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: TROPANE ALKALOIDS AND TERPENES SYNTHASE GENES OF DATURA STRAMONIUM (SOLANACEAE)
Review round: 1
Reviewer: 1

Output:
Basic reporting: The data presented in this manuscript attempts to investigate the evolution of genes involved in tropane alkaloid and terpene biosynthesis in the Solanaceae family. The study is far too preliminary to be considered for publication and interpretation of the data is flawed. The authors claim to be using sequences derived from a genome sequence of Datura stramonium that is not publically available and none of the sequences are provided in the manuscript or submitted to Genbank. Hence there is no possibility for anyone to independently confirm their findings. In addition, throughout this study, accession numbers or gene identifiers are not provided for any of the sequences used to construct the phylogenetic trees. It is impossible for the reader to understand the identity of the genes that are being compared. No raw data is shared.

Furthermore, the study is focused on investigating gene duplications and losses and one of the claims states that TRII has expanded in the Solanaceae, particularly Datura stramonium, while TRI copy number has contracted. There is absolutely no evidence to support these data. TRI and TRII are both members of the short-chain dehydrogenase / reductase superfamily and plant genomes contain many sequences that are related to these genes. They encode enzymes that are catalytically promiscuous and act on many ketones to produce alcohols. The authors provide no biochemical evidence to indicate that the sequences they have identified actually encode enzymes with TRII activity. Hence the assumption that these enzymes contribute to tropane alkaloid biosynthesis is flawed. Indeed it is highly likely that these enzymes catalyze other reactions. Similarly, analysis of the terpene synthase sequences is also fundamentally flawed. Numerous other studies have utilized phylogenetic analyses to place terpene synthases into several subfamilies, which can be used to infer function. For example, tomato contains close to 50 terpene synthase genes whereas the current study presents five sequences from Datura and is therefore almost certainly incomplete. The phylogenetic framework used by the authors has not taken into account the sub-groupings adopted by the terpene field.

Finally, specialized metabolism is inherently complex. You cannot infer gene function based on phylogeny alone. The data presented by the authors represents a potential start of future studies to investigate their findings and the function of the genes they have identified. As presented, this data alone is not suitable for publication.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: No additional comments


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

Input:
Title: TROPANE ALKALOIDS AND TERPENES SYNTHASE GENES OF DATURA STRAMONIUM (SOLANACEAE)
Review round: 1
Reviewer: 2

Output:
Basic reporting: Topic of great interest and the article is original. Clear study written in professional English with sufficient bibliographic references. The article structure was conform. However, document may be better presented.
Experimental design: The study meets the requirements of the journal with clearly defined objectives. The scientific approach was rigorous.
Validity of the findings: Research results were clear and well discussed. They linked to the original research question.
Additional comments: Topic of great interest and the article is original. However, document may be better presented.

Abstract:
Specify, in the abstract, that the study is carried out using bioinformatics tools.

Main text:

   All abbreviations should be clarified on first instance (for example: line 77, CDS);

   Correct the title on line 115: Conserved protein motifs and exon/intron structure de TAs and TPSs genes;

   Homogenize the references in the main text, for example, in the line 57: (Wang et al 2012), in the line 68: (Brille et al., 2019), in the line 80: (Xu et al. 2017) and in the line 100:  (Mulder N.J et al., 2005);

   Reference for line 91 dated 2022?! (Drummond, et al., 2022). Rectify.


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: TROPANE ALKALOIDS AND TERPENES SYNTHASE GENES OF DATURA STRAMONIUM (SOLANACEAE)
Review round: 1
Reviewer: 3

Output:
Basic reporting: THIS STUDY COULD BE COMPLEMENTED WITH CHEMISTRY AS WELL. The authors are using sequences derived from a genome sequence of Datura stramonium that is not publically available and none of the sequences is provided in the manuscript or submitted to Genbank. Lack of  accession numbers or gene identifiers to confirm phylogenetic trees.
please revise data.
Experimental design: please add raw data
Lack of  accession numbers or gene identifiers to confirm phylogenetic trees.
Validity of the findings: please GenBank data
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: TROPANE ALKALOIDS AND TERPENES SYNTHASE GENES OF DATURA STRAMONIUM (SOLANACEAE)
Review round: 2
Reviewer: 1

Output:
Basic reporting: Clear study written in professional English with sufficient bibliographic references. The article structure was conform.
Experimental design: The scientific approach was rigorous.
Validity of the findings: Research results were clear and well discussed.
Additional comments: The manuscript should be accepted as is.


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: ALLELESHIFT: AN R PACKAGE TO PREDICT AND VISUALIZE POPULATION-LEVEL CHANGES IN ALLELE FREQUENCIES IN RESPONSE TO CLIMATE CHANGE
Review round: 1
Reviewer: 1

Output:
Basic reporting: The paper provides a very useful package for application by researchers in a hot development area. The output files look great.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Here are some suggestions for consideration:
22-23 alleles unrelated to adaptive traits may also change under migration so this needs some rewording.
33-36 This is critical but a little dense to follow. Given that RDA is used it might not be apparent to a reader why negative values are avoided.
103-105 Isn’t this a circular argument though? A linear analysis is more suitable for a linear pattern. I’d reword this.
106-108 There is of course plenty of evidence in the literature that clinal patterns in alleles along climate gradients can be non linear as well as linear.  Might be worth a comment and I suspect that researchers will end up testing both approaches.
148-149 This could be restrictive given that SNP approaches typically do lead to some missing data. Bioclimatic data can always be interpolated but perhaps some guidance might be provided for the extent to which missing genetic data might be tolerated.
249-256 Not sure of the value of this! We know that the approach is correlative so as an exercise I suspect that this is not particularly useful.
258-275 The obvious challenge here is to think about how much difference evolutionary adaptation through allele shifts could make to shifts in species distributions as predicted by SDMs as per other papers like Aguirre-Ligouri and Bush et al (Ecology Letters) though the latter is based on heritability estimates. I suspect the authors could more clearly introduce this context here.
277 -285. There are quite a few papers on this that pre-date Aitken and Whitlock and discuss the broader issue of approaches around provenancing (see for instance Broadhurst, Prober etc in Frontiers in Ecology and Evolution). And of course, the challenge and application here is much wider than just considering plants, see recent Hoffmann et al paper in Evolutionary Applications.
General. There are of course some major limitations with the approach generally that go well beyond what is mentioned here. We don’t really know whether the correlative approach will tell us much about future adaptation in the absence of much validation. There have been critiques such as Fitzpatrick et al 2018 Science which should probably be mentioned somewhere.


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: ALLELESHIFT: AN R PACKAGE TO PREDICT AND VISUALIZE POPULATION-LEVEL CHANGES IN ALLELE FREQUENCIES IN RESPONSE TO CLIMATE CHANGE
Review round: 1
Reviewer: 2

Output:
Basic reporting: This is a very nice piece reporting a new R package that allows modeling allele frequencies as a function of environmental or explanatory variables and, assuming adaptation or dispersal under conservative geographical patterns, to project the future expected allele frequencies under climate change. The paper is in general well written and I’m sure that a new nice and friendly user R package may trigger further applications of this area and highlight the need for more studies coupling genetic variation and ecological scenarios for climate change.
Experimental design: it is not an experimental paper
Validity of the findings: the script will be quite useful indeed
Additional comments: This is a very nice piece reporting a new R package that allows modeling allele frequencies as a function of environmental or explanatory variables and, assuming adaptation or dispersal under conservative geographical patterns, to project the future expected allele frequencies under climate change. The paper is in general well written and I’m sure that a new nice and friendly user R package may trigger further applications of this area and highlight the need for more studies coupling genetic variation and ecological scenarios for climate change.

The package is well designed, with nice functions and great visualization tools, and, most important, well compatible with distinct dataset formats commonly used in population genetics packages. Also, it is based on and actually uses several functions from Vegan, which is the most important package (in my opinion) for data analysis (especially multivariate analysis) in Ecology.

Thus, I have just to minor concerns that hopefully may help authors improve a bit the manuscript.

First, although I understand the worry of the author about the (slight) changes in respect to the other protocol proposed by Blumstein et al., it is weird to go into such details in a paper describing a package. Perhaps a few lines in the introduction and discussion are enough, and my impression is that author is perhaps “over-reacting” to previous comments or suggestions. If so, I suggest deleting most of the final part of the introduction and Table 1. So, the paper would be more focused on the package. Perhaps the author would like to compare the two protocols or approaches, but I think this would be a completely different paper, analyzing empirical data and, even better, simulated data (and if author decides to go with this idea here, the R-package would be a supplement or something like this). Anyway, regardless of the strategy, presenting the package and comparing approaches are two different ideias.

Another minor issue, in the “methods”, “data analysis” section, I would like to see a bit more detail on projections. There is a lot of emphasis on calibration, and I understand this, but I barely see something on projection. Also, I’m not sure I followed why goes with an RDA and the use of GAM, it is not clear (perhaps my fault).


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: ALLELESHIFT: AN R PACKAGE TO PREDICT AND VISUALIZE POPULATION-LEVEL CHANGES IN ALLELE FREQUENCIES IN RESPONSE TO CLIMATE CHANGE
Review round: 1
Reviewer: 3

Output:
Basic reporting: Language: There are a few minor corrections to the language that do not change the meaning of the text:

Line 26-27 - “AlleleShift of predicting changes in allele frequencies at populations’ locations.”
- Perhaps: “at the population level or for each location”

Line 40 - “These include ‘dot plot’ graphics”
- These outputs include…
- Also, in several instances, you use "This" and "These" at the begging of a sentence relating to the subject of the sentence before. Although English is not my native language, perhaps you should always write down what "this" and "these" are related to in each sentence as in the suggestion above.

Line 204 “Various options of fitting smoothed regression surfaces are available through providing”
- Change to: by providing

Line 225 – “…negative frequency predictions as can occur with the CCA approach”
- Maybe rephrase to: as what/it may occur with the CCA approach?

Figure 1 - Add what variables are the acronyms in the figure.

You have done a good job of providing updated references and figures, tables and raw data that are meeting the standards of the journal.
Experimental design: Line 104-105 – Perhaps add a caveat here or in the discussion that not all changes are expected to be linear.

Lines 130-131 – There are also the CHELSA and CLIMOND databases with current and future predictions of climatologies.

Line 148-149 – “1). I further advise to remove any individuals with partially missing
genetic or (bio)climatic data prior to the analysis.”
- Would you advise to estimate missing allele states for these individuals using the LEA package or something else?

Lines 156-157 – “What is important to check in the ordination graphs is whether populations shift in a similar fashion”
- And what if they don’t change in a similar fashion? Will the method still provide reliable results? Perhaps add a sentence to help the user.

Lines 226-228 – “My methodology however faces the same limitations of data requirements as discussed by Blumstein et al. (2020) for their protocol in terms of the initial identification of responsive markers.”
- Shouldn't this limitation be mentioned in the methods? If I understood you correctly, you have to remove neutral loci, right? Maybe also advise which methodologies one could use to solve this caveat prior to applying AlleleShift

Lines 233-234 – “I recommend reducing both the explanatory variables and the response variables to subsets of data with a maximum Variance Inflation Factor (VIF) of 20 or less”
- Here you should provide a reference. I have seen values of 5 or 10 for the VIF procedure in the literature.

Lines 236-237 – “if VIF would be larger than 20, I would use function VIF.subset in an iterative procedure whereby earlier selected subsets of alleles are excluded”
- I understood that VIF.subset would only remove environmental variables, right? Perhaps include/expand this explanation in the methods section instead of in the results.

Lines 244-245 – “it is warranted to consider the transferability of the calibrated models and ideally to provide ‘transferability metrics’ that quantify prediction uncertainty (Yates et al., 2018).”
- Could this be provided in the AlleleShift documentation/scripts as well with the dataset that you are using?

Lines 258-261 – “As an approach to obtain a better handle on the transferability of AlleleShift predictions into future climates, I recommend to also estimate the suitability (and ideally the transferability, for instance via evaluation strips as proposed by Elith et al., 2005) of target species via species distribution models (SDMs).”

- I am not sure that I understand how to use the SDM results to handle the transferability of AlleleShift predictions. How would you actually do it? Or would you just compare/visualize both outputs across the geographical space? It would be nice to make this clearer for the user. For example, when would you not calculate the AlleleShift predictions depending on the evaluation strips that you mentioned? Finally, could you do the same for the dataset used in your manuscript to show that the data you are using is transferable?
Validity of the findings: Lines 167-168 –  "This procedure ensures that predictions are within the realistic interval for frequencies between 0 to 1.”

I understand the need for having allele frequencies in the interval between 0 and 1. I can see that the RDA and GAM results with your dataset are very similar. But as a speculation, is it possible to estimate known changes in allele frequencies with a virtual dataset just to check whether AlleleShift is providing the “true” answer? Or at least test these results with an additional dataset to see if the results are always similar between RDA and GAM?
Additional comments: I believe that your manuscript is interesting, the methodology is useful, and that there are mostly small corrections to be made. Here some general comments:

Lines 23-  - “with adaptive traits are expected to change in future climates through local adaption and migration”.
- As you mention migration here, perhaps indicate in the discussion that migration is not being considered in your methodology (or indicate whether migration could mask the effects of predicted changes in allele frequencies not related to adaptation).

Abstract – Methods: I think you should start with the input data, for example, current climate data, predicted climates, SNPs/genomic data.

Lines 31-32 - “As such, the procedure is fundamentally different to an alternative approach
recently proposed to predict changes in allele frequencies from canonical correspondence analysis (CCA).”
- Here it is missing a sentence with some information on why this procedure is better than CCA (as you indicate in the introduction/methodology).

Line 38 - Perhaps: “AlleleShift provides data sets with predicted frequencies, several visualization methods..”

Line 55 – “There is clear evidence of climate change”
- When? Perhaps add: There is clear evidence of anthropogenically induced climate change.

Line 56 – “many countries are developing National Adaptation Plans (NAPs)”
- Here a small explanation is needed at the end of this sentence, what is a NAP?

Line 63 – I am missing a sentence or two providing a link between climate change and changes in the genome/allele frequencies.

Line 70 – Here I believe that you should first introduce the method in a separated sentence and then compare it with Blumstein et al. (2020). What is AlleleShift? Is it already online and being widely used?

Line 261-262 – “utilizing SDMs to predict shifts in species habitat”
- I would not say for species habitat but for species habitat suitability.

Table: “Fit superellipses and conduct RDA to check for niche differentiation between baseline and future positions of climates”
- Niche differentiation sounds like something different, perhaps check for changes in climate conditions across locations between baseline and future climates?


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: ALLELESHIFT: AN R PACKAGE TO PREDICT AND VISUALIZE POPULATION-LEVEL CHANGES IN ALLELE FREQUENCIES IN RESPONSE TO CLIMATE CHANGE
Review round: 2
Reviewer: 1

Output:
Basic reporting: I think this is fine.
Experimental design: My earlier comments have been addressed.
Validity of the findings: Earlier comments have been addressed.
Additional comments: Thanks for the revisions, happy to see this published.


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

Input:
Title: ALLELESHIFT: AN R PACKAGE TO PREDICT AND VISUALIZE POPULATION-LEVEL CHANGES IN ALLELE FREQUENCIES IN RESPONSE TO CLIMATE CHANGE
Review round: 2
Reviewer: 2

Output:
Basic reporting: .
Experimental design: .
Validity of the findings: .
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: ALLELESHIFT: AN R PACKAGE TO PREDICT AND VISUALIZE POPULATION-LEVEL CHANGES IN ALLELE FREQUENCIES IN RESPONSE TO CLIMATE CHANGE
Review round: 2
Reviewer: 3

Output:
Basic reporting: The manuscript has improved a lot in this new version. The language is clearer and the text is nice to read. References are updated and more than enough to provide the basis for research using the package described. All figures and tables are useful and well designed.
Experimental design: The methodology is well described and all concerns that I had before regarding the comparison of RDA and GAM allele frequencies in a simulated or additional dataset were satisfactorily answered. Now the goals of the manuscript appear clear to me, with a better focus on explaining the new method instead of exhaustively comparing it with others. The supplementary material and the material on GitHub allow direct access to the raw data and all steps needed for the analyses. It was clear to me how this research fills the gap on the topic of predicting allele frequencies into distinct scenarios of environmental change.
Validity of the findings: I believe that the new package and methodology will be very welcomed and the author clearly indicated the impact and novelty of it in the manuscript. The results with the test datasets are very interesting, the underlining data was provided, and it looks easily reproducible, given that every step is documented on the package webpage provided. The results were well linked with the conclusions and with the suggestions on how/when to use or not use this method. The reviewing processes really made a difference for the improvement of this manuscript.
Additional comments: The manuscript has improved a lot in this new version. I am glad to see how easily I could read it this time, almost without any interruption, understanding every step. You did a great job of incorporating the many suggestions of the reviewers. As a potential user, I could also follow well the online material on GitHub and it really does look easy to apply to any other datasets. I do not even have any specific comments on language and structure this time, so these are all my 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: DENOUNCING THE USE OF FIELD-SPECIFIC EFFECT SIZE DISTRIBUTIONS TO INFORM MAGNITUDE
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: The survey results do not seem to extend or add to the theoretical statements made.
Additional comments: The manuscript titled “Denouncing the use of field-specific effect size distributions to inform magnitude” argues that the interpretation of effect sizes should not use field-specific effect size distributions (DES), but instead provide interpretations within the context of the study. Further, to support the claim that effect size interpretations are increasingly based on field-specific effect size distributions, the authors conducted a survey on how often papers (Hemphill, 2003; Gignac & Szodorai, 2016; Rubio-Aparicio et al., 2017) that promote this perspective are cited and for what purpose.

The paper is written in a clear style, and this perspective is an interesting take toward the interpretation of effects sizes. The paper is relatively short and thus was limited in its theoretical development as well as interpretation of empirical results. Below, I highlight several general points that come across as weaknesses of this current version of the manuscript.

THEORETICAL CONTRIBUTION. The paper makes the strong claim that effect sizes should be interpreted within the context of the study and not according to DES. The primary reason for this position offered is that there is no rationale for the DES approach. Unfortunately, the authors have not shown why there is no rationale for this position when making a declarative statement.

The rationale for collecting effect sizes within a field must have been provided within the key articles of Hemphill (2003), Gignac & Szodorai (2015), and Rubio-Aparicio et al. (2017). It would be helpful to review the motivation of these authors to provide an effect size distribution. Were effect size distributions provided for the interpretation of other effect sizes? Were they provided to obtain a combined meta-effect of the field? Or were these effect sizes collected to provide some basis of power analysis? Effect sizes and their distributions can serve many purposes – meta-analysis, power analysis, and interpretation. It would be helpful to clarify what the original purpose of effect distributions are before narrowing it to the interpretation of effect sizes.

To state that there is no basis to interpret effect sizes using DES seems like general statement that requires substantiation.

The interpretation of effect size magnitudes are straightforward in applied areas of research (e.g., public health, education). Such examples are provided in the arguments against using DES to interpret effect sizes of a study. However, magnitudes in effect sizes in basic research (e.g., persuasion and attitude change, stereotype threat) are more elusive. The difference between applied versus basic research has to do with measurement of the constructs of interest (Blanton & Jaccard, 2006). For instance, the examples about practical significance is straightforward in applied research (e.g., change in blood pressure, number of suicides, number of cigarettes smoked). But, measures of constructs in basic research are not well-established. How “large” is a correlation of r = 0.2 in terms of a mood manipulation on attitude change? Is a d = .5 small or large for the relationship between positive emotions and novel thought-actions? If correlations in a specific area does not ever reach .6 in reality, shouldn’t adjustments be made to the interpretation of the magnitude of the effect size according to what is observed in the field of research? This rationale is consistent with DES and seems to be theoretically based. If DES are not to be used, how does one interpret an effect size with no consensus on what is large vs. what is small? It could be argued here that interpreting effect sizes according to their theoretical range is unhelpful to understand the practical significance of an effect. An empirical example within basic science would be extremely helpful to illustrate the point of how interpretations of effect sizes within the context of the study are to be conducted. There is no consensus on the magnitude of even standardized effect sizes because of the nature of basic research where interest is in the direction of an effect and not on the size of an effect (e.g., see Jones & Tukey, 2000).

If all effect sizes are to be interpreted within the context of the study, then how can a cumulative science be built? Wouldn’t a single effect size be nested within a distribution of relevant effect sizes in a discipline? A deeper theoretical treatment of the interpretation of effect sizes for what purpose (e.g., specific to the study, specific to the field, etc.) can address this concern.
In general, there are many contexts with which effect sizes can be interpreted. More clarity on what is meant by context can help the theoretical development of the ideas presented in the paper.

EMPIRICAL DATA. The survey was conducted to quantify the popularity of the DES approach to interpret effect sizes. How is this purpose related to the theoretical argument that the DES approach should not be used? Stated differently, how does these data add to the argument that the DES approach should not be used?

By focusing on numbers and not frequency, we run into the reference group problem. There are an increasing number of journals, and so the increasing number of citations may reflect the increasing number of publications and not an increase in the DES approach.  To show that DES is increasing, it could be helpful to show that the recommended approach to interpret effect sizes is decreasing or remaining constant. If not, then the empirical results provide little evidence to support the argument being made in the paper.

Additionally, articles citing primary articles are categorized into direct interpretations, conceptual interpretations, theoretical/methodological and irrelevant. How do these categories help with the theoretical argument made in the paper?

Minor point:
A bar chart for Figure 2 can allow readers to better make comparisons between the size of the categories.

References
Blanton, H., & Jaccard, J. (2006). Arbitrary metrics in psychology. American Psychologist, 61, 27–41. https://doi.org/10.1037/0003-066X.61.1.27
Jones, L. V., & Tukey, J. W. (2000). A sensible formulation of the significance test. Psychological Methods, 5, 411–414. https://doi.org/10.1037/1082-989X.5.4.411


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: DENOUNCING THE USE OF FIELD-SPECIFIC EFFECT SIZE DISTRIBUTIONS TO INFORM MAGNITUDE
Review round: 1
Reviewer: 2

Output:
Basic reporting: Pie charts provide a minimal amount of information. It would be better to include the summary information in the text or use an alternative approach to data visualisation.
https://scc.ms.unimelb.edu.au/resources-list/data-visualisation-and-exploration/no_pie-charts

I believe the authors confuse or conflate two distinct issues, the distribution of effect sizes and the categorising or defining of relative effect sizes from available data. I will expound on this issue in the general comments section.

Jacob Cohen’s research is quoted throughout the paper, but none of his work is listed in the references. This seems to be an honest mistake. But, while it is clear one of the references is his power analysis book, it is not clear what the 1992 reference should be. Just in case the authors have missed Cohen’s discussion papers on this topic, I have always found these articles to be very helpful.

https://psycnet.apa.org/record/1995-12080-001
https://psycnet.apa.org/doiLanding?doi=10.1037%2F0033-2909.112.1.155

It is not clear why “Rationale” is a subsection. It consists of four sentences with the first two being transition sentences. It seems the authors would be better served incorporating the rationale within the regular text of the introduction.

Please refrain from defining an abbreviation as a plural, and maybe use an apostrophe s to make it plural, i.e., use “effect sizes (ES)” and “the magnitude of ES’s”. I would also suggest not making any other abbreviations, e.g., use “ES distribution”. I do not feel that ES magnitude has any real meaning since these are usually singular values, i.e., not a vector, and the sign is usually made positive, i.e., direction is often ignored.
Experimental design: No comment
Validity of the findings: No comment
Additional comments: The authors make very strong claims in the title, abstract and text against the use of effect size distributions. My initial impression was they are wholeheartedly wrong which I can demonstrate. However, after a careful read through, I believe their arguments have a lot of merit. Let me explain.

First, there is nothing wrong with effect size distributions. The authors state in the abstract they “are devoid of a theoretical framework”. The distribution of an effect size like, say, Cohen’s d, is related to the t-distribution for normally distributed data. This framework is used to compute p-values and construct confidence intervals, which can be for the mean, mean difference, or standardised mean difference like Cohen’s d. Maximum likelihood estimators, which form the theoretical basis for much of statistical inference, have an approximate normal distribution (by Central Limit Theorem) and can be used to provide similar information.

Hedges did a good job explaining the standardised mean difference distribution, but there are other really good sources out there.

https://doi.org/10.3102%2F10769986006002107

The term “distribution” can mean a probability distribution (as discussed above) but can also apply to a sample of observations. The authors need to clearly delineate these two.

Note that Cohen’s d is a “biased” ES for delta, the true ES. So, the statement on line 92 is not correct unless using Hedge’s bias adjustment for Cohen’s d, or argue for asymptotic unbiasedness.

After a careful reading, I believe the authors are not advising against ES distributions, but instead the somewhat arbitrary use of relative effect sizes. This needs to be made clear in the manuscript and I strongly urge the authors to clearly define these issues and use more standard terminology. Kelley & Preacher (2012), cited by the authors, do a good job discussing the different ways “ES” can be viewed. Others have used this framework to define ES in three ways – (1) ES measure, (2) the observed ES, and (3) relative ES. I believe a fourth use, ES distribution, could also be defined.

https://www.tandfonline.com/doi/abs/10.1080/03610926.2015.1134575

It is the use of the third definition, relative ES, by some authors that the authors are railing against, and I agree with them. Other authors have collected ES’s through systematic reviews which they divide into equal thirds which are labelled as “small”, “medium” and “large” relative ES’s. This approach makes the very strong and unverifiable assumptions that all ES’s are non-zero and they are important in some sense. This is tantamount to moving the goalposts after the kick is made, and the authors are correct in “denouncing” their use.

There will always be a desire to declare an observed ES as large or small. Cohen argued for “operational” ES as medium if “likely to be visible to the naked eye of a careful observer”, small as “not so small to be trivial”, and large as above a medium ES the same distance as small to medium. Without more information about a problem, Cohen’s relative or operational ES’s can be argued for on this basis and has made them very popular over the years.

Cohen was not a big fan of his own approach, but his reasoning has one huge benefit over the “three equal quarters of observed ES” approach. Cohen’s relative ES’s are independent of the observed ES’s. No goalposts are being moved and not all ES’s are important. At a bare minimum, relative ES’s should be chosen to allow for null effects with no constraint of equal numbers of small, medium, and large ES’s. Also, some consideration should be given to the consequences of mislabelling ES (e.g., is labelling a tiny ES generated from an ES distribution with 0 mean as “medium” going to provide misleading information for those in a research area?).

Another issue with creating relative effect sizes from published ES’s is publication bias. We often have a truncated view of observed ES’s since it has been well established that “significant” (i.e., p<0.05) studies are more likely to be published than negative results (i.e., p>0.05).

Can the authors summarise the results visually? I think their points would be clearer if readers could view the distribution of observed ES’s in a histogram, boxplot or other figure.

This paper reminded me of discussions on magnitude-based inference, which has similarly been used to argue that seemingly unimportant results are really “important” based on speculative methods. There have likewise been many critics of this approach.

https://pubmed.ncbi.nlm.nih.gov/25051387/


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: DENOUNCING THE USE OF FIELD-SPECIFIC EFFECT SIZE DISTRIBUTIONS TO INFORM MAGNITUDE
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: Thanks for thoroughly addressing my earlier concerns.

I would still like a visual display of the data but do not want to hold up publication. The authors are correct that I would like to see the raw effect sizes which are then put into thirds or quarters (as per Hemphill and others). It is a shame Hemphill did not supply this data and this is no fault of the current authors. But I do believe this would make it much easier for the authors to explain how effect size distributions work and are used to interpret magnitude. Perhaps the authors could simulate data for demonstrative purposes?


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: EXPANDING STANDARDS IN VIROMICS: IN SILICO EVALUATION OF DSDNA VIRAL GENOME IDENTIFICATION, CLASSIFICATION, AND AUXILIARY METABOLIC GENE CURATION
Review round: 1
Reviewer: 1

Output:
Basic reporting: This is a well-written, clearly presented and cohesive manuscript, with appropriate references to literature, background and excellent figures. I found it an exciting read and an absolutely necessary piece of work to further the field of viromics.

For basic reporting I have only a couple of minor comments:
Line 120: There is no Table S1.
Line 123: typo “links is listed”
Line 129: Please deposit the in-house script in an appropriate repository (eg. GitHub) to make it available to other researchers.
Experimental design: Materials & Methods: Some of the figures (Fig3 and S1) are showing significance values, but no statistics are explained in the M&M section. Please add this information. From the figure legend of Fig3, it seems like there was an ANOVA performed with t-tests. Given that there are multiple values compared, what was the correction used? Were the data normally distributed? In the legend of Fig S1, the authors describe the use of a non-parametric comparison of means (Kruskal-Wallis with post-hoc Dunn test and Bonferroni correction). Why was this a different test? Please clarify.
Validity of the findings: I have a couple of major comments on the description and interpretation of the analyses and the databases used, that should be addressed to prevent misinterpretation of the usefulness of the benchmarked tools and suggested workflows.

Firstly, the virosphere comprises more than viruses of microbes (as mentioned in the first line of the introduction), and viruses of microbes are much more than just dsDNA viruses. But many of the tools and the analyses presented here, seem to be specific for dsDNA bacteriophages and to a lesser extent archaeal viruses (eg. VirSorter, vConTACT2). This is an important issue that needs to be clarified across the manuscript, as there are many publications and research groups exclusively working on, for example, RNA viromes or circular ssDNA viromes.
The mock dataset viral fractions are all derived from the viral RefSeq database which includes all types of viruses, ssRNA, dsRNA, ssDNA, dsDNA, segmented, linear, circular with vastly different genome sizes. How does this influence false discovery, recall, MCC? Or is the mock community solely derived from dsDNA viruses of microbes? There is a big difference between a 3 kb fragment of a 150 kb Caudovirales phage genome, a 3 kb geminivirus genome or a 5 kb microvirus bacteriophage genome. If the first one does not get recognised as viral, that is less of an issue than the latter two for which the fragment is the whole or >50% of the genome length.
It would be great if the authors could narrow down whether the benchmarking performed here can be generalised for all viromes and the whole virosphere, or whether it is for a specific subset, and if so which subset with which qualifiers.

My second main point follows on from the first point. The authors use a different version of the RefSeq database for classification using gene-sharing networks. This section of the manuscript should present more data backing up the claims of classification accuracy. Again, the RefSeq database contains all virus types the gene-sharing network from Jang et al 2019 only comprises bacterial and archaeal viruses. How can a virus be correctly classified if its reference genome is not represented in the network? What version of the taxonomy release was used and was it from the same year as the RefSeq release? At what ranks (species, genus, family, …, realm) were the sequences assigned and how did this rank affect the accuracy of the assignments? The authors show that the accuracy improved with increased fragment length, but what about short complete genomes? Maybe the short fragments were not robustly placed in the network because their reference counterparts weren’t present?

I find the AMG workflow and suggestions hugely helpful, but I come back to my previous points. I have only ever heard of AMGs in the context of bacteriophages. Are they relevant for other viruses? Is this workflow generalisable for all viromes?
Additional comments: General comment on the use of the viral RefSeq database: This database is a curated subset of all viral genomes available from GenBank and does not represent full viral diversity. NCBI maintains two validated datasets, RefSeq and neighbor genomes (https://www.ncbi.nlm.nih.gov/genome/viruses/about/assemblies/). The RefSeq database does not contain many viral genomes that are extremely similar to each other, and a mock community based on RefSeq alone might deviate from reality in which many viral populations may co-exist that are extremely similar. Since the benchmarking starts from assembled genome fragments, this is not necessarily an issue for the data analysis here, but the authors might want to address this as a limitation.

Minor semantics issue: the term viral sequence is used a lot, which can be anything (sequence reads, contigs, genomes). What the authors are really presenting here are assembled sequences and mock contig datasets, which should be made more clear to the audience.


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: EXPANDING STANDARDS IN VIROMICS: IN SILICO EVALUATION OF DSDNA VIRAL GENOME IDENTIFICATION, CLASSIFICATION, AND AUXILIARY METABOLIC GENE CURATION
Review round: 1
Reviewer: 2

Output:
Basic reporting: In this manuscript the authors perform benchmarking analysis of different tools for virome analysis. Specifically, they test the ability of these tools to identify viral genomes in metagenomes/metaviromes, the ability to correctly assign taxonomic classification to these genomes, and to identify their auxiliary metabolic genes. The topic is extremely relevant as, to my knowledge, no studies have previously performed analysis such as the one presented by the authors. The experiments are well designed and the tutorial like structure of the discussion will likely be helpful to researchers trying to decide on the best tools for their specific needs. Nevertheless, some issues need to be addressed to make this manuscript suitable for publication.
Experimental design: Ln 110-114: The the authors did not analyze the performance of in silico host predictions methods for viruses, for which many approaches are currently available [1–4]. I understand that this might not be within your scope and therefore I will not insist that you include such analysis (although I do believe it would increase the relevance of the manuscript). Nevertheless, given the importance of these methods for studies of viral ecology I believe at least a brief mention to these methods is necessary in the introduction section as well as an explanation for not including them.

Ln 118-123: The dataset selection is inadequate. The viruses from RefSeq are the same that were used to train the majority of the tools for viral identification. Meaning that tools such as VIBRANT, MARVEL and DeepVirFinder which rely on machine learning models should be able to easily identify these sequences as viral. To make it really challenging for these tools it is necessary to use a test set consisting of only sequences that are divergent from those in the training set. Ideally, to mimic real world scenarios, the use of uncultured viral sequences from metagenomes should be applied, preferably covering multiple different ecosystems, such as marine, freshwater, soil, gut etc. This would ensure that the results are representative of multiple ecosystems since the diversity of viruses within each of them should be different.  To avoid any circular logic, I recommend that the authors perform manual identification of viruses from metagenomes from this ecosystems and then use these bona fide viral sequences, fragmented or not to estimate the performance of the tools.

Ln 174-183: There are other methods for viral genome classification that were not analysed [5–8]. Specifically those that rely on phylogenomic approaches. These are important and popular tools that are very likely to be affected by genome fragmentation and therefore a proper benchmarking of the performance of this tools should also be included.

Ln 174-183: Regarding the analysis with vCONTACT 2.0: The setup seems to follow a somewhat circular logic. If the “unknown” fragmented genomes are just a subset of the full RefSeq it should be relatively easy for this tool to classify the genome fragments because the whole genome version is also included in the set. Again, the use of metagenomic viruses would be a more realistic scenario. Furthermore, this type of analysis should be performed with leave-one-out cross validation to answer questions like: How is a genome fragment classified when there are no representatives of its taxa in the reference database?
Validity of the findings: Some of the findings are not valid due to the issues mentioned in the previous section. I will re-evaluate the validity of the findings once these issues are corrected.
Additional comments: Ln 72: Silveira et al., 2017 and Coutinho et al., 2017 actually refer to the same publication. And the correct citation is the one for Coutinho et al., 2017

Ln 106: Edwards et al., 2016 performed benchmarking for in silico host prediction approaches and not for diversity estimation

Ln 160: It would be useful to evaluate the performance of VIBRANT also without the –virome flag

Figure 2: You need to explicitly mention what the error bar represent in the bar plots. Also, from reading the methods I understood that there were only 4 mock communities and no replicates. Hence, it is unclear to me why there are error bars in this plot.

1. Galiez C, Siebert M, Enault F, Vincent J, Söding J. WIsH: who is the host? Predicting prokaryotic hosts from metagenomic phage contigs. Birol I, editor. Bioinformatics [Internet]. 2017;33:3113–4. Available from: https://academic.oup.com/bioinformatics/article-lookup/doi/10.1093/bioinformatics/btx383
2. Wang W, Ren J, Tang K, Dart E, Ignacio-Espinoza JC, Fuhrman JA, et al. A network-based integrated framework for predicting virus–prokaryote interactions. NAR Genomics Bioinforma [Internet]. Oxford University Press; 2020;2:505768. Available from: https://academic.oup.com/nargab/article/doi/10.1093/nargab/lqaa044/5861484
3. Edwards RA, McNair K, Faust K, Raes J, Dutilh BE. Computational approaches to predict bacteriophage–host relationships. Smith M, editor. FEMS Microbiol Rev [Internet]. 2016;40:258–72. Available from: https://academic.oup.com/femsre/article-lookup/doi/10.1093/femsre/fuv048
4. Villarroel J, Kleinheinz KA, Jurtz VI, Zschach H, Lund O, Nielsen M, et al. HostPhinder: A phage host prediction tool. Viruses. 2016;8:1–22.
5. Coutinho FH, Edwards RA, Rodríguez-Valera F. Charting the diversity of uncultured viruses of Archaea and Bacteria. BMC Biol [Internet]. BMC Biology; 2019;17:109. Available from: https://www.biorxiv.org/content/10.1101/480491v1.full
6. Meier-Kolthoff JP, Göker M. VICTOR: genome-based phylogeny and classification of prokaryotic viruses. Kelso J, editor. Bioinformatics [Internet]. 2017;33:3396–404. Available from: http://academic.oup.com/bioinformatics/article/doi/10.1093/bioinformatics/btx440/3933260/VICTOR-genomebased-phylogeny-and-classification-of
7. Rohwer F, Edwards R. The Phage Proteomic Tree: a Genome-Based Taxonomy for Phage. J Bacteriol [Internet]. 2002;184:4529–35. Available from: http://jb.asm.org/cgi/doi/10.1128/JB.184.16.4529-4535.2002
8. Nishimura Y, Yoshida T, Kuronishi M, Uehara H, Ogata H, Goto S. ViPTree: the viral proteomic tree server. Valencia A, editor. Bioinformatics [Internet]. 2017;33:2379–80. Available from: https://academic.oup.com/bioinformatics/article-lookup/doi/10.1093/bioinformatics/btx157


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: EXPANDING STANDARDS IN VIROMICS: IN SILICO EVALUATION OF DSDNA VIRAL GENOME IDENTIFICATION, CLASSIFICATION, AND AUXILIARY METABOLIC GENE CURATION
Review round: 2
Reviewer: 1

Output:
Basic reporting: The writing is clear and professional. However, some of the new paragraphs have grammar mistakes that should be corrected prior to publication. This could be addressed by the editorial team.

Something went wrong with the referencing. For example: lines 273-277 all the references for the tools are wrong and jumbled, VirSorter referring to EasyFig, PHASTER to VIBRANT, etc. I think this is occurring all through the manuscript, but I haven’t cross-checked all entries. Please correct.
Experimental design: In my previous round of review, I asked about versioning of the RefSeq and the Taxonomy database, and whether they matched. The authors provided updated information with the date of use.
RefSeq Database & Table S1: The choice of input sequences is still confusing. The authors used 1,213 genomes of viruses of bacteria from the RefSeq database, but the number of classified dsDNA bacteriophage genomes published in May 2020 was 1,991 using a quick search of the ICTV Viral Metadata Resource (version May 1st, 2020). What happened to the >700 genomes? Were these deliberately omitted? Or did the authors use a previous version of the taxonomy release.

Lines 363-376: VIRIDIC is not a virus classification tool in itself, even though it is often used in virus classification. It is a pairwise whole genome comparison tool meant to be used with complete genomes on a user-selected subset of genomes suspected to be related, with user-specified thresholds for phage species (default 95%) and genus (default 70%) clustering. So using fragmented genomes to assess the performance of this tool in inherently flawed. If you use a reference genome and a fragment of that reference genome, the tool will state that they are not related enough to be classified together. There will be a similar issue with VipTree, which is also made and optimised to classify full genomes. I’m not familiar enough with VPF-class to know whether the same goes here.
Validity of the findings: The findings are novel and an important contribution to the literature.

The classification section was reworked and now focuses on the different classification of known genome fragments versus full genomes. This is a big improvement, and provides important information. However, this needs to be made clear in the abstract as well, where it seems like classification of any virome is discussed.
Additional comments: This is an important and timely contribution to the literature and I congratulate the authors on this excellent piece of work.


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

Input:
Title: EXPANDING STANDARDS IN VIROMICS: IN SILICO EVALUATION OF DSDNA VIRAL GENOME IDENTIFICATION, CLASSIFICATION, AND AUXILIARY METABOLIC GENE CURATION
Review round: 2
Reviewer: 2

Output:
Basic reporting: All my comments have been properly addressed. This manuscript is now suitable for publication.
Experimental design: The updated experimental design is adequate.
Validity of the findings: Conclusions are supported by the findings.
Additional comments: Ln 122: Change “retain” to “retained”

Ln 128: Change “been” to “be”

Ln 273-277: References in this section are switched

Caption for Figure S2 is wrong

Figure S4: This analysis refers to the combine results of all tools?


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: GENETIC CONNECTIVITY BETWEEN ATLANTIC BLUEFIN TUNA LARVAE SPAWNED IN THE GULF OF MEXICO AND IN THE MEDITERRANEAN SEA
Review round: 1
Reviewer: 1

Output:
Basic reporting: The present study was same to those of previous studies.
Experimental design: There are a few studies to assess using "larvae" but fundamental methodology is similar to previous studies.
Validity of the findings: Same and similar results have been found in proviso studies.
Additional comments: The current study tried to understand stock discrimination between Atlantic bluefin tuna larvae by means of molecular genetic analyses. As authors stated and cited, there are a number of similar research have been published, although there is no study have been examined in larvae. However, there are no difference between present and previous studies, and I do not see any novelty except for authors used larval samples. I know larval collection is not easy as it needs to allocate plenty of time and expenditure for research cruise. However, stock analysis is not necessary to use larval samples only as previous studies used juvenile and adult samples. I suppose authors need to collect larvae during spawning period to assess temporal variation because spatial assessment has been done by previous studies. I feel the present study does not overwhelm previous same, similar and relevant studies by means of molecular, otolith and behavioural analyses in the Atlantic bluefin tuna. Although authors stated “Larval early life stages have scarcely been studied to decipher ABFT population structure despite providing the genetic signal from successful breeders” in the first sentence of Discussion, stock discrimination and connectivity research are not necessary examined for larval samples. Furthermore, the current discussion is redundant, e.g., L307—L323 should be in Introduction and/or Results and/or Materials methods. I do not understand why authors still debate about molecular methodology in L324—L341 because there have been done similar or same research in the species and other fish species. Such the discussion seems to be no longer interesting. Authors should focus on population structure, divergence and connectivity only together with conservation and management of the species (L342 onwards are useful discussion). Overall, I cannot recommend the paper is published in Peer J because of lacking of novelty.


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: GENETIC CONNECTIVITY BETWEEN ATLANTIC BLUEFIN TUNA LARVAE SPAWNED IN THE GULF OF MEXICO AND IN THE MEDITERRANEAN SEA
Review round: 1
Reviewer: 2

Output:
Basic reporting: Language is appropriate, clear and unambiguous.  Introduction is partially to be revised as stated in the comments to authors. Literature references are adequate. Iconography and structure of article is adequate.
Experimental design: Adequate and sound. Hypothesis to be tested and aims are clearly stated. The described research fits the aims and scope and standard of the journal.
Validity of the findings: No comment. Please refer to the Comments to Authors.
Additional comments: Johnstone et al. carried out a population genetic study on Atlantic bluefin tuna (ABFT) larvae using microsatellite and CR sequence markers, providing a substantial corroboration of already known ecological and population dynamic features of ABFT but also providing new evidence on the occurrence of a panmictic or near panmictic population in the Gulf of Mexico (the well-known western spawning area) and, with some but not all the statistical tests, a possible, very weak but detectable, reproductive connectivity between the eastern Mediterranean population and ABFT inhabiting the eastern part of the Gulf. The strengthen of this work is the unprecedented exclusive use of ABFT larvae that were collected according a very solid and informative sampling design (all main ABFT spawning areas covered within the same spawning season and year). Results are largely consistent in terms of levels of genetic differentiation and of significance patterns and authors appropriately the use of a limited number of neutral microsatellite loci that have been repeatedly proven and used for polymorphisms and replicability by numerous previous research works. However, the use of a mtDNA partial sequence marker that is highly-variable but less informative for population genetic purposes appears to weak the analytical power of their analyses and issue from phylogeographical analyses are effectively redundant with respect to those already published by several other authors. Results were adequately and deeply discussed highlighting advances and confirmation of already known ABFT population genetics’ patterns.
Therefore, the research paper contributes to shape ABFT population dynamics and connectivity providing some foreseen regarding additional features to those already scientifically consolidated.

Major and Minor comments
Abstract
l. 31 “supporting connectivity between larvae”: larvae cannot reproductively interact. Spawning populations or mature individuals can interact reproductively. Rephrase the sentence more appropriately and with correct meaning.
ll. 38-39, “leading to a strong connectivity between populations”. Statistical data and results obtained did not speak in favor of a strong connectivity but rather of a weak but detectable connectivity. Also, not all statistical results accounted for this issue and this should be mentioned in the abstract.
l. 40. Change “management units” to “populations”

Introduction
ll.  61-72. Several population genetic studies carried out along with 15 years have accounted for the genetic and reproductive separation of ABFT into two populations spawning predominantly in the Mediterranean and Gulf of Mexico. Such genetic issues supported those retrieved from a huge number of observations obtained with multidisciplinary approaches (i.e. reproductive biology, ecology, standard, satellite and microchemical tagging that have been integrated since 2011-12 by ICCAT-GBYP program and surveys). Even if the levels of genetic differentiation assessed between population samples collected from the two spawning populations were very low (from ~1‰ to ~1%), they resulted mostly significant and the structure appeared and was interpreted as robust (see all the works from BBlock group and those produced within the ICCAT-GBYP framework). Such low levels of differentiation were interpreted as due to the relative recent origin of the Atlantic ABFT populations (see Alvarado-Bremer et al. 2005) and to the very large population size that strongly reduces genetic drift detectable by neutral markers. I recommend to authors to summarize better and more consistent with results already obtained the ecological-biological and genetic state-of-the-art of ABFT. For example, Block et al. 2005 and Rooker et al. 2008 revealed the intermingling of individuals from the two spawning populations in the Atlantic feeding grounds (e.g. Mid Atlantic Bight, Bay of Biscay, Coast of Maine etc) but also demonstrated that the any ABFT born in the Mediterranean visited the Gulf and viceversa. This should be accounted to provide a scenario more coherent to existing and published data.
ll. 85-87. This sentence is correct but I recommend to authors to acknowledge here that at least four popgen surveys were based on early-life stages (larvae and YOY): Carlsson et al., 2004, 2007; Puncher et al., 2018; Rodriguez-Ezpeleta et al., 2019, almost possessing the same robustness given by the exclusive use of larvae.
ll. 106-108. This info is irrelevant to the purpose of the paper.
ll. 109-111: same criticism pointed for ll. 38-39: change strong with a more prudent and soft adjective. Let be more cautious in postulating that data “support the hypothesis of mixing of breeders belonging to different stocks”. “Could support” or “seem to support” could be better for consistency with the results. In addition, “Statistical analysis” change in “Individual-based clustering analyses”.

Materials and Methods
ll. 135-136. Instead of sentencing “A minimum number of ….” provide here and not in the Results the number of larvae analysed” and coherently provide in the previous paragraph the number of larvae collected.
ll. 153-154. Authors gave in the manuscript the GenBank accession numbers of the deposited mtDNA sequences; however deposited sequences are not yet accessible. On the contrary, any statement on microsatellite data deposition was given by authors. Authors must provide availability of these data.

Results
ll. 266-268. This sentence is strongly consistent with the results obtained and represents most of the statistical issues obtained and is strongly discordant with the sentences in the abstract and at the end of the Introduction. I recommend to homogenize the issues provided along with the ms. At any rate, the paragraph (from ll. 264-272) should be moved in the Discussion.
ll. 301-305: I disagree and these results can be also explained by an incomplete sorting of the gene pool due to the very low genetic drift affecting these large sized populations.
ll. 356-358. This is incorrect. MED is not intermediate. The intermediate genetic cluster refers to eGOM.
l. 382 “GOM” changes into “eGOM”
l. 396 “Fixation indices and clustering analysis indicated connectivity between ABFT that breed in the MED and in the eGOM was stronger… “ changes into “Fixation indices and clustering analysis indicated weak but detectable connectivity between ABFT that breed in the MED and in the eGOM was stronger…“


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: GENETIC CONNECTIVITY BETWEEN ATLANTIC BLUEFIN TUNA LARVAE SPAWNED IN THE GULF OF MEXICO AND IN THE MEDITERRANEAN SEA
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript of Johnstone et al is a new study on the population genetic connectivity of ABFT between Mediterranean and Atlantic. The study is well defined with a remarkable extensive effort in the sampling of larvae.
The paper is well structured, well written with affectable structure. No major flaws were detected in the structure, English, Figures and Tables
Experimental design: Several flaws were detected in the design and analyses of the manuscript.
I cannot find the rationale of comparing two submaples within the Gulf of Mexico. All the literature accepts the GoM as a single reproduction are, thus a better description why the authors decided to compare the two samples of GoM is needed.
Accordingly, the authors should better define the objective of the study. Similarly, the author should better describe the sampling procedure. It is really confusing. The section of “Genetic Analysis of ABFT larvae” should be improved. It is difficult to find how many larvae have been analyzed in each location. For instance, the number of specimens analyzed is described in the results section, but it should be also included in material and methods (a summary of the sampling would be appreciated).
The analysis proposed is correct, although the interpretation of some results is confusing. In Table 2, no correction of multiple tests is done, with a possible invalidation of the results and conclusion drawn from the results.
Validity of the findings: The result obtained here are in concordance with the current acknowledge of the population structure of ABFT, which has been extensively published in the literature. Some flaws in the analyses are detected (see previous section) and thus with the possibility of invalidating the results and conclusions.
In addition, the DAPC analysis depicts a weak differentiation between Med and GoM, which is contradictory with the title of the manuscript.
Additional comments: Other changes should be done along the manuscript
Line 45. “They are managed by regional fisheries organizations as four stocks”. This sentence is really confusing. The authors confused stocks and species.
Line 266. There is a conclusion in the results section. Conclusions should be detailed in the discussion and conclusion section.
Table 3. I cannot find the Johnstone et al., 2019 study. This refence is not necessary. Keep only “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: GENETIC CONNECTIVITY BETWEEN ATLANTIC BLUEFIN TUNA LARVAE SPAWNED IN THE GULF OF MEXICO AND IN THE MEDITERRANEAN SEA
Review round: 2
Reviewer: 1

Output:
Basic reporting: Comments are summarized in the General Comments for the author
Experimental design: Comments are summarized in the General Comments for the author
Validity of the findings: Comments are summarized in the General Comments for the author
Additional comments: Authors addressed all requests and comments I posed to the submitted version of the manuscript, adequately motivating and detailing changes introduced.


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: BIOLOGICAL CONTROL OF IMPORTANT FUNGAL DISEASES OF POTATO AND RASPBERRY BY TWO BACILLUS VELEZENSIS STRAINS
Review round: 1
Reviewer: 1

Output:
Basic reporting: English language used in the manuscript seems to be clear, professional and understandable.
Literature is well referenced and relevant.
Structure conforms to PeerJ standards.
Figures are relevant, well labelled and described.  Figure S2 is high quality. Figure S1 seems to have too low resolution.
Custom check. I have successfully downloaded all the files mentioned in the article. I also have successfully performed DNA data checks.
Can I access the deposited data? – Yes.
Has the data been deposited correctly? – Yes.
Is the deposition information noted in the manuscript? – Yes.
Experimental design: The results presented in the manuscript are original primary research within the scope of the journal.
Research questions are well defined, relevant and meaningful from the point of biological control of phytopathogenic fungi Rhizoctonia solani and Didymella applanata. The research fills knowledge gaps regarding the use of Bacillus for biological control of phytopathogenic fungi in various environmental conditions. The authors have demonstrated the effectiveness of southern bacterial strains as biological control agents for fungal infections of potatoes and raspberries in more severe environmental conditions. The results are valuable both for agricultural biotechnology and for microbial ecology.
The study adheres to technical and ethical standards.
Methods are described with sufficient details and information to replicate, with some exceptions (see comments below).
Validity of the findings: All underlying data have been provided; they are robust, statistically sound and controlled.
Conclusions are well stated, linked to original research question and limited to supporting results.
Additional comments: Line 129. Error in the text. 104-106 CFU/mL instead of exponent.
Lines 153-154. Insufficient information about the fertilizer (how much nitrogen, phosphorus,  potassium,  magnesium, and  sulfur is in its composition)?
Lines 236-241, 245, 246. Do the authors mean percentages or percentage points? I would recommend use both percentages and percentage points.
Statistical approach. In the future, I would recommend that authors pay special attention to multivariate statistics (Principal components analysis, Discriminant analysis and other very useful methods for analysis multivariate data).


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

Input:
Title: BIOLOGICAL CONTROL OF IMPORTANT FUNGAL DISEASES OF POTATO AND RASPBERRY BY TWO BACILLUS VELEZENSIS STRAINS
Review round: 1
Reviewer: 2

Output:
Basic reporting: - There are some problems in the English language throughout the manuscript.
- The study presents interesting data and has the potential for being an important contribution regarding biological control after being revised.
- Some references in the text are missing and they were pointed out in my revision.
- Too many tables (9) are presented.
Experimental design: Please check the general comments for the author
Validity of the findings: .
Additional comments: The manuscript “Biological control of important fungal diseases of potato and raspberry by two Bacillus velezensis strains” investigated the biological control potential of these B. velezensis strains against fungal diseases in potato and red raspberry cultivated in Russia. The study presents interesting data and has the potential for being an important contribution regarding biological control. However, the manuscript provides an insufficient description of the used methods and the data analysis presented in the manuscript should be improved in order for the manuscript to reach its full potential. The English language should be corrected in different parts of the manuscript. My detailed comments regarding my assessment of the presented manuscript can be found below. I hope that my comments will be constructive and that they will help to improve the manuscript.
Introduction
Line 53 – Explain (or provide a reference) why the full restoration of nature from the negative effects of chemicals is impossible in Western Siberia. There are other places worldwide that short growing seasons occur. Is this true or not?
Line 54 – Can you give an estimative of the sharp changes in temperature and humidity during a year?
Line 79 - by using biological control that corresponding conception of organic agriculture – do you mean “that corresponds…”? Biological control fits into the concept of organic farming – is that you mean?
Line 81 - Biological control of a common potato disease caused by R. solani, have been used by bacteria of the genus Bacillus… Biological control has been used? Other meaning? Please rewrite the sentence.
Line 82 - Biological control of red raspberry disease caused by D. applanata, was much less known? It was less known but now it is not anymore? Please rewrite the sentence.
Line 98 - in another locality with different environmental conditions. What are the main environmental differences between South and Western Russia? And the type of soil?
Line 104 - We can find in the literature many papers where Bacillus velezensis is used in the biocontrol of different genera of phytopathogenic fungi  in crops of high agricultural importance such as potato, mango, avocado, papaya, citrus, tomato, blueberry, blackberry, raspberry, zucchini, melon, cucumber, watermelon and others (Galindo et al., 2013; Balderas-Ruíz et al., 2020; Cui et al., 2020; and others). Therefore, your statement is not true.
Line 108 – what do you mean with much more severe climate conditions? In table 1, the temperature is presented in Celsius degree varying from 17.2 to 20.2. These are not low temperatures. And the rest of the year? How much do the climate conditions of the rest of the year influence the growing season?
Materials and methods
Line 118 - potato-glucose agar - Inform the formula or supplier.
Line 119 - For each experiment, fresh liquid cultures of the strains were prepared on the original optimized nutrient medium – How can we know what is the original optimized nutrient medium?
Line 121 – why the strains were grown in different temperatures and time of incubation?
Line 127 – What were the conditions of Western Siberia?
Line 129 - How were the cell numbers determined? The experiment was carried out by the agar blocks techniques. Provide a reference.
Line 133 – media instead of medium.
Line 144 - The average temperature of these months of 2015 was 18.6 °C, and in 2016 - 19.1 °C. Are these temperatures considered low ones?
Data presented in Table 1 can be shown here in the text. Table 1 is not necessary.
Line 159 - During the growing season – how long did it take? How many measurements?
Line 161 - At each site of the field, 10 plants were counted in four replications. Site of the field – Do you mean the plot of 30 m2?   – counted for what? Four replications of what? Please, clarify.
Tables 2 and 3 – the colony diameters – show average with standard deviations.
Line 225 - the inhibitory effect was not so much – was not so evident? Please clarify.
Table 4 and the following ones – substitute Bacillus subtilis for B. velezensis
Line 231 - the disease incidence in both years of research was at least 80% on two cultivars in control (Table 4). – something is missing in this sentence.
Line 234 - and weather conditions of the year…. How did you conclude that?
Line 236 - in all cases except the strain BZR 517 on the cv. Svitanok kievskiy (P> 0.05) (Table 4). How can we see this result in Table 4? In all columns, data are statistically different.
Line 239 – these data are not shown in table 4.
Line 248 - In 2016, with less air humidity, the effect of bacilli on the disease incidence and severity was statistically less than in the previous year (Table 4). Which evidence you have to correlate humidity with the results obtained?
Line 256 - In daughter tubers of cv. Yuna, the results for both strains were similar in 2015 (Table 4). However, in 2016, a greater value was obtained for the strain BZR 517 (P <0.05) (Table 4).  Are these results correct? The results seem to be similar in 2016.  A greater value of what? Explain
Tables 5, 6 and 7 (titles) – potato cultivars differed by disease resistance? What do you mean? In materials and methods, you presented the cultivars as two potato cultivars of different maturity groups: early maturity cv. Yuna and mid-early cv. Svitanok kievskiy. Do they naturally differ in disease resistance? Or there is a problem in the titles?
Table 5 – standard deviations are important to be presented.
Overall, in 10 weeks after planting, the treatment with both strains for two years on two potato cultivars led to a significant increase of morphometric characteristics of plants (Table 5). - Also in Yuna with BZR 517?
Table 6 – How to explain the results observed with Yuna in 2016, mainly in the medium fraction?
Can Table 7 be incorporated in Table 6? Also add standard deviations.
Line 285 - due to the weather conditions. How this correlation was done? What happened?
Line 295 - both B. velezensis strains significantly reduced the disease severity on cv. Kirzhach (P<0.05), the difference between 336 g and 517 strains was not statistically significant (P>0.05). Please rewrite to make the sentence clear.
Line 309 - This fact may be related both to the different susceptibility of cvs. Kirzhach and Kolokolchik, and to the different weather conditions in 2015 and 2016. This statement is quite speculative.
Discussion
Line 328 - However, no such experiments have been conducted using B. velezensis. Minimize affirmative.
Line 333 - This showed the antagonistic activity of B. velezensis strains against R. solani in the region differs by environmental conditions from the location where they were isolated. Please correct the English language.
Line 340 - the dependence of the bacilli efficacy on abiotic factors? Please explain. This is in accordance with data that some Bacillus strains offer additional benefit to the plants, such as more tolerance to abiotic stress, and growth promotion. I agree with the last sentence but it has nothing to do with the previous one, unless the meaning of the phrase is not that.
Line 345 - two B. subtilis strains – B. velezensis
Line 351 - that less than usually applied by researchers in other localities. Do you mean that increasing the amount of bacteria you could have observed a better result?
Line 357 - the strain BZR 336 g contributed more significantly to this value. Which value?
Line 380 - this was confirmed by their influence on crop yields. Considering ISR is merely speculative in this case.
Line 383 - are able to realize their potential – what does this mean?
Line 410 - For the first time, we demonstrated B. velezensis strains potential for use as biological control agents on potato and on red raspberry. Although the results are important, other studies have already been performed using B. velezensis strains in the biological control of potato and red raspberry, as mentioned before.


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: BIOLOGICAL CONTROL OF IMPORTANT FUNGAL DISEASES OF POTATO AND RASPBERRY BY TWO BACILLUS VELEZENSIS STRAINS
Review round: 1
Reviewer: 3

Output:
Basic reporting: This is a solid research demonstrated B. velezensis strains as biological control agents against R. solani on potato, and against D. applanata on red raspberry.

Except for the writing that need to be improved, the research was solid and very well organized and designed.
Experimental design: Line 158, Before  planting,  potato  seed 158tubers  were dipped for a few  minutes in a suspension of the  strains  BZR  336g  and  BZR  517159(106 CFU  mL-1).   Please specify how many minutes for the coating (dipping)?
Validity of the findings: Please present Figure 1S as Figure 1 (not as supplemental figure).
Additional comments: The researches and finding are very interesting. However, the writing could be organized and presentated in a better way, for instance, line 321 to 328 could be moved to introduction section.
This just one example, the discussion part needs to be reorganized.

Ideally, please present some images showing the field crop growth and disease symptom (control/suppression).


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: BIOLOGICAL CONTROL OF IMPORTANT FUNGAL DISEASES OF POTATO AND RASPBERRY BY TWO BACILLUS VELEZENSIS STRAINS
Review round: 2
Reviewer: 1

Output:
Basic reporting: Clear and unambiguous, professional English used throughout.
Experimental design: Research question well defined, relevant & meaningful.
Validity of the findings: Ok
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: BIOLOGICAL CONTROL OF IMPORTANT FUNGAL DISEASES OF POTATO AND RASPBERRY BY TWO BACILLUS VELEZENSIS STRAINS
Review round: 2
Reviewer: 2

Output:
Basic reporting: In general, the authors answered accordingly to the requests made during the review. However, I have still a few comments presented here.
Experimental design: Methods are now described with more details.
Validity of the findings: The findings are valid and contribute to the research field.
Additional comments: In general, the authors answered accordingly to the requests made during the review. However, I have still a few comments:
1.	I do believe that the text would benefit after being corrected by a native English specialized professional.
2.	The authors mention in the abstract that “for the first time, they demonstrated B. velezensis strains potential for use as biological control agents against R. solani on potato”; in the introduction section that “no other research concerning biocontrol of R. solani in potato”; and in conclusion section “For the first time, we demonstrated B. velezensis strains potential for use as biological control agents against R. solani on potato”. All these statements have to be minimized. There are other studies already published demonstrating the effect of Bacillus velezensis against R. solani on potato. Only one example: Cui et al. (2020), Biological Control 141, 104156.
3.	Table 4. Effect of B. subtilis strains on morphometric characteristics – although you stated that the title has been corrected, Bacillus subtilis is already in the title.
4.	Note in Table 6 – please correct.
5.	Figure 1 – title should be presented in English
6.	Charon-Lamoureux V, Thérien M, Konk A, Beauregard PB. 2020. Bacillus subtilis and Bacillus velezensis population dynamic and quantification of 2 spores after… correct: quantification of spores


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: DEVELOPMENT AND VALIDATION OF FERROPTOSIS-RELATED LNCRNAS SIGNATURE FOR HEPATOCELLULAR CARCINOMA
Review round: 1
Reviewer: 1

Output:
Basic reporting: Overall, the manuscript is well written. However, there are few minor issues that should be corrected

1. 1.Line 53: substitute “Immune checkpoint inhibitors and Kinase” by “Immune checkpoint inhibitors and kinase inhibitors” Do this if you refer also to inhibitors of kinases. Otherwise, the term “kinase” alone does not make sense.
2. Lines 65 - 66: substitute “defined as an autonomous” by “are autonomous”.
3. Lines 235 - 236: “Tumor immunotherapy utilizing ICB had gradually become a promising strategy for therapy of advanced HCC”. Please, add a proper reference supporting this statement
4. Lines 270 - 271: “ICB immunotherapy acted as emerging strategies has revealed treatment effects in HCC and other cancers”. Please, rephrase the sentence for a better understanding.
5. Line 293: Substitute “PART1 involved in cell migration and invasion” by “PART1 is involved in cell migration and invasion”
6. Line 299: substitute “possessing” “by possess”
Experimental design: No comment
Validity of the findings: Did the authors assess the probable influence of confounding factors like other comorbidities or alcohol consumption on both the robustness and accuracy of the FRlncRNAs signature?  I consider that this should be assessed by virtue that other non - cancer liver diseases are also related with ferroptosis such as acute liver failure, alcoholic liver disease, NAFLD and immune-mediated hepatitis [Mao et al. Cell Death Dis. 2020;11(7):518]. Indeed, NAFLD has been related also with diabetes, insulin resistance, obesity, and overweight. Thus, the influence of these morbidities like confounding factors may be also tested.
Additional comments: It was found that the FRncRNAs signature may be a predictor of the overall survival of HCC patients; the higher the expression of FRncRNAs, the lower survival. Indeed, the score seems to be useful to measure the success of treatment with ICI. On the other hand, HCC can be resistant to conventional chemotherapeutic due to induction of ferroptosis resistance [Galmiche A. (2019) Ferroptosis in Liver Disease. In: Tang D. (eds) Ferroptosis in Health and Disease. Springer]. Does the FRncRNAs signature may be used to predict the resistance to classical chemotherapy via ferroptosis -induction? I wonder if ferroptosis may be easier to induce with conventional chemotherapy in patients with a higher FRncRNAs signature (i.e., more probability of liver ferroptosis induction) than in those with a lower one (i.e., lower probability of liver ferroptosis induction). I think that this may be an important question to address since ICI therapy is way expensive and has the risk of immune-related adverse events [Galle et al. J. Hepatol. 2018;69:182–236]. Please, discuss this possibility.


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: DEVELOPMENT AND VALIDATION OF FERROPTOSIS-RELATED LNCRNAS SIGNATURE FOR HEPATOCELLULAR CARCINOMA
Review round: 1
Reviewer: 2

Output:
Basic reporting: The paper has some grammatical errors
Experimental design: No comment
Validity of the findings: no comment
Additional comments: This study is aided to establish a ferroptosis-related lncRNAs (FRlncRNAs) signature in predicting the prognosis of patients with HCC. The work was well designed and written and the conclusions offered are consistent with the results. Although the work has several remaining issues as stated by the authors, it contains very valuable information related to FRncRNAs and its use as a signature of the patients with HCC, which could be used as a powerful tool in predicting the prognosis of patients with this type of cancer.

Minor points:

1.- Line 36, please add “overall survival” before “OS”.
2.- Line 139, change “were positive” with “had positive”.
3.- Line 222, please write “was implicated”.
4.- Line 247, delete “had”, in the same line, change “played” to “play”.
5.- Line 250, change “participated” to “participate”.
6.- Line 255, change “had” by “have”.
7.- Line 260, please change “this study existed several shortages” with “that study had several limitations”.
8.- Line 264, please remove “in”.
9.- Line 268 please change “might be served” with “might serve”.
10.- Line 270-271, please rewrite “acted as emerging strategies has revealed” to “emerged as a strategy to reveal”.
11.- Line 272, change “had” to “has”
12.- Line 273, change “response” to “respond”.
13.- Line 278, change “possessed” to “have”.
14.- Line 279, please rewrite “roles for measurement in response to ICB therapy” to “to be used to measure the response to ICB therapy”.
15.- Line 281, change “implied” to “implies”.
16.- Line 285, please change “has” to “have”.
17.- Line 292, change “had” to “has”.
18.- Line 299, change “possessing” to “possess”.
19.- Figure 8, panels A-F, a break in axis “X” at the Risk score value of 5 would help to amplify the plots to better appreciate the tendency of the data.
20.- Figure 6, please exchange the labels “D” and “C” in the panels.


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: DEVELOPMENT AND VALIDATION OF FERROPTOSIS-RELATED LNCRNAS SIGNATURE FOR HEPATOCELLULAR CARCINOMA
Review round: 1
Reviewer: 3

Output:
Basic reporting: no comment
Experimental design: 1. the AUC of risk score in Figure 3B is different from that in figure 3D. Please improve it.
2. In multivariate Cox regression analysis, should be adjusted  some risk factors, including family history, and Hepatitis C/B, which can be also obtained from the TCGA database.
3. Calibration curves should be consisted with the nomogram of 1-, 3-, and 5-year survival.
4. The cut-off of validation cohort should be provided reasonably .
5. The AUC of risk score for one, two, three in validation cohort. Please provide the AUC for one, three, five years.
6. Authors should investigate the effect of cross-talk between the risk scores and immune checkpoint genes on the prognosis.
7. The role and mechanism of ferroptosis and the ferroptosis-related genes in development and prognosis of HCC should be discussed.
Validity of the findings: please see 2.Experimental design.
Additional comments: MAJOR REVISIONS


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

Input:
Title: NCOV2019: AN R PACKAGE FOR STUDYING THE COVID-19 CORONAVIRUS PANDEMIC
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: Overall, the flow of the paper is easy to follow and the work is very meaningful. I have a few specific comments:

Title: "An analytics tool ..." The wording "analytics tool" is a bit unclear. After reading the paper, my understanding is that nCov2019 is an R package that provides historical covid infection data and visualization. It also provides some forecast utility. I suggest the authors consider making the title more precise.

Abstract Line 30: "Although there are a large number of public tools that provide statistics data" (Line 46): "Although there are many tools and web services
provide epidemic data, there are still fewer tools provide historical data for retrospective
analysis"
-> I wonder if authors can provide more context on what data is available/easily accesible before this work and what is not available/easily accessible. Some references will be extremely helpful. By providing more context of the existing public tools, the contribution of this paper will be clearer and more appreciated. Authors should provide more details about what is missing in the current tools such as refs 3,10.


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: NCOV2019: AN R PACKAGE FOR STUDYING THE COVID-19 CORONAVIRUS PANDEMIC
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript by Tianzhi Wu et al “nCov2019: An analytics tool for studying the COVID-19 coronavirus pandemic” present  nCov2019 an R package and a Shiny application for data visualization and analysis to follow the SARS-CoV-2 epidemic at country-level. Data analysis and visualization is an essential tool for exploring and communicating findings in medical research. I find the shiny web application intuitive and responsive.

Major comments:

My main concern is related to method and discussion section:

In lines 139 to 144, the authors explain which data and statistics are included in the app but no definitions, details or formula are provided neither epidemiological jargon is used. For example, according to Rothman et al [Rothman K, Greenland S. Modern epidemiology. Philadelphia, PA: Lippincott-Raven Publishers; 1998.] the epidemiological term for “Mortality rate was calculated by dividing total deaths by total confirmed case” should be the case fatality rate. Please, add a definition for each term, specify which graphs are provided, and use epidemiologic terms.

In lines 145 to 150, the authors explain the forecast module. Statistical details on this module are scarce and mixed with results. Please, specify statistical details about the model and parameters used for forecasting. If authors have performed validation on models with data from Italy, the validation method should be explained in the Methods section and the results of this validation in the Results section. The use of data only from Italy for validation should be justified. Moreover, verify citation format and think if it's redundant to cite the paper and the web page of the paper's author.

In figure 2 the nCOV2019 dashboard is presented. However, the image does not coincide with the linked app in the manuscript (http://www.bcloud.org/e/). Please, verify and correct.

Author’s vignette (https://guangchuangyu.github.io/nCov2019/) on nCov2019 package is full of interesting details and good examples. The authors could include the vignette as supplementary material.

On line 72, 114 and 180, the authors defend that they provide data to establish predictive models. In this reviewer's opinion, this statement should be lowered. One thing we have learned after 9 months is the complexity of this epidemic. At present, we can estimate incidence trends but we are far from having reliable predicting models. Forecasting of a pandemic can only be done based on a wide variety of parameters such as transmission rate, incubation period, the number of carries (symptomatic or asymptomatic), quarantine impact, and many other environmental and social factors.

The discussion should be expanded in terms of the strengths and limitations of the provided analytic tool. Moreover, the authors mention some examples of worldwide applications of the COVID-19 epidemic but do not provide a comparison. It would be valuable to show the differences between them to provide a comparison and emphasize the main contribution of the present work.

Figure 1, 2 and 3: Figures are blurred. Please provide a better quality of the graph.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Minor comments:

Introduction
On lines, 55 to 57 authors presented a list of top countries in relation to the number of confirmed cases. A rate, cases divided by population, would result in a fairer comparison. Otherwise, you are only reporting the number of cases of the highest populated countries.

In line 63, CDC is mentioned for the first time. Please, provide what the CDC acronym stands for.

In line 64, the authors mention “and most of them are presented using online dashboards”. Please, could you cite and explain the most relevant examples of these online dashboards from WHO or from an specific country?

On lines 66 to 70, in relation to what is convenient to analyze of this epidemic, the authors said: “Thus, more specific historical data is needed. However these data are not well obtained at present, as many data sources only provide real-time aggregated data. There is still a lack of clean, easily accessible historical data source and analysis tool”. I agree with this point. However, I miss a proposal on how data should be.  Please, could you provide, in your point of view, how should this data be? You can focus your explanation on a specific country or in a specific type of epidemiological analysis.
Results
On line 153 “According to data calculations from the nCov2019 package, as of August 10th,” and in Figure 3 data is from October 10th. Please clarify.

On line 161 “In these 43 countries, case 161 dead rate ranges from 0.17% (219/127778) in Qatar to 10.34% (36140/349494) in Italy”. According to Rothman et al the epidemiological term should be case fatality rate.

On line 164 “By exploring the latest growth rate, we found that although the United States, India and 165 Brazil have received public attention due to the huge total number of confirmed cases”. A date is necessary to contextualize. In a few weeks, this statement could false. Moreover, authors should explain how they have estimated the latest growth rate. The explanatory sentence on line 144 on the methods section seems to cut off.

Discussion
On line 176 “There are currently lots of data sources 3,10”. References are about online interactive dashboards one from WHO and another from Johns Hopkins University. There are many open data sources, most listed on this web page from the National Institute of Health –United States (https://datascience.nih.gov/covid-19-open-access-resources). And several online interactive dashboards such as those cited or: https://covid19.healthdata.org/, https://ubidi.shinyapps.io/covid19world/.

On line 184 “Our package was collected in several 185 resource list and served as backend for several plotforms”. Please, correct typo on platforms. Moreover, reference 11 on “Coronavirus Tracker. https://waiter.john-coene.com” is not about an online interactive dashboard. Please, correct the link or delete the reference.