than mention of minimal requirements listed in the user guide, I did not see any performance data associated with running the algorithm.  It might be advisable to give some indication of the performance (i.e. run times) relative to the number of feature sets and to compare this with the performance of ECMPred.  Are there trade-offs here that would become relevant over larger datasets, say, all sequenced genomes?

Main text Line 278-279:  Per “ECMPred is taken as a representative of the previous tools”.  Is it the case that ECMPred was the best of the preceding tools?  Or, is it simply that it is the only one available and you propose it is a suitable stand in?  I do not see how ECMPred can represent any other tool than itself, nor does it necessarily need to.  Simply state why the other tools are unsuitable (or unavailable) and scope your conclusions to the comparison you were able to perform.

Main text Line 299-300:  There is no data presented to support the observation that 263 ECMPride predicted proteins contain at least one ECM specific domain.  Nevertheless, does this really represent an improvement in prediction due to ECMPride? Or, could it perhaps reflect a subsequent change in the underlying domain annotations since the last Matrisome update?
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: ECMPRIDE: PREDICTION OF HUMAN EXTRACELLULAR MATRIX PROTEINS BASED ON THE IDEAL DATASET USING HYBRID FEATURES WITH DOMAIN EVIDENCE
Review round: 2
Reviewer: 1

Output:
Basic reporting: These aspects are correct.
Experimental design: Changes included in the revised version have substantially improved the manuscript.
Validity of the findings: Raised concerns are adequately answered and corresponding changes included in the revised version.
Additional comments: Raised concerns are adequately answered and corresponding changes included in the revised version.


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: ECMPRIDE: PREDICTION OF HUMAN EXTRACELLULAR MATRIX PROTEINS BASED ON THE IDEAL DATASET USING HYBRID FEATURES WITH DOMAIN EVIDENCE
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors clearly explain their rationale in light of previous work on this data set and have used acceptable professional English language throughout.

Concerns:

1. Line 330 contains a misspelling of “ECMPrirde”
2. Lines 238-242 is a little vague.  Rather than saying “better” I recommend walking the reader through the specific results shown in the corresponding table.
Experimental design: No additional concerns.
Validity of the findings: The authors have adequately responded to the concerns raised in the initial review.

Additional Concerns:

1.  Line 266 – Perhaps the limitation is with the GO annotation itself ?
2.  Lines 268-270 – You do not discuss the (very) large non-overlap with the additional data sets in Fig 3b,c (Naba and Ahrman) which are striking!  Are they merely false positives?
Additional comments: I congratulate the authors on their thoughtful and resourceful responses to the initial review.  Overall, this is a much more sound and informative paper in its present form.


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: REASSESSING THE OBSERVATIONAL EVIDENCE FOR NITROGEN DEPOSITION IMPACTS IN ACID GRASSLAND: SPATIAL BAYESIAN LINEAR MODELS INDICATE SMALL AND AMBIGUOUS EFFECTS ON SPECIES RICHNESS
Review round: 1
Reviewer: 1

Output:
Basic reporting: This is a well presented and scholarly article describing the results of a re-analysis, using new statistical techniques, of existing datasets describing acid grassland species richness, with a view to detecting and attributing the impacts of nitrogen deposition. I found the English clear and the article well written and easy to follow, especially given the technical statistical nature of content. The article structure was logical, and figures appropriately selected and clearly labelled.
I had only a few minor comments on the presentation of the text:
1.	Reference citations in the text don’t seem to be consistent about the number of authors before ‘et al’ or the way the names are presented (some have both first and family names) this should be checked throughout the document.
2.	Ln 133. The number 320 should refer to plots not sites.
3.	Figures 1, 2 & 3 – It’s instructive to compare these different models to see how they change between datasets and with the inclusion of different parameters – this would be easier if the 3 panels could be presented side by side on a landscape format page.
4.	Figure 4 – what are the units of species richness change?
Experimental design: The study describes the application of a new statistical approach to existing datasets with the aim of comparing the outcome of different approaches to the analysis of these data. The new approach aims to be more rigorous and transparent in the model building process. The rationale behind this re-analysis is clearly explained and a rigorous investigation has been carried out to explore the effects of N deposition using this new approach. The statistical methodology is well described, and appears appropriate, although please note that I am not an expert in Bayesian statistics and so I am unable to comment in detail on this aspect.
One thing I did note is that in the analysis of the SEA04 dataset the authors made the decision to combine four of the climatic variables, which were closely correlated, by using the first two components of a PCA of these data instead. It would be useful to see a fuller description of the correlation between all of the variables used in the models, since e.g. total N and total S deposition are likewise usually closely correlated, was there a cut-off used to decide how correlated variables could be before they were no longer considered independently?
Validity of the findings: The conclusions drawn from the work appear valid and are clearly linked to the results. One thing that struck me was that the spatial random fields had quite different structures between the two datasets. You mention in the discussion that these fields may include effects related to the limited resolution of some of the co-variables (e.g. N deposition data are represented only at the 5km grain size) and other processes such as dispersal. But, given the general similarities of the vegetation composition and explanatory variable datasets, why would the spatial structure in the data be so different? It may be worth expanding on this slightly in the discussion.
I also think it would be worth adding some discussion on how these results compare to those derived from experimental studies (since most other analyses of observational datasets are subject to the criticisms being made by the present manuscript). Do the small effects of N on richness match responses to experimentally applied N? I was particularly curious about this given the large effect of soil %N SEA04 model 2.
Additional comments: In general, I think that this is a well written paper, which makes a useful contribution to the debate around attribution of N impacts on biodiversity. The observations made in the paper are widely applicable across a range of studies and will make a useful and valuable contribution to the literature.


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: REASSESSING THE OBSERVATIONAL EVIDENCE FOR NITROGEN DEPOSITION IMPACTS IN ACID GRASSLAND: SPATIAL BAYESIAN LINEAR MODELS INDICATE SMALL AND AMBIGUOUS EFFECTS ON SPECIES RICHNESS
Review round: 1
Reviewer: 2

Output:
Basic reporting: No comment
Experimental design: My only comment would be about imputed mean values (Line 164 in the Methods section). Did you do any sensitivity analysis of using other imputation approaches - are these plots likely to be close to the mean value? Is there an option for multiple imputation approach (perhaps to look at the sensitivity of the model to these imputed values)? This is a minor comment.
Validity of the findings: It would be good to have your R code so that the analysis can be directly replicated. I assume this will be available to the readership online in some form (OSF, Github, etc)
Additional comments: Wow! It was a pleasure to read this manuscript. Thank you for the opportunity. Excellent, well written with appropriate referencing, style and structure. The logical and detailed approach to reassessing the published data was very impressive. The world needs more papers like this! I do have a few minor comments that I think might improve the manuscript (I would wouldn't I!) Throughout you seem unable to believe your own findings! For example 235:242, you wait until the last line to point out the lack of support for this model that you describe in quite some detail. In line 296:324 you suggest how your approach is "deficient" (potentially). I am supportive of caution, but I think you could be more positive about your findings. I find it hard (I am not knowledgable about ndep) to understand 326 where you say that you do not dispute the negative impacts of nitrogen but you show that there is little evidence of such an effect. I think I understand this but it would help (me at least) to have an understanding of what is a "heavy" nitrogen deposition. Is this something we are likely to see in Agricultural settings in the UK? Or is it only possible in controlled (lab) experiments.  Related to this is that there is a need (again for me) to have a paragraph on the consequences of your finding for policy or Research priorities. If our new Government asks you what to do about the problem of ndep in the UK what will you advise them?


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

Input:
Title: REASSESSING THE OBSERVATIONAL EVIDENCE FOR NITROGEN DEPOSITION IMPACTS IN ACID GRASSLAND: SPATIAL BAYESIAN LINEAR MODELS INDICATE SMALL AND AMBIGUOUS EFFECTS ON SPECIES RICHNESS
Review round: 2
Reviewer: 1

Output:
Basic reporting: I had only a couple of small comments on the revised text:
1. Line 100 – presumably pH, Al and C:N refer to soil parameters? This needs to be specified as it is the first mention of them.
2. Line 106 – after N dep it should be ‘than’ not ‘that’.
3. Line 109 – hyphenate pre-treatment.
Experimental design: No comment
Validity of the findings: No comment
Additional comments: The authors have dealt satisfactorily with all of my comments on the previous version of their manuscript. Their additions to the discussion particularly have much improved the paper by placing it into better context of both the experimental and observational evidence for N deposition impacts.


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: REASSESSING THE OBSERVATIONAL EVIDENCE FOR NITROGEN DEPOSITION IMPACTS IN ACID GRASSLAND: SPATIAL BAYESIAN LINEAR MODELS INDICATE SMALL AND AMBIGUOUS EFFECTS ON SPECIES RICHNESS
Review round: 2
Reviewer: 2

Output:
Basic reporting: No additional comments
Experimental design: No additional comments
Validity of the findings: No additional comments
Additional comments: Great paper - no additional comments. The authors have done a great job and answered all my queries


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: BI-LEVEL VENTILATION DECREASES PULMONARY SHUNT AND MODULATES NEUROINFLAMMATION IN A CARDIOPULMONARY RESUSCITATION MODEL
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: The research question is well defined in the introduction. However, the authors should develop the choice to compare these two particular strategies among others. The specific literature on bi-level ventilation during CPR should help to put this research question in context, especially in light of the conclusions from the study of Kill et al. (Crit Care Med. 2014 Feb;42(2):480-1.).
The investigation is rigorous and performed with high technical and ethical standard. The authors should comment on their choice to apply no PEEP during IPPV. The difference observed in this study could indeed be related to differences in end expiratory pressure level and/or in ventilation mode.
The choice of 15-17cmH2O during Bilevel should also be justified better.
Validity of the findings: The values of IL-6 and TNFa expression within the lungs, as well as histology figures should be shown, at least in supplemental material.
The measurement of IL-6 and TNFa in cortex seems to show opposite results as compared to hippocampus, even if not statistically significant. This trend could be related to a decrease in brain perfusion due to bi-level. This should be discussed in light of studies from Dr Lurie’s Lab showing that lower intrathoracic pressure during CPR is associated with better brain perfusion (Moore et al. Resuscitation. 2017 Dec;121:195-200).
Additional comments: This is a well conducted study on a very important topic. However, several end-points are not convincing, especially regarding the neuroinflammation (discrepeancies between hippocampus and cortex). The title should be reworded in order to be less categorical about the effects of bi-level ventilation on inflammation


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: BI-LEVEL VENTILATION DECREASES PULMONARY SHUNT AND MODULATES NEUROINFLAMMATION IN A CARDIOPULMONARY RESUSCITATION MODEL
Review round: 1
Reviewer: 2

Output:
Basic reporting: This is a well written and structured experimental study investigating an important part of resuscitation.
Literature is sufficient and inculdes recent studies.
Experimental design: Adaquate experimental design and relevant well defined question
Validity of the findings: Validity and interpretation of findings correct and conclusive.
Additional comments: There are some relevant points that should be clarified:

1. Methods and results:" Statistical analyses were performed using 2-way ANOVA inter-group tests with post-hoc Bonferroni correction for repeated measurements as well as Mann-Whitney U test for
single measurements with Student-Newman-Keuls post-hoc analysis via GraphPad  Prism 8 software (GraphPad Software Inc., La Jolla, CA, USA). Data in the text are presented as mean (standard deviation)", but it remains unclear in the Figuers, wich test was applied as well as how the p values resulted. Please provide p values for all measuerments in Figures and tables and declare the tests (ANOVA or U-test) and explain the bar ends (SD or percentiles?).
2. Discussion: As IPPV was performed without PEEP and BILVEL with PEEP of 5, the results are not surprising me. You should discuss this limitation.
3. You cite Ref 23 as follows: "Additionally, this mode has been shown to produce excessive inspiratory pressures, when applied incorrectly during CPR(23). Although we could not observe these pressure peaks in our study and have no indication of resulting pulmonary damage, further evaluation would be necessary." What do you mean with "incorrectly applied"? How did you measure airway pressure in your study (which sampling rate)?
4. As you cite several studies dealing with CCSV you also should discuss this ventilation mode in relation to your results

If you would revise your manuscript conidering these comments I would appreciate to be allowed to review your work again.


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: BI-LEVEL VENTILATION DECREASES PULMONARY SHUNT AND MODULATES NEUROINFLAMMATION IN A CARDIOPULMONARY RESUSCITATION MODEL
Review round: 2
Reviewer: 1

Output:
Basic reporting: No additionnal comments
Experimental design: No additionnal comments
Validity of the findings: No additionnal comments
Additional comments: The authors addressed all my previous comments. I have no further questions.


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: BI-LEVEL VENTILATION DECREASES PULMONARY SHUNT AND MODULATES NEUROINFLAMMATION IN A CARDIOPULMONARY RESUSCITATION MODEL
Review round: 2
Reviewer: 2

Output:
Basic reporting: As metioned in my initial review, I strongly recommend to show all p-values eiteh in the graphs or in the table.
Experimental design: OK
Validity of the findings: OK
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: ABERRANT EXPRESSION OF TWO MIRNAS PROMOTES PROLIFERATION, HEPATITIS B VIRUS AMPLIFICATION, MIGRATION AND INVASION OF HEPATOCELLULAR CARCINOMA CELLS: EVIDENCE FROM BIOINFORMATIC ANALYSIS AND EXPERIMENTAL VALIDATION
Review round: 1
Reviewer: 1

Output:
Basic reporting: 1. Another round of language editing and revision might be required.
2. Line 76. The listed conditions are risk factors and not causes of HCC. Also there are no references to support the statement.
3. The authors calculated the adjusted p-values to account for multiple testing but they chose p-value < 0.05 along with fold-change as criteria for significance! A similar cutoff was used to select significant functional categories with no attempt at controlling for multiple testing.
4. Only the platform/server for performing the differential expression and enrichment analysis are mentioned in the text, GEO2R and Enricher. The exact method for performing the analysis on these platforms should also be mentioned. In addition, the method for standardizing probe intensities (Line 202) and co-expression analysis (Line 290) are not mentioned at all.
5. Figure S1. It is not clear what the bars show or what the colors mean. The figure legend and axes labels are also missing.
Experimental design: 1. The choices to use top n features either in the differential expression, to construct PPI networks,  to filter hub genes or to perform enrichment analysis are arbitrary. The authors may present the reasoning behind this filtering or show that these decisions improves the results.
2. Being a known target of differentially expressed microRNA doesn’t necessarily mean the gene itself is regulated, up or down. The reasoning for constructing regulated lists (Line 128) or the PPI network (Line 135) is not sound, or at least misapplied. These lists would be valuable to explore where the known target come from, but the wouldn’t directly indicate whether the categories (gene sets) that they come from are functional or not in the HCC context.
3. miR-222-3p has almost as many targets in the “hub genes” as miR-221-3p including 4 shared targets, but it was dropped from the analysis completely. Please, justify.
Validity of the findings: The study demonstrated the effect of two microRNAs and some of their target genes on the HCC proliferation and invasion. That much is clear, however, several analysis choices were taken to arrive at the importance of those two features in the first place. The choices were not sufficiently explained or justified.
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: ABERRANT EXPRESSION OF TWO MIRNAS PROMOTES PROLIFERATION, HEPATITIS B VIRUS AMPLIFICATION, MIGRATION AND INVASION OF HEPATOCELLULAR CARCINOMA CELLS: EVIDENCE FROM BIOINFORMATIC ANALYSIS AND EXPERIMENTAL VALIDATION
Review round: 1
Reviewer: 2

Output:
Basic reporting: n/a
Experimental design: n/a
Validity of the findings: n/a
Additional comments: In this study by Liu et. al. have reported that they have identified several up- and down-regulated mi-RNAs in HCC tumors. The authors focused on two miRNAs (miR-221-3p and miR-375) and claimed that they promoted proliferation, HBV amplification, migration, and invasion of HCC.

The involvement of miR-221 in HCC has been studied before and many of the conclusions of this paper were previously reported. For example, Fu Y et. al. (2019) have already shown that miR-221 promoted HCC by targetting PHF2, which in turn is a known regulator of p53 (Lee et.al., 2015). The authors failed to mention this study completely in this manuscript. Here are just a few peer-reviewed studies that have reported the involvement and effects of miR-221 in HCC:

1. https://www.ncbi.nlm.nih.gov/pubmed/31214616

2. https://www.ncbi.nlm.nih.gov/pubmed/31069760

3. https://www.ncbi.nlm.nih.gov/pubmed/30370582

4. https://www.ncbi.nlm.nih.gov/pubmed/31698701

5. https://www.ncbi.nlm.nih.gov/pubmed/31258758

Now the other miRNA that has been investigated in this report (miR-375) is also quite well known as a regulator of HCC proliferation. Upregulating miR-375 has shown to have an inhibiting effect in HCC by Lil L et. al. (2018). Here are a few among many other studies that looked at this relationship:

1. https://www.ncbi.nlm.nih.gov/pubmed/29858089

2. https://www.ncbi.nlm.nih.gov/pubmed/29555460

3. https://www.ncbi.nlm.nih.gov/pubmed/31681610

4. https://www.ncbi.nlm.nih.gov/pubmed/31406139

5. https://www.ncbi.nlm.nih.gov/pubmed/30302825

6. https://www.ncbi.nlm.nih.gov/pubmed/30127930

Could the authors comment on why this study is novel in light of these papers? Somehow authors have neglected to cite many of these papers which very clearly showed the involvement of miR-221 and miR-375 with HCC.

In my view, this manuscript has some novel aspects. However, this study suffers severely by failing to mention the already published literature and try to claim some already known facts as new findings. It is possible to review these (and many others) materials as the starting block and build a stronger paper which may include some of the experiments reported in 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: ABERRANT EXPRESSION OF TWO MIRNAS PROMOTES PROLIFERATION, HEPATITIS B VIRUS AMPLIFICATION, MIGRATION AND INVASION OF HEPATOCELLULAR CARCINOMA CELLS: EVIDENCE FROM BIOINFORMATIC ANALYSIS AND EXPERIMENTAL VALIDATION
Review round: 1
Reviewer: 3

Output:
Basic reporting: In the manuscript titled ‘Aberrant expression of two miRNAs promoted proliferation, HBV amplification, migration and invasion of hepatocellular carcinoma cells: Evidence from bioinformatic analysis and experimental validation’ the authors identify key miRNAs influencing the progression of hepatocellular carcinoma.

Although the manuscript is generally understandable, there are a lot of grammatical errors. I suggest authors proofread the manuscript. It might also be useful to have the manuscript reviewed by a native English speaker or edited through one of the English grammar software.
Example:
Line 90-91: the sentence is not well constructed.
During the last decades,  Microarray technology have the advantages of high throughput, high sensitivity, and automatically.
Experimental design: Computational analysis and experiments are performed with proper controls. However, several important details are lacking in the Materials and Methods section.
Please rename ‘Materials and furthermore methods’ as ‘Materials and Methods’.
In several instances the authors provide ‘manufacturer’s instruction’ as the protocol for the experiment. However, the details of products/kits are not identified (other than the manufacturer’s name). Further details, such as the detailed name of the kit, catalog or a reference number available are necessary. Furthermore, provide a brief summary of the experimental procedure (if the detailed protocol is easily accessible). Also, cite other published work describing the protocol in detail.
Rewrite the entire Materials and Methods section to include appropriate details (both for computational work and the experimental work). This is necessary to make the data generation transparent and reproducible. For instance, not details have been provided regarding the source and nature of the antibody used, incubation conditions, and other details necessary to make this experiment reproducible.

Include the details of statistical analysis in the figure legend.
Please rewrite all the figure legends to make figures understandable and informative. In figure legend include:
1.	The overall point/evidence the figure is providing
2.	Provide the details what each figure panel is representing
3.	Explain the observation
Example:
Figure 4B: The comparison of miR-221-3p expression between HCC tissues and matched paratumor tissues from 20 clinical samples indicate… (explain observation here).

Provide the details of the statistical test used in the figure legend.
Validity of the findings: It is critical that the appropriate details are provided in the Materials and Methods section to make the data acquisition transparent and reproducible.
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: ABERRANT EXPRESSION OF TWO MIRNAS PROMOTES PROLIFERATION, HEPATITIS B VIRUS AMPLIFICATION, MIGRATION AND INVASION OF HEPATOCELLULAR CARCINOMA CELLS: EVIDENCE FROM BIOINFORMATIC ANALYSIS AND EXPERIMENTAL VALIDATION
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The revised addressed some of the issues raised in the review. However, a few issues remain which could be addressed by the authors in the manuscript, if not by revising the analysis itself.

1. Regarding the adjustment for multiple testing, the specific technique used to calculate the adjusted p-values and the cutoffs need to be specified.
2. The authors explained that, on several occasions, they used the top n features to “limit the scope of target research.” This issue here is that the decision is completely arbitrary, this might result in loss of important signal. The authors may address this point in their discussion or employ an alternative approach. A data-driven decision might be a good solution for example by using object functions or natural divisions in the results.
3. The term “regulated lists” is misused as it should refer to features that are expressed differently between conditions, not just the targets of regulated microRNAs. Therefore, it would be beneficial if the authors rephrase this section of the manuscript. to reflect the reasoning they provide in the review.
4. Regarding choosing one microRNA to verify but not the other despite the fact that they have similar numbers of targets is understood. However, this needs to be presented as such in the manuscript, for example, as a limitation.


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: INTANSV: AN R PACKAGE FOR INTEGRATIVE ANALYSIS OF STRUCTURAL VARIATIONS
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: In the manuscipt titled ‘intansv: an R package for integrative analysis of structural variations’, the authors provided an R package of integrating structural variations (SVs) from multiple SV-genotyping programs. The versatile package can be used to process raw results from some SV-genotyping programs, to merge formatted results, to annotate SV’s’ effects, and to visualize the SVs’ distribution. Considering the complexity of SV-genotyping, the package is very useful for biologists or geneticists. The manuscript is generally well written and the supplied data is sufficient. I still have two minor comments which the authors should be concerned.
1.	As a proof, the SV results were obtained based on the NGS data of Zhenshan 97 using the ‘intansv’ package. It is suggested that the authors should provide some description or statistics for the genotyped SVs of Zhenshan 97 in the ‘Results’ part.
2.	 ‘versatile’ should be ‘versatility’ (Line 90).


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: INTANSV: AN R PACKAGE FOR INTEGRATIVE ANALYSIS OF STRUCTURAL VARIATIONS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The article is explained in clear and unambiguous English.

The figures and tables are properly labelled and referenced in the article.
Experimental design: In the "Materials and Methods" section, the method used to filter out the Low-quality predictions can be added.

The process is explained well with a code sample.
Validity of the findings: In the discussion section, the benefits from the visualization of the results can be explained. As it was mentioned as a key improvement in the abstract (please refer to lines 39 and 40).

Please rephrase line number 214 - 216 in the conclusion section explaining the improvements made in this article and the future scope of this package.
Additional comments: This article talks about the addition of a new package to the open-source R software for the Integrative analysis of structural variations. The source code and various methods available in the package are provided. The visualization generated in this package is helpful to understand the results.


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

Input:
Title: INTANSV: AN R PACKAGE FOR INTEGRATIVE ANALYSIS OF STRUCTURAL VARIATIONS
Review round: 1
Reviewer: 3

Output:
Basic reporting: Good introduction, provides good background.
Clear english... Some sentences uses "you". change those.
Discussion could be expanded.
Some figures (for instance figure 2) need to be higher quality. Some parts are blurry.
Experimental design: Interesting package. The difficulty of using multiple programmes to SV detection is very real, if this package simplifies this it is a huge benefit.

Methods could use more a lot more detail. How were SVs deemed overlapping in from different programme outputs?
How many individuals used?
size of genome? GC content?
A bit more detail is needed.
There should also be:
- How SVs from different programes clustered? merged? annotated etc?

It is hard to see what was actually done .....
Validity of the findings: Result section is lacking. Reads more like a methods section with some raw output here and there. Summarise findings etc. Remove raw output. Maybe put in supplemental data...

The use of “you” may not be the best way to present results. Try using for example “All files should be put in the same directory….”

Very unclear what the findings were? final results? run time? etc...

A lot of the results section should actually be in the methods section.
Additional comments: The methods section need expanding overall. very little detail on what was actually done. See comments in methods section and figures. Unclear how SVs from different platforms were merged, clustered, formatted?

Results section could benefit from being re-organised and detailed.

What does the final output look like? how were SVs formatted?

Discussion to be expanded to emphasise the novelty of a package that uses multiple programmes and outputs a single, standardised, final output.


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: INTANSV: AN R PACKAGE FOR INTEGRATIVE ANALYSIS OF STRUCTURAL VARIATIONS
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 of my concerns. In my opinion, the manuscript can be accepted for publication.


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: INTANSV: AN R PACKAGE FOR INTEGRATIVE ANALYSIS OF STRUCTURAL VARIATIONS
Review round: 2
Reviewer: 2

Output:
Basic reporting: The article is explained in clear and unambiguous English.
The figures and tables are properly labelled and referenced in the article.
Experimental design: The method to filter the low-quality predictions are described in detail.
Validity of the findings: The use of visualization is explained in the Conclusion part.
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: STOMATAL CONDUCTANCE BEARS NO CORRELATION WITH TRANSPIRATION RATE IN WHEAT DURING THEIR DIURNAL VARIATION UNDER HIGH AIR HUMIDITY
Review round: 1
Reviewer: 1

Output:
Basic reporting: I have had serious problems when reading the manuscript. I cannot really find a guideline in the presentation of results. After reading the paper, I am not really sure of the real goal of the research.
I encourage the authors to rewrite the manuscript with a deep change in the sequential presentation of arguments and results prior discuss them and propose a conclusi
Experimental design: I am concerned with the idea of LHR, MHR and HRH. In my experience, the stomata response is affected by the HR (or VPD) in the chamber of the gas exchange system. In fact, it is hard to elucidate the effect of environmental (I mean the atmospheric HR or VPD) and chamber HR/VPD in the stomatal response. Can you rescue the VPD values in your measurement? Most of the gas exchange systems allow to modufy or not the HR of the air inlet by using dessicators. However, the a`pporttation of water vapour by the leaf induces a new equilibrium.
Validity of the findings: I encourage the authors to reordinate the results in order to clarify tehe reading. Do authors consider the internal regulations of stomatal sensitivity to HR or is merely a matter of pressure gradient? I am not really sure of the depth of their finding due to difficulties when reading the paper. In some sentences of the paper, really evident statements are done...The results should be written according to the hypothesis that guide the research to highlight the main goals
Additional comments: I encourage the authors to establish a different order when writting the paper. It is need to elucidate between hypothesis and results step by step, as the current presentation does not allow to appreciate clearly the real goal of the research. I propose to check the validity of gas exchange data by analysisng the real conditions during the measurement. Please, also check the relationship between the highly correlated HR, VPD and air temperature and discuss the functional differences (specially among the two firsts) in terms of stomatal sensitivity. A priori, I am not able of elucidate the real difference, but it may be a real and inetresting goal of the research. Moreover, I miss some comments about the specific sensitivity to HR of stomata and influence on the process under study.


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

Input:
Title: STOMATAL CONDUCTANCE BEARS NO CORRELATION WITH TRANSPIRATION RATE IN WHEAT DURING THEIR DIURNAL VARIATION UNDER HIGH AIR HUMIDITY
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The relation between PN, Tr and gs is here dealt in six wheat genotypes. According to the reviewer’s opinion, the current manuscript suffers from the following severe limitations, ought to be dealt by the authors with due caution: (1) environmental conditions during  plant growth are not reported; (2) treatments refer to short term changes in environmental variables (nowhere mentioned); (3) the RH range is too small (15–36%). Deserves to be noted that high RH in other studies is above 85%; (4) VPD cannot be compared with either T or RH, since VPD is calculated based on T and RH; (5) the effect of long-term soil water deficit is nowhere to be seen.

Lines 44, 177: what do you mean by uniform?
Lines 46, 95: correct term is relative air humidity (abbreviated as RH). Please correct it here, and elsewhere in the text.
Lines 47–49 & 95–97: report the values of RH. What do you mean by low, moderate and high RH? Please provide the values.
Lines 54–55: not correct to compare VPD with either T or RH, since VPD includes both T and RH. You may compare T with RH, but not VPD with these two environmental variables.
Line 113: Table 1 reports the genotypes, and not environmental conditions. Besides environmental conditions during measurement (reported in Table 2), it is of outmost interest to report the environmental conditions during the whole growth period. This is missing from the study!
Lines 154–156: Thus treatments are actually environmental conditions during growth. This is not reported in the abstract, results and discussion section! The way the paper is written, it gives the erroneous notion that the RH treatments refer to the growth period. However, what is really meant, and nowhere can be seen, is that short-term changes in environmental conditions. Please clarify this everywhere on the text.
Line 233: high RH in the literature is above 85%. In this study, high RH is only 36.7%. In the remaining literature 36.7% is rather low RH.
Line 235: soil moisture affects plant transpiration through regulation of stomatal opening.
Line 236: “Leaf transpiration rate during cultivation is dominated by RH, with gs exerting minor role” in Fanourakis D, Giday H, Hyldgaard B, Bouranis D, Körner O, Ottosen C-O (2019) Low air humidity during growth promotes stomatal closure ability in roses. European Journal of Horticultural Science 84, 245–252.
Line 243–245: This study deals with another issue, as compared to the one dealt in this study. For sure, mentioning the long-term effects (i.e., throughout growth) of high RH is well-deserved. Note that in this case high RH means higher than 85%. Plants grown under high (≥85%) RH develop stomata that are not responsive to closing stimuli. In the growth environment, transpiration rate is dominated by factors other than gs, which is continuously high. Please check and report the following reviews dealing with this subject: Fanourakis et al 2016 (Journal of Plant Physiology 207, 51–60) & Fanourakis et al. 2015 (Acta Horticulturae 1064, 195–204). These reviews present the role of stomatal opening in determining transpiration in the growth environment, as well as in environments with low (40%) RH.
Line 264–269: Did the authors assess Ci? Why is this relevant here?
Discussion: the effect of soil water deficit is missing!!


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: STOMATAL CONDUCTANCE BEARS NO CORRELATION WITH TRANSPIRATION RATE IN WHEAT DURING THEIR DIURNAL VARIATION UNDER HIGH AIR HUMIDITY
Review round: 1
Reviewer: 3

Output:
Basic reporting: The study investigates the correlation of stomatal conductance with photosynthesis rate and transpiration rate under varying air relative humidity in wheat. It is concluded, that under low and moderate air relative humidity, stomatal conductance and transpiration rate are significantly correlated, but under high relative humidity no correlation occurs and the variation in transpiration rate under high relative humidity is rather explained by varying environmental factors.
The main strength of the study is that it investigates an interesting topic concerning sustainable crop production. The results of this study have a considerable contribution to its field and provide new and interesting knowledge. Sufficient field background is provided and it can be understood, how the work fits into the broader field of knowledge.
Relevant prior literature is referenced, although the referencing and the reference list does not entirely comply with the journals’ style. For example, there are missing publication years on lines 237, 243, 263, 265. The reference list is not uniform, some journal names are in full, some are abbreviated, in some article titles each word is written with a capital letter. One reference (Mott on line 263) is missing from the reference list. The paragraph on lines 94-101 and sentence on line 241 needs a citation.
The English language used in the manuscript is not clear and unambiguous. Throughout the manuscript, there are sentences, that are not easy to understand and the meaning of the sentence remains unclear. The manuscript should undergo a thorough and professional proofreading. A few examples of non-professional English and hard to understand sentences are on lines 59 (“higher effected”), 80-82, 122-127, 133-135, 138-149 (mixed up and incomplete sentences, repetitions).
There are no Figures in the manuscript. I strongly recommend to add graphs showing the diurnal variation of temperature, air relative humidity, VPD, stomatal conductance, photosynthesis rate and transpiration rate. Adding these figures would greatly improve the manuscripts’ quality.
Tables are incorrectly cited. For example, it is referred to Table 1 on line 113, but in fact the data are in Table 2. In my opinion, there should not be any citations to Tables in the Statistical analysis section (lines 168-172), all the more the citations are incorrect (citation to Table 4 refers to Table 3; Table 5 to Table 4).
In Table 3, it is unclear, how many genotypes were analyzed – in the title it is said fifteen but in the legend, it is said six. No units are given in Table 3, 4, 5 and 6. In Tables 3 and 4, an abbreviation “A” is used, but it is not defined (should there be PN instead?).
Experimental design: The submission is original primary research within Aims and Scope of the journal, but research questions are not defined. The knowledge gap is somewhat identified on lines 70-71, 80-82 and 102-105, but the aim of the study and hypotheses are absent. The last two sentences of Introduction (lines 105-107) should be omitted, as they give a conclusion of the findings in the current study and should be placed in the Conclusions.
In general, methods are described sufficiently, if the language usage problems are not taken into account. Some questions still arise. The altitude of the experimental site is important to know, so it should be added (line 113). It is not stated, how many plants were measured for stomatal conductance, in order to identify genotypes with a wide spectrum of gs values (line 127). Due to the language usage problems, it is not clear, how many replications were used in the experiment. On lines 162-165, the models and the producers of the used apparatuses are not given. In the statistical analysis section, it is not stated, whether the normality and homogeneity of the data are checked.
Validity of the findings: No comment.
Additional comments: The sentence on lines 180-182 does not belong to Results but should rather be placed in the Discussion.


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: STOMATAL CONDUCTANCE BEARS NO CORRELATION WITH TRANSPIRATION RATE IN WHEAT DURING THEIR DIURNAL VARIATION UNDER HIGH AIR HUMIDITY
Review round: 2
Reviewer: 1

Output:
Basic reporting: reviewer's comments were adequately dealt
Experimental design: reviewer's comments were adequately dealt
Validity of the findings: reviewer's comments were adequately dealt
Additional comments: The authors dealt with my comments respectfully.
I just realized that one point is still missing in the discussion and ought to be incorporated.
RH was found to be the main driver of environmental differences.
Having said that, it must be highlighted that the RH effect on transpiration rate also stimulates differences in leaf temperature (Giday et al. (2015) Cultivar differences in plant transpiration rate at high relative air humidity are not related to genotypic variation in stomatal responsiveness. Acta Horticulturae 1064, 99–106.) Please integrate this at your discussion to further strengthen the noted RH effects.


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: THE MITOCHONDRIAL PHYLOGENY OF LAND PLANTS SHOWS SUPPORT FOR SETAPHYTA UNDER COMPOSITION-HETEROGENEOUS SUBSTITUTION MODELS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Review of “The mitochondrial phylogeny of land plants shows support for Setaphyta under composition heterogeneous substitution models (#43726)”

The phylogenetic relationships among three bryophyte lineages and tracheophytes have received a great amount of attention recently, especially from scientists who obtained nuclear genomic and transcriptomic data. Because some fundamental issues regarding gene orthology and the fit between genes with different evolutionary rates and the scope of phylogenetic have not been seriously addressed in studies that used the nuclear data, it is difficult to take the results from those nuclear gene studies seriously. The organellar genes do not have the orthology issue, but their rates can be quite variable and thus are not all suitable to reconstruct the basal land plant phylogeny. In this regard, the authors of this paper, after obtaining three new green alga mitochondrial genomes, performed more careful analyses before discussing the phylogenetic results. My main concern is on the validity of the theoretical standards that can be used to evaluate appropriateness of certain genes and data types for reconstructing the basal land plant phylogeny. I am worried that even a good fit between a model and the data only indicates that the data is OK with the model, but it does not say anything about representativeness of the particular genes/data types and the ENTIRE data universe of the organisms, which should be the result of organism evolution. In this regard, I suggest that the authors should experiment with permutation analyses of different taxon sampling schemes to further evaluate the effect of taxon sampling on performance of certain genes in reconstructing the basal land plant phylogeny. GenBank does have more data than what they have included in this study.
Experimental design: Review of “The mitochondrial phylogeny of land plants shows support for Setaphyta under composition heterogeneous substitution models (#43726)”

The phylogenetic relationships among three bryophyte lineages and tracheophytes have received a great amount of attention recently, especially from scientists who obtained nuclear genomic and transcriptomic data. Because some fundamental issues regarding gene orthology and the fit between genes with different evolutionary rates and the scope of phylogenetic have not been seriously addressed in studies that used the nuclear data, it is difficult to take the results from those nuclear gene studies seriously. The organellar genes do not have the orthology issue, but their rates can be quite variable and thus are not all suitable to reconstruct the basal land plant phylogeny. In this regard, the authors of this paper, after obtaining three new green alga mitochondrial genomes, performed more careful analyses before discussing the phylogenetic results. My main concern is on the validity of the theoretical standards that can be used to evaluate appropriateness of certain genes and data types for reconstructing the basal land plant phylogeny. I am worried that even a good fit between a model and the data only indicates that the data is OK with the model, but it does not say anything about representativeness of the particular genes/data types and the ENTIRE data universe of the organisms, which should be the result of organism evolution. In this regard, I suggest that the authors should experiment with permutation analyses of different taxon sampling schemes to further evaluate the effect of taxon sampling on performance of certain genes in reconstructing the basal land plant phylogeny. GenBank does have more data than what they have included in this study.
Validity of the findings: Review of “The mitochondrial phylogeny of land plants shows support for Setaphyta under composition heterogeneous substitution models (#43726)”

The phylogenetic relationships among three bryophyte lineages and tracheophytes have received a great amount of attention recently, especially from scientists who obtained nuclear genomic and transcriptomic data. Because some fundamental issues regarding gene orthology and the fit between genes with different evolutionary rates and the scope of phylogenetic have not been seriously addressed in studies that used the nuclear data, it is difficult to take the results from those nuclear gene studies seriously. The organellar genes do not have the orthology issue, but their rates can be quite variable and thus are not all suitable to reconstruct the basal land plant phylogeny. In this regard, the authors of this paper, after obtaining three new green alga mitochondrial genomes, performed more careful analyses before discussing the phylogenetic results. My main concern is on the validity of the theoretical standards that can be used to evaluate appropriateness of certain genes and data types for reconstructing the basal land plant phylogeny. I am worried that even a good fit between a model and the data only indicates that the data is OK with the model, but it does not say anything about representativeness of the particular genes/data types and the ENTIRE data universe of the organisms, which should be the result of organism evolution. In this regard, I suggest that the authors should experiment with permutation analyses of different taxon sampling schemes to further evaluate the effect of taxon sampling on performance of certain genes in reconstructing the basal land plant phylogeny. GenBank does have more data than what they have included in this study.
Additional comments: Review of “The mitochondrial phylogeny of land plants shows support for Setaphyta under composition heterogeneous substitution models (#43726)”

The phylogenetic relationships among three bryophyte lineages and tracheophytes have received a great amount of attention recently, especially from scientists who obtained nuclear genomic and transcriptomic data. Because some fundamental issues regarding gene orthology and the fit between genes with different evolutionary rates and the scope of phylogenetic have not been seriously addressed in studies that used the nuclear data, it is difficult to take the results from those nuclear gene studies seriously. The organellar genes do not have the orthology issue, but their rates can be quite variable and thus are not all suitable to reconstruct the basal land plant phylogeny. In this regard, the authors of this paper, after obtaining three new green alga mitochondrial genomes, performed more careful analyses before discussing the phylogenetic results. My main concern is on the validity of the theoretical standards that can be used to evaluate appropriateness of certain genes and data types for reconstructing the basal land plant phylogeny. I am worried that even a good fit between a model and the data only indicates that the data is OK with the model, but it does not say anything about representativeness of the particular genes/data types and the ENTIRE data universe of the organisms, which should be the result of organism evolution. In this regard, I suggest that the authors should experiment with permutation analyses of different taxon sampling schemes to further evaluate the effect of taxon sampling on performance of certain genes in reconstructing the basal land plant phylogeny. GenBank does have more data than what they have included in this study.


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

Input:
Title: THE MITOCHONDRIAL PHYLOGENY OF LAND PLANTS SHOWS SUPPORT FOR SETAPHYTA UNDER COMPOSITION-HETEROGENEOUS SUBSTITUTION MODELS
Review round: 1
Reviewer: 2

Output:
Basic reporting: This is a well-written and logically structured article. In general I found no faults in the basic reporting, although I did note a few typos, listed below:

Lines 60-61: "...between a clade uniting mosses and liverworts and other embryophytes...". On first pass I read this as clade of all three: mosses, liverworts, and other embryophytes, but I assume the authors mean the clade contains only mosses and liverworts. I would suggest rephrasing this for clarity, to something like: "...between a clade uniting mosses and liverworts as sister to other embryophytes...".
Lines 97-98: There is no Cox et al. (2018) listed in the references; should this be Cox et al. (2014), or Cox (2018)?
Line 182: "non-monphyly" - missing 'o'.
Line 252: "was a not a good" - additional 'a' should be deleted.
Line 261: "ccm" should be in italics.
Line 264: "other" - should be 'others'.
Line 348: "toplogies" - missing 'o'.
Line 369: "relatively to" - '-ly' should be deleted
Line 391: "if there were" - should be 'they'.
Supporting information: Tree legends should be checked carefully for typos - I noticed "composition" was frequently missing one 'i', while "predictive" frequently had an extra 'i'.
Experimental design: This is a coherent study and in general the methods are presented well. Although I think the sampling is a little too sparse and would have liked to see at least a few more representatives in each bryophyte lineage (discussed below), the authors do a good job of justifying their restricted sampling due to the computational complexity of the analyses.

I do think there is some inconsistency with the discussion (and selection) of taxonomic sampling, and I think the sampling strategy could be better explained. In the abstract and introduction the authors imply that they are interested in six lineages, three each in bryophytes and tracheophytes (Lines 29-30, 44, 47-48), and in the methods state that they had a deliberately restricted sampling to provide a “balanced selection” between the bryophytes and tracheophytes (Lines 107-108). However, the remainder of the paper treats the tracheophytes as a single lineage, and bryophytes as three distinct lineages (Lines 180-181, 291, 299), which suggests that for a "balanced selection" a similar sampling for each of the four lineages under investigation would be required. The authors also stress that the addition of fern taxa within the tracheophytes might reduce the possibility of artefacts relating to long-branch attraction (Lines 370-372), but don’t address this within the bryophyte lineages.
Therefore, I think the paper would benefit from a better explanation of the breakdown of each lineage -- why so few hornworts for example? Why no representatives of the Jungermanniidae or Hypnanae (the most speciose groups of liverworts and mosses, respectively)? Is this simply due to data availability?
I understand that with each additional sample the increase in computational complexity is not linear, but I feel a few more samples to balance each bryophyte lineage with the tracheophytes would have been useful, without the analyses becoming intractable. I'm not suggesting the authors revisit their alignments and analyses at this point, but I do feel a more thorough explanation would improve clarity.

Some additional minor queries on the methods are listed below:

Line 156: What was the cause of missing genes (incomplete genomic coverage, genes transferred to the nucleus in some lineages, etc)?

Lines 181-183: Why were aberrant genes considered unsuitable (it seems there is an assumption of the monophyly of each lineage, but I'm not sure that sources supporting this were explicitly cited previously?)

Line 223: Will the ML trees be included among the files on Zenodo? It would be good to have them available somewhere, although including them in the supporting information file would be preferred.
Validity of the findings: No comment.
Additional comments: I enjoyed reading this article on the mitochondrial phylogeny of land plants, which adds to a growing body of work using genomic data to attempt to resolve land plant relationships, and ties in well with other recent studies based on nuclear and plastid genomic data sets. This study is particularly useful in that it adds to the relatively limited literature on mitochondrial phylogenomics of land plants (compared to the more abundant nuclear and plastid studies; although please see the very recent article in ‘early view’ in AJB as an additional study of land plant relationships using plastid and mitochondrial data sets: https://doi.org/10.1002/ajb2.1397), as well as further investigating the techniques appropriate for analysing these sorts of data, particularly with reference to computational limitations which will continue to be an issue for phylogenomic analyses as richer data sets become available.

While relationships within lineages is not the focus of the paper, I think it would be worthwhile for the authors to briefly discuss the anomalous results inferred in some of their trees, namely the recovery of Marchantia, rather than Treubia, as sister to the other liverworts in several analyses, and the apparent paraphyly of the gymnosperms (both situations shown in Figure 2 with maximum support, but contrary to a growing body of evidence -- e.g. Forrest et al. 2006 - The Bryologist 109: 303-334; Liu et al. 2014; Wickett et al. 2014). If this apparent mis-inference within lineages is the result of saturation effects in long branches, then it somewhat undermines the argument that these artefacts can be better dealt with by more complex modelling in restricted sampling than by increasing taxonomic coverage to break up long branches. As was suggested with ferns in the tracheophytes here (Lines 370-372), additional sampling within each bryophyte clade may have helped reduce these effects (e.g. see Pollock et al. 2002 - Syst Biol 51: 664–671; Hedtke et al. 2006 - Syst Biol 55: 522–529).

Some additional comments not addressed above:
Lines 35, 105-107, 372-373:
Zygnematales were represented in the sampling of Turmel et al. (2013). Additionally, Bell et al. (https://doi.org/10.1002/ajb2.1397) sampled several ferns and Zygnematales in their mitochondrial analyses.

Lines 99-101:
Or in conflict with different analyses from within the same genomic compartment! Most of the cited studies did recover conflicting topologies for the same genomic compartment, depending on how they treated the data.

Lines 384-387:
The short branches linking each subsequent split among the four major lineages here, relative to the long branch between algae and embryophytes (Fig. 2 and supporting information) may also be a factor here -- is this something the authors have considered? Is there scope to develop more suitable models to tackle the saturation effects which may mislead inference due to this extreme long branch situation?


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: THE MITOCHONDRIAL PHYLOGENY OF LAND PLANTS SHOWS SUPPORT FOR SETAPHYTA UNDER COMPOSITION-HETEROGENEOUS SUBSTITUTION MODELS
Review round: 1
Reviewer: 3

Output:
Basic reporting: In this study, the authors using the mitochondrial (mt) genome data to test the controversial question about the deep phylogenetic relationships of early land plants. They newly sequenced mt genomes from three algae, including two from Zygnematales, the putative closest outgroup of land plants. They also include ferns that were not sampled in the previous study by Liu et al. 2014. With a dataset of 21 land plants and 5 algal outgroups, they performed phylogenetic analyses of nucleotide, amino acid, and codon-degenerate nucleotide data, under the tree-homogeneous or tree-heterogeneous composition models, respectively. Their results indicated that under the tree-homogeneous model the three bryophyte lineages are paraphyletic, while under the tree-heterogeneous model, the three bryophyte lineages are paraphyletic or mosses and liverworts form a clade, i.e., Setaphyta is recovered. However, a monophyletic relationship of bryophytes was never recovered by the mt genome data. The research carried out intensive analyses to test the phylogeny of early land plants using the mt genome data, which are informative and worth to publish, however some issues as listed below should be addressed.

1. Although the authors explained due to the computational restriction, only a small size of samples (21 land plants) were included, they should still discuss about this limitation, considering more than 100 land plant mt genomes are available in GenBank. The drawbacks of employing reduced taxon sampling have been long discussed.

2. As I understand, the purpose of degenerating codons is to reduce the effects of base composition. It is amazing the data still cannot pass the homogeneity test. The logic of using a heterogeneous model on a heterogeneous “treated” dataset seems odd, please explain.

3. Both the codon-degenerate data and amino acid dataset cannot pass the homogeneity test, that means some lineages are biased on GC composition, but the authors did not point out which lineages stand out on GC compositions, i.e., what exactly caused the conflict between the two (hetero- and homo-) models.

4. When the tree-heterogeneous composition NDCH2 model was used to model the concatenated protein data, the two separate runs did not converge on the same tree topology, what is the alternative topology? Does it mean the sampling of the parameters is not sufficient? The authors should consider run the MCMC for more generations.

5. RNA editing is common in plant mt genomes, some lineage such as lycophytes host >2,000 editing sites. Dose RNA editing impact phylogenetic analysis? This point should be discussed.
Experimental design: The new data the authors generated in this study were from three algae, including two Zygnematales, but they did not discuss the question about which lineage of charophytes share the closest common ancestor with land plants. From the trees presented in the manuscript, it is not clear if this question is resolved, as the trees were not rooted. The data the authors generated and the question they focused seem not fit, the authors should explain.
Validity of the findings: no comment
Additional comments: no comment


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

Input:
Title: THE MITOCHONDRIAL PHYLOGENY OF LAND PLANTS SHOWS SUPPORT FOR SETAPHYTA UNDER COMPOSITION-HETEROGENEOUS SUBSTITUTION MODELS
Review round: 2
Reviewer: 1

Output:
Basic reporting: No further comment.
Experimental design: No further comment.
Validity of the findings: No further comment.
Additional comments: The authors have addressed all concerns raised by the reviewers adequately, and I think the updated text will be a valuable addition to the literature on this topic.


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: THE MITOCHONDRIAL PHYLOGENY OF LAND PLANTS SHOWS SUPPORT FOR SETAPHYTA UNDER COMPOSITION-HETEROGENEOUS SUBSTITUTION MODELS
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors’ response is appreciated, but with regards to the argument on the sparse sampling in this study, the authors’ explanation is still not convincing. The study by Liu et al. (2014) focused on a same question as this study, and they took similar analytical strategies. Actually this study is more or less a reduced set of that study’s sampling (ca. 20 vs 60). The main difference is this study included three newly sequenced algae, and two recent published ferns. But as the authors pointed out adding branches in tracheophytes should not impact the resolution of relationships among major clades.

Since Liu et al. (2014) did not recover a Setaphyta with a larger dataset, what makes the current study recovered it? In other words, which result is more reliable and should be accepted? Isn’t it worth to test whether removing the newly added algal outgroups will lead to a constant result, or including the new algal outgroups in Liu et al. (2014)’s dataset will result in a Setaphyta as in this study? I worry about the consistency and robustness of the novel result (i.e. a Setaphyta) found in this study.
Experimental design: NA
Validity of the findings: NA
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: IDENTIFICATION AND EXPRESSION ANALYSIS OF THE DREB TRANSCRIPTION FACTOR FAMILY IN PINEAPPLE (ANANAS COMOSUS (L.) MERR.)
Review round: 1
Reviewer: 1

Output:
Basic reporting: Literature references, sufficient field background/context provided.
Experimental design: Rigorous investigation performed to a high technical & ethical standard.
Validity of the findings: All underlying data have been provided; they are robust, statistically sound, & controlled.
Additional comments: Dear All
This paper was writen as good and detailed. It contributes to understanding of DREB genes in plants. Bioinformatics and wet-lab studies were used sucessfully in this study. Some minor problems were indicated as red in pdf file.


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 AND EXPRESSION ANALYSIS OF THE DREB TRANSCRIPTION FACTOR FAMILY IN PINEAPPLE (ANANAS COMOSUS (L.) MERR.)
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Chai et al's manuscript entitled "Identiﬁcation and expression analysis of DREB transcription factor family in pineapple (Ananas comosus (L.) Merr.)" first identifed 20 DREB genes in pineapple and characterized their gene's struture, tissure specific expression profile and expression profile under different stresses. This study provides new insight for future studies to ﬁnd out the functions of DREB genes in pineapple. The manuscript is well written in English clearly. I recommend for publish in Peer J.


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 AND EXPRESSION ANALYSIS OF THE DREB TRANSCRIPTION FACTOR FAMILY IN PINEAPPLE (ANANAS COMOSUS (L.) MERR.)
Review round: 1
Reviewer: 3

Output:
Basic reporting: This paper is focusing on identification and characterization of DREB genes in pineapple. Authors have used various in silico and in vivo techniques to achieve their goal. I would like to appreciated the effort put forward by the authors dissecting the role DREB family members in pineapple.
While appreciating the contribution made by the authors I would like to provide following suggestions to improve the manuscript.
1. There are considerable amount of formatting and sentence structure issues throughout the manuscript (Please see the attached annotated manuscript).
2. Most of the references were excluded through out the manuscript. Please back all the claims/facts you are pointing with valid references. Introduction and discussion need to be further strengthen while minimizing overlaps.
3.  Figure titles needs to be revisited. Some suggestions are included in the annotated manuscript. Also, Fig 7. please include a colored text strip to indicate tissue/organ for O, S and G so readers can easily understand what tissue each abbreviation is referring to.Figure 4, - I would like to suggest a single line to indicate the promoter and use arrows to indicate the orientation of the cis-elements.
5. Gene conventions and formatting, abbreviation standards needs to be followed through out the manuscript. Please carefully revisit the manuscript.
Experimental design: The research presented here in the manuscript suit the aims and scope of the PeerJ.
1. Authors need to further elaborate the application of the research outcome in broader context.
2. Though the methods used in the study are appropriate, I noticed the following.
a. relevant references of tools  are cited. There are deadlinks. Also it is important to include software versions in order to increase the reproducibility of the outcome.
b. Statistical analysis should be carried out for qPCR data in order to use the term "significantly" describe expression differences observed.
3. Consensus cis-element sequences and relevant references need to Incorporated either in methods or as a supplement.
Validity of the findings: Authors have generated comprehensive set of data in characterizing DREB family. However stress induced gene expression results need to be statistically assessed  before make conclusions. There is a room to improve discussion incorporating and interpreting major advances made in DREB genes characterization in other plant spp. Conclusions need to address the objectives of the study and it is lacking in the current version of the manuscript.
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 AND EXPRESSION ANALYSIS OF THE DREB TRANSCRIPTION FACTOR FAMILY IN PINEAPPLE (ANANAS COMOSUS (L.) MERR.)
Review round: 1
Reviewer: 4

Output:
Basic reporting: The article English text is clear but needs some improvements in the highlighted regions.
The introduction and background is sufficient to display the matter, but the references are not sufficient in the material and method section and also should be prepared based on the journal style.
The article structure is provided in a professional style and figures are sufficient to display your results, but the raw/supplementary data should be inserted to your paper for more justification.
Experimental design: The research was significantly within the Aims and Scope of the journal.
Research questions were well defined and expressed.
Methods were well described but some highlighted sections should be prepared in sufficient detail.
Validity of the findings: The description and conclusions sections can be improved with some unrepeated sentences during the manuscript.
Additional comments: Totally, the manuscript can be publish in the PeerJ, but after revisions; there are four general comments:
1. the paper text including headings and sub-headings should be prepared based on the journal template
2. the English text should be improved during the highlighted regions specifically in the materials and methods section
3. the supplementary tables and figures should be submitted
4. the references should be cited in the highlighted sections as well as should be prepared based on the journal style.


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 AND EXPRESSION ANALYSIS OF THE DREB TRANSCRIPTION FACTOR FAMILY IN PINEAPPLE (ANANAS COMOSUS (L.) MERR.)
Review round: 2
Reviewer: 1

Output:
Basic reporting: While appreciating the authors for incorporating changes as requested, I like to point out that the manuscript is still require language improvement. This is essential for passing the significance of the study to targeted audience. I highly suggest authors to obtain help from a professional language editing service. That way authors will be able to improve the structure and flow of the manuscript.
Experimental design: Authors addressed most of my concerns regarding experimental design. However, things like how the qPCR data was analyzed is not given and relevant details are not specified in in the figure as well. I appreciate if authors can go through the manuscript again giving special consideration to methods in order to address deficiencies.
Validity of the findings: Authors have done fairly good job after the revision. However, impacts of the study can be further elaborated.
Additional comments: Please revisit figure legends (Fig 8 and 9). They need be further improved
"Figure.8: Heat-map for validation of DREB genes RNA-seq.
Validation of 16 genes at nine different tissues through qRT-PCR. Heat-map was constructed
from relative gene expression in different tissues (qRT-PCR) data." First part is on RNASeq while description is talking about qRT-PCR.


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 AND EXPRESSION ANALYSIS OF THE DREB TRANSCRIPTION FACTOR FAMILY IN PINEAPPLE (ANANAS COMOSUS (L.) MERR.)
Review round: 3
Reviewer: 1

Output:
Basic reporting: The article must be written in English and must use clear, unambiguous, technically correct text.
Experimental design: Original primary research within Aims and Scope of the journal.
Validity of the findings: Conclusions are well stated, linked to original research question & limited to supporting results.
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 AND EXPRESSION ANALYSIS OF THE DREB TRANSCRIPTION FACTOR FAMILY IN PINEAPPLE (ANANAS COMOSUS (L.) MERR.)
Review round: 3
Reviewer: 2

Output:
Basic reporting: The authors has improved the writing style and structure of the manuscript.
Experimental design: Authors failed to include the statistical analysis they used to indicate significance of qPCR results (fig 9) in methods and figure legend is also incomplete even though it was requested under earlier review.
Validity of the findings: Section on qPCR is need to be improved to match with the comments made under experimental design.
Additional comments: No 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 AND EXPRESSION ANALYSIS OF THE DREB TRANSCRIPTION FACTOR FAMILY IN PINEAPPLE (ANANAS COMOSUS (L.) MERR.)
Review round: 3
Reviewer: 3

Output:
Basic reporting: The article is written in English clearly and contains technically correct text
Experimental design: The submission clearly defined the research question, which is relevant and meaningful.
Validity of the findings: All underlying data have been provided; they are robust, statistically sound, & controlled.
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: LONG-TERM CONTINUOUSLY MONOCROPPED PEANUT SIGNIFICANTLY CHANGED THE ABUNDANCE AND COMPOSITION OF SOIL BACTERIAL COMMUNITIES
Review round: 1
Reviewer: 1

Output:
Basic reporting: .
Experimental design: .
Validity of the findings: .
Additional comments: I appreciate the hard work on the manuscript entitled “Long-Term Continuously Monocropped Peanut Significantly Changed the Abundance and Composition of Soil Bacterial Communities”. The following comments would be helpful for this paper.
1. The object of this MS was focused on the changement of bacterial community based on the background of soil sickness caused by continuously monocropped. However, no related peanut sickness index was recorded in this MS. Meanwhile, I strongly suggested that more plant-growth data (e.g., weight, length, pod number) and the soil physichemical properities should be included. If not, the relationship between microbial community difference and the peanut soil sickness is so weak.
2. To the best of our knowledge, papers from authoritative journal have already suggested that the soil sickness in peanut continuously monocropped regime was mainly attribute to the imbalance of soil pathogenic fungal community. Hence, why the authors regard the difference of soil bacterial community assembly as the mainly reason of peanut soil sickness?
3. From the perspective of bio-informatic analyse, the presentation way of this MS was too traditional. I suggested that LefSE (LDA analysis) should be performed to further compare the significantly differential bacterial taxa among each treatment. Meanwhile, the PERMANOVA (Adonis) analyse should be calculated to further certify the significant level of community difference in PCA analysis.
4. In the terms of sampling processing, the authors have not clearly stated that the collecting soil samples is belonged to the peanut rhizosphere or the bulk soil.
5. As an nitrogen fixing crop, the community of rhizobia plays important role in the healthy growth of  peanut. Since the MS only mentioned about the soil bacterial community, I suggested the authors should deeply discussed this problem.


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: LONG-TERM CONTINUOUSLY MONOCROPPED PEANUT SIGNIFICANTLY CHANGED THE ABUNDANCE AND COMPOSITION OF SOIL BACTERIAL COMMUNITIES
Review round: 1
Reviewer: 2

Output:
Basic reporting: The MS investigated the feedback responses of soil bacteria to short- and long-term continuous monocropping of four different peanut varieties using high-throughput sequencing techniques, and found Long-term consecutive monocropping could lead to a general loss in bacterial diversity and remarkable changes in bacterial abundance and composition, and inferred simplification of bacterial communities, especially beneficial communities, and bacterial community function simplification with long-term peanut monocropping could be the main cause of peanut soil sickness. It is a good MS, and some suggestions are expected to help further improve the quality of manuscripts.
It is better to simplify the results descriptions (including figures ) and enhance bacterial function discussions (beneficial or pathogenic ). Some merging of unimportant types or without specific functions will increase the readability since it was not clear in the current version.
Line 94, I am not sure ”this is the first study”.  It perhaps occurred in a specific region or soil condition.
Since varieties had little effect on soil microorganisms, what is the mechanism of sensitive or tolerant peanuts to monocropping?
Experimental design: Line 105, what is mean 1m high ?  What’s kind of soil ?  How to drain on rainy days?
Was one peanut variety continuously planted for 11 or 12 years without variety change?
Besides soil microorganisms, main physical and chemical properties of soil should be list in a Table(S)
Validity of the findings: no comment
Additional comments: No additional comments


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

Input:
Title: LONG-TERM CONTINUOUSLY MONOCROPPED PEANUT SIGNIFICANTLY CHANGED THE ABUNDANCE AND COMPOSITION OF SOIL BACTERIAL COMMUNITIES
Review round: 1
Reviewer: 3

Output:
Basic reporting: Chen’s manuscript entitled “Long-Term Continuously Monocropped Peanut Significantly Changed the Abundance and Composition of Soil Bacterial Communities” investigated the response of soil bacterial community to the peanut variety, growth stage and continuous cropping time, and found that the microbial communities were changed among continuous cropping times. The authors attributed the soil sickness caused by continuous monocropping to the declined abundance of the beneficial bacterial taxa.
Experimental design: From which rhizocompartment the soil was collected? Need a more detailed description of how and why the soil sample was taken.
Validity of the findings: It is still hard to find a clear conclusion of the manuscript. The structure of the soil microbiome has changed over time, this could be due to too many factors, such as raining, irrigation, fertilization et al. What is the main reason for the changes in microbial community structure should be discussed.
Additional comments: The language of the manuscript need to be revised by a native English speaker. There is a lot of confusing statements and mistakes of the basic concepts in the manuscript. For instance, the first sentence of the Abstract “Soil sickness is the progressive loss of soil quality due to continuous monocropping”. Soil sickness could be introduced by a number of practices other than monocropping.


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: LONG-TERM CONTINUOUSLY MONOCROPPED PEANUT SIGNIFICANTLY CHANGED THE ABUNDANCE AND COMPOSITION OF SOIL BACTERIAL COMMUNITIES
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Line 187: “The soil pH in samples from 2-year monocropped plots (7.20 - 8.05) were slightly higher than that in 1-year monocropped plots samples (8.30 - 8.59)”, Check it, please.
What is “Y1.H20…..Y12H917”? It should be described when the codes first emerged.
Line 357 “These analyses indicated that soil pH, available P, K could be closely related with soil bacterial communities under long-term monocropping of peanut”, but there was no description in the MS.