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: EFFECT OF UNDERWATER VISUAL SURVEY METHODOLOGY ON BIAS AND PRECISION OF FISH COUNTS: A SIMULATION APPROACH
Review round: 1
Reviewer: 1

Output:
Basic reporting: The paper will be a very good contribution on the topic once some of the roughness in model description, interpretation of results, and general language is dealt with. I found the description of model parameters to be extremely lacking and it took several reads to sort things out.  The first three paragraphs of discussion do not touch on your results in any sense.  I found this odd as it should be the showcase.  Not some long discussion that really reads like an introduction.  I want to know what it is you found that should captivate me as a reader, and then get into the literature supporting it.  Structurally I was searching for the point until I hit paragraph four when you actually start talking about what your model performance.

In my mind this is a Gerritsen-Strickler encounter probability model and the diver speed is just confounding within the area sampled issue.  The way point samplers deal with this is by using habitat based designs and ramping up sample to collect increase information over area. I think this needs to be dealt with in paragraph one of your discussion.  It’s the main point of your results and to me it’s pretty clear in your results. The explanation of the model results in the discussion really needs to be rethought and reorganized because it doesn’t flow as written and I struggled making sense of what you believe were the most important outcomes.
Experimental design: The fundamental design is fine.  The execution of describing the model is lacking.

For instance there are few biological or mathematical descriptions of fish traits.  What does diver avoidance = 10 actually mean?  I have no idea, is this a function of some kind?  I can’t decipher how this manifests in behavior in the model?  What is patch gathering?  What is centre?

You describe some pretty common ‘behaviors’ such as schooling or cryptic but you don’t actually tell the reader how that is modeled.  For instance schools really act as a single individual in space, but from what I could tell you’re modeling individuals to simulate a density of 0.3 per meter.  Are you simply distributing the school spatially?  Do they aggregate in the model?  Schools by definition are extremely patchy because all the individuals occur in one sighting.  At one point you state that abundance apparently does not affect bias.  Yet when I think about counting a school of a thousand fish as a diver, with no permanent record, all I can think of is bias.  Memory in the sense of what you modeled should dramatically reduce the ability of a diver to detect ‘individuals’ in a school and bias these counts severely.  Yet I don’t understand how that’s handled because don’t know how individuals in schools move relative to one another, how they’re actually spatially distributed on your grid, and how that might manifest itself in ‘counting and density errors’ in your model.

Units are difficult to sort out relative to time-step, fish movement speed, cell size, and count-memory.  I have no idea how the perception distance, perception angles etc are actually used in the model to understand how those may impact model results (i.e. fish response to vehicle is not really described other than to say that you have some parameters).  This all needs better description, not just redirection to another paper or effort somewhere else.  It’s not the readers job to hunt down supporting arguments if those can’t be easily explained within the context of a stand-alone effort.
Validity of the findings: I don’t really understand the estimation of density on the point count census.  I understand it from a transect point of view because its distance.  However you state the for the point count, the thing is rotating, but do not describe if the swept area of that cube is additive or static?  More importantly, in either sampling case, time is confounded with area and speed.  You don’t really address this in a straight forward way.  Think about the stationary point count, as an extremely slow vehicle in which area basically doesn’t change and yet survey time is increased incrementally.  Thus encounter probability in the volume is increasing, yet area/volume is static.  Nobody in their right mind should be deriving densities in this manner from empirical data, specifically for highly mobile species.  You’ve got that correct in here.  Now think about the moving diver case.  When you increase speed and decrease distance all you’ve really done is decrease encounter probability.  Vice versa, decrease speed and increase transect length, you’ve increased encounter probability.  Hence time is confounded within the transect information.  Whereas you explicitly deal with it on the point count.  In my mind this is a Gerritsen-Strickler encounter probability model and the diver speed issue is confounded with the area issue.  The way point samplers deal with this is by using habitat based designs and ramping up sample (i.e. get data over more area).  I think this needs to be dealt with in paragraph one of your discussion.  It’s the main point of your results and its pretty clear in your results (heat plots).

Line 253.  I think this is critical and needs to be in the results or somehow highlighted.  Don’t think it’s enough to just call it bias.  I like the heat plots, but maybe you can incorporate color to determine positive/negative bias results?  Or 3 dimensional.

Line 254-255.  Maybe that’s the case in the literature but I would not have assumed that these were overestimates at all, particularly in the case of mobile schooling species with strong ‘boldness’ factors.

Line 285-287.  No idea what this actually means.

Line 301.  You approach the detection probability relative to movement of the sampler/diver (i.e. non-instantaneous sampling) but don’t put it into a single package.  A stationary point count, is just a really slowly executed transect.  The more you increase time, the higher the encounter probability gets and the worse the estimate.  Transects are just confounded with speed and distance.

Line 307-308.  Fits with just what I said, but in this case you increase the area and thus relieved some of the assumptions about encounter probability all of which is confounded by the fish movement.

All of this doesn’t really deal with attraction and avoidance, where there is active ‘seeking’ behavior where divers are trailed or avoided.  It’s sort of dealt with as shy or bold in approach distance, but that doesn’t really describe the behavior of fishes that follow (e.g. barracudas).
Additional comments: I definitely believe this is an effective model and deserving of publication but not in it's current state.  Model description needs to be sorted out so that the interactions dealt with above can be sorted out by the reader.  Otherwise it's sort of a mixed bag of things that take a lot of effort by the reader to understand the point.


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: EFFECT OF UNDERWATER VISUAL SURVEY METHODOLOGY ON BIAS AND PRECISION OF FISH COUNTS: A SIMULATION APPROACH
Review round: 1
Reviewer: 2

Output:
Basic reporting: The introduction was very clear and laid out the problem with designing surveys for reef fish very well. The literature appeared relevant. The structure of the paper followed normal patterns. I may have missed it, but there didn’t appear to be any raw data provided? The model parameters for the simulations are provided. The tables were informative and followed from the text. A couple of comments on the figure.
1)	The manuscript would be improved by showing some of the relationships in graphical form that are observed in the S1 and S2 tables. Patterns in relationships among variables using the raw simulation results would be nice to see here. I suggest adding some multi-panel figures that show some of the interesting results from the bias analyses, at least as additional supplementary material.
2)	The lone figure is difficult to interpret. I would recommend splitting this figure by species type and showing the results for each type in a single figure (with CV and bias). This would greatly simplify the information and make it easier to follow the patterns being described in the results.
3)	Could the authors present an additional table with the recommended (based on the simulation) sample design and the most attainable values for bias and CV? The current Table 4 is interesting in that the values for bias and precision on average are quite high when combined and likely none of them (on average) would be a sutiable level of bias or CV. It would be more informative to present the best version of survey design and the likely bias and CV.
Experimental design: The research seems appropriate for PeerJ. The research question is well defined and survey design for underwater visual surveys is has a number of knowledge gaps for which this simulation provides a nice tool to examine. The experimental design seems appropriate for the simulations, the authors do a nice job of presenting information in to back their choice of species groups/parameter combinations. Two main comments here
1)	It is not quite clear where the choice of density of fish was derived from. Also, I couldn’t tell if this was the overall density of fishes across all 4 groups or whether this was the density of each fish group.
2)	There could be few more details provided on the mechanics of the simulation model. For example, the reader has to refer to the original contribution to understand where the stochasticity in the simulation came from.
Validity of the findings: The simulations presented in this manuscript appear to be robust and represent the natural variability in conditions the authors have observed in reef fish. As the authors recognize in the discussion, the specific results of this simulation are relevant to the characteristics of the fishes they have specified parameters for. The value of this paper is to demonstrate how the simulation model can be used to explore other types of fishes with other characteristics. As such it is novel, but the more novel contribution would be in the original paper that set out the simulation (i.e. Pais and Cabral 2017). It also seems that a fair amount of work has been done previously on these questions, but the previous results are well integrated into this manauscript. The conclusions that the authors reach appear logical given the data and simulations presented and the data seem robust and statistically sound.
Additional comments: I enjoyed reading this paper, it was an interesting look at a simulation model for an important survey question and the methods here could be applied elsewhere.
1)	The abstract is a bit confusing to the reader that has not read the paper. It was not initially clear to me what “bold” and “sly” fish were or what a “slow swim” referred to. Probably because I was not aware the authors were talking about diver surveys until reading the introduction (as there are other types of underwater visual surveys). I would suggest defining this in the abstract, as well as some of the other terminology that appears in the abstract, such as by saying “bold fish (fish attracted to divers)”.
2)	It was interesting that a single density was chosen for all the simulations. It would seem from other studies of this type that the true density has a large effect on CV and potentially bias (for rarer species). Also, some of these species groups are likely to have very different underlying densities on reefs. For example, pelagic schoolers might be an order of magnitude more dense than cryptic species. I would be interested to hear a bit more about justification of this number across the different species groups.


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: EFFECT OF UNDERWATER VISUAL SURVEY METHODOLOGY ON BIAS AND PRECISION OF FISH COUNTS: A SIMULATION APPROACH
Review round: 2
Reviewer: 1

Output:
Basic reporting: The revisions and supplemental material have addressed my primary concerns about the basic reporting. The figure is still difficult to interpret.
Experimental design: The supplemental text in the manuscript and supplemental material has improved the description of the experimental design.
Validity of the findings: The improvements to the manuscript have addressed any concerns about the validity of the findings.
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: MORE OF THE SAME: ALLOPATRIC HUMPBACK WHALE POPULATIONS SHARE ACOUSTIC REPERTOIRE
Review round: 1
Reviewer: 1

Output:
Basic reporting: Very well written, with only a few general comments:

Line 56: Perhaps define pinniped and cetacean upon initial use as this journal is aimed at a general audience.

Line 75: Remove “and”, and start a new sentence with “Further”.

Lines 120-127: This is slightly confusing. Perhaps note that Frederick Sound is in SEAK and is where the 1976 recordings were made and not just have that information in the figure alone.

Line 165: For the reader that is not versed in acoustics, it might be helpful to provide the call types that fit into the category of harmonic sounds vs pulsed sounds here as well as Table 3. I find it helpful to read short descriptions of calls as well as looking at spectrograms. I understand there might be word limit or other constraints, but it is nice to know additional information about these calls if it is available (always given in bouts? ever variable?)

Line 192: Remove the word “with”.

Line 221: Should be “pitched” not “pitch”.

Line 258: See also Parks et al. 2016 "Noise impacts on social sound production by foraging humpback whales" for a more recent citation.

Line 308: Insert the word “the” between “understanding” and “role”.

Figure 2:  The x axis label (Time) is not centered, and the x axes on each separate panel are a bit confusing - two separate calls should have separate time tick marks. The headers "Pacific" and "Atlantic" are not centered above their spectrograms. It seems "teepee" should be plural if "swops" is. In the actual uploaded figure file, the axes are not present.

Figure 3: It would be nice to have significance noted on the figure with asterisks, perhaps next to the call type name. Also, again, if "swops" is plural, "teepees" should be as well.

Figure 4: Same regarding “teepees” being plural.

Table 1: Some of the rows in this column don't line up well with the rows in the last three columns - they are just slightly off enough to disrupt the flow while reading. This is likely due to the words being centered in the cell. Consider adding grid lines to facilitate reading.The words “unknown” and “unavailable” should be capitalized for consistency with the rest of the table.

Table 2: Labelled incorrectly in the caption (labelled as Table 1 again). Also, PC2 should be defined as “rotated” principal component, rather than “rotate” principal component.

Table 3: The broad call type classification (harmonic vs pulsed) is not really discussed elsewhere in the text. I like to see broad categories, as often call types are often graded and not always as well split with CART and RF as they are here. I would suggest perhaps adding this briefly to the main text.

Table 4: Capitalize “Regression” and “Tree” in the caption.
Experimental design: Line 169: I appreciate citations in methods for brevity, however this reference is for your thesis which is not yet accessible. Is it possible to include either an additional reference to work you have already published, or to provide some information on the quantitative classification methods used here?

Line 171: Could you please provide the package information? Additionally, it would be helpful to provide a brief explanation of what each of these analyses offers statistically, and more specifically, why you chose to use each.

Line 188: Why did you use an older version of R? Does this mean that the packages used were also older versions? Packages can sometimes be updated to reflect advances in statistical methods.
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: MORE OF THE SAME: ALLOPATRIC HUMPBACK WHALE POPULATIONS SHARE ACOUSTIC REPERTOIRE
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comments
Validity of the findings: no comments
Additional comments: The authors studied two allopatric humpback whale populations (in the North Pacific Ocean and North Atlantic Ocean) and showed that both populations shared five call types, which provides evidence that these call types may be innate. The spectrograms in Fig. 2 showed that both populations used call types with a very similar structure. But their analytical approaches are more confusing than helpful (see below).
1) General question (L50-64): “What is less common among mammalian vocal learners, however, is the coupling of stable sound types, which may be innate, with a dynamically changing repertoire of sound types whose variation appears to be culturally driven.”
That is not true. Similar like birds, mammalian vocal learner have a rich repertoire of innate calls. This is true for mice (vocal learners?), pinnipeds, bats or elephants. It is less clear for some cetacean species because most studies were done to emphasize their vocal flexibility. Insofar this is an interesting study but it is wrong to say that it would be surprising that mammalian vocal learner have innate calls.
2) It remains unclear how the calls from the North Pacific recorded in 1976 differ from the 2007 recordings and whether this has an influence on the results.
3) L. 147-49: “Frequency parameters were log-transformed to account for the mammalian perception of pitch, which is approximately logarithmic rather than …”.
If such a transformation is necessary at all, the authors should use a transformation related to the pitch perception of humpback whales. Mammalian pitch perception could be very different.
4) L.170-73: The validity of forest analysis remained unclear, especially in combination with an additional observer validation. I think it would be more objective to use just the assignment result of this analysis, or any other procedure which are able to compare the similarity of two samples (population).
5) Table 2:
Aggregate entropy is under such conditions not a measure of total disorder in the call, because this measure is mainly influenced by recording conditions, like distance of caller, different equipment, etc. I think it makes no sense to use a variable which is mainly influenced by the unknown distance of the signaler.
6) Results in Table 3:
The authors have no knowledge about the number of subjects. Therefore the use of Kruskal-Wallis test or similar tests is incorrect. There are possibilities to deal with such data sets (e.g. Vester et al. 2016 Physical Review). In addition, it remains unclear what the authors like to show with these tests. There are high significant p-values. Should this mean that both populations differ in their call types?
6) L-197-98: “PCA output indicated that the use of two principal components was adequate to encompass the variability of the data (χ2= 683.29, p < 0.00001).”
It remains unclear what this result should tell. And why did the authors use in addition to the two PCs also the other acoustic variables? They just showed that the two principle components were adequate to encompass all the variability.
7) L. 210-12: “The variables most important for splitting decisions in the random forest analysis were bout, end frequency, duration, entropy, lower frequency, PC1, PC2, and frequency trend.”
Again, why using both PCs the other acoustic variables. What means most important in case nearly all acoustic variables a most important?
8) L. 213-14:
What is the value of this mixture between analytical assignment (CART) and observer validated assignment. The call type categories were established by observer criteria. Therefore one should be able to achieve an agreement between different observers, especially by omitting critical calls.


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: MORE OF THE SAME: ALLOPATRIC HUMPBACK WHALE POPULATIONS SHARE ACOUSTIC REPERTOIRE
Review round: 2
Reviewer: 1

Output:
Basic reporting: Resubmission.
Experimental design: Resubmission.
Validity of the findings: Resubmission.
Additional comments: The authors gave answers to all my comments, but ignored my suggestions. Therefore I cannot really say that the manuscript has improved. But as I mentioned in my first review, the conclusions are supported by the results. Humpback whales of the North Pacific Ocean share call types with conspecifics in the North Atlantic Ocean.
But I do not agree with following explanations:
1) L 154-57: “...; all recordings were made with 16 bit resolution; once resampled recording were technically comparable in all aspects.”
Just use the same amplitude accuracy to resample the recordings does not mean to have technically comparable recordings. The recordings remain different in relation to recording equipment and conditions.
2) L 234-47; Wilcoxon rank-sum test:
I do not see a good reason why the authors used this test. They do not know their real sample size; therefore it is not possible to use this test.

3)  L 259-60:
The classical description of the quality a factor analysis is KMO, Bartlett's test, and the explained variance of the factors. A p-value of 0.00001 is not very informative. It would be difficult to produce a non-significant factor solutions.


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: BTW—BIOINFORMATICS THROUGH WINDOWS: AN EASY-TO-INSTALL PACKAGE TO ANALYZE MARKER GENE DATA
Review round: 1
Reviewer: 1

Output:
Basic reporting: The language used is fine.
The MS is well structured - apart from a clear hypothesis-support/rejection approach.
It should more clearly (in the Abstract) and earlier (in the main text) stated that "bioinformatics" so far only refers to a comparatively narrow window of application.
Experimental design: It is a valuable endeavor to facilitate NGS data analysis for Windows users.
Just one single comparison of performance appears to me insufficent.
As an Ubuntu cluster user I cannot judge the usability for the target Group.
Validity of the findings: The single test outcome seems to be plausible, but see above.
Additional comments: As much as I appreciate the attempt of broadening the application of NGS data analysis, it remains unclear who is intended to read the article. For newcomers to the field, you would have to get into much more detail. Mentioning the actual programs in the figure would not be enough.


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: BTW—BIOINFORMATICS THROUGH WINDOWS: AN EASY-TO-INSTALL PACKAGE TO ANALYZE MARKER GENE DATA
Review round: 1
Reviewer: 2

Output:
Basic reporting: see below
Experimental design: see below
Validity of the findings: see below
Additional comments: In this manuscript, the authors introduce a software package which allows users of the Windows operating system to run a variety of bioinformatics analysis. They compare its performance to native Linux-based software and find it to be similar.
I think the manuscript is well written and the presented software has the potential to be useful for a wide audience. I have one major concern though, which is the targeted audience for this paper: If it is aimed mainly at complete bioinformatics and Linux environment beginners, then it lacks a comprehensive introduction of the field of bioinformatics, in particular why it is simply not possible to continue using a standard desktop computer for most high-throughput sequencing analysis. Even people familiar with microsatellite or Sanger sequencing data often think they can just continue with the tools they’re used to. So in my opinion it is important to highlight and illustrate the main feature of these data - hundreds of thousands to millions of reads – which is the reason for why it can’t be analyzed manually or visually (like for example Sanger data).
In addition it would be helpful if the used example (16S rRNA analysis pipeline) would be described / explained in more detail and without jargon. Again, for bioinformatics newcomers, many of the terms and steps might be completely unknown.
People already familiar with bioinformatics might still find the software helpful for teaching purposes, but also then a more extensive introduction is needed.

So in my opinion, to be the intended guide towards bioinformatics, a more comprehensive introduction and illustrative examples should be added to the manuscript.

Detailed line-by-line comments:

Line 30: ‘…is a Bash script and is available on GitHub.’  For people not familiar with the Linux / Bioinformatics environment this sentence might already be too cryptic.

Line 49: ‘… is typically developed for Unix Shell…’  This might be a good starting point to explain why the software is typically developed for Unix shell (and not point-and-click GUI)

Line 52: It is debatable whether a graphic interface is more user-friendly than a command line interface. (Just a comment)

Line 73: ‘Running a complete 16s rRNA data analysis pipeline…’  Please explain this pipeline and also the biological background in more detail, since it might not be familiar to all readers.

Line 82: ‘…using the RDP classifier…’ Please explain abbreviation.

Line 96: ‘…and 32 GB of RAM…’ This amount of RAM is quite above the average of a standard desktop computer, so readers might ask themselves why so much is required.

Line 113: ‘…of having avoided that most daunting first contact barrier…’ To me it is not entirely clear what that barrier is. Is it the need to switch the operating system? Because performing different analysis in BTW still requires using the command line interface right?
-


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

Input:
Title: BTW—BIOINFORMATICS THROUGH WINDOWS: AN EASY-TO-INSTALL PACKAGE TO ANALYZE MARKER GENE DATA
Review round: 1
Reviewer: 3

Output:
Basic reporting: no comment
Experimental design: The script 'peerj-24722-Illuminabash.sh' should be documented in the same fashion as the
'win_bmp.sh' script provided in GitHub repo.
The same about the installation procedures. A README file containing all the instructions is missing on the github repository.
I strongly suggest to the authors do make a PDF file containing all the instructions available in the website (http://www.brmicrobiome.org/tutorialbtw and http://www.brmicrobiome.org/win16s), and make it available on the package provided in the Github repo, and also including as supplementary material of this manuscript.
This will guarantee that the describe methods are with sufficient detail and information to replicate.
Validity of the findings: -most important issue
On lines 106 to 109, the authors state that:
"Furthermore, WSL is still in the beta phase, meaning that some Bash scripts and
tools currently used for bioinformatics will not work perfectly. For instance, we experienced fails [segmentation fault (core dumped)] while running QIIME Uclust-based commands and the USEARCH package (Edgar, 2010) which seems to be an issue related to the compilation process. "

The authors should propose a possible solution to overcome this potential issue until the final version of WSL be released.
Indeed this will attest the reliability of the BTC pipeline for everyone .
Additional comments: This manuscript describe a new pipeline designed for processing marker gene data, including 16S rRNA.
Although several efforts and tools were developed during the last years for this purpose, this may be the first fully automated pipeline and easy-to-install package as well as a complete pipeline for microbial metagenomic analysis for Windows operational system.
Therefore, this pipeline can be very useful for both students and professionals in biological sciences which generally struggles when facing the command-line interfaces.


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: BTW—BIOINFORMATICS THROUGH WINDOWS: AN EASY-TO-INSTALL PACKAGE TO ANALYZE MARKER GENE DATA
Review round: 2
Reviewer: 1

Output:
Basic reporting: -
Experimental design: -
Validity of the findings: -
Additional comments: I see that the authors sufficiently addressed the issues raised by the reviewers.
In the manual, they should not only ask for reference to their work and to the underlying programs, but should also provide the respective appropriate formulations ("Miller AB 2016 Super Program. Bioinformatics 3, 4-5.") including the program version used.


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: BTW—BIOINFORMATICS THROUGH WINDOWS: AN EASY-TO-INSTALL PACKAGE TO ANALYZE MARKER GENE DATA
Review round: 2
Reviewer: 2

Output:
Basic reporting: see below
Experimental design: see below
Validity of the findings: see below
Additional comments: The authors have addressed all my comments and concerns to my satisfaction and substantially improved the manuscript. Therefore I can now recommend it for publication.


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

Input:
Title: BTW—BIOINFORMATICS THROUGH WINDOWS: AN EASY-TO-INSTALL PACKAGE TO ANALYZE MARKER GENE DATA
Review round: 3
Reviewer: 1

Output:
Basic reporting: -
Experimental design: -
Validity of the findings: -
Additional comments: thanks for providing details on the underlying programs


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: BTW—BIOINFORMATICS THROUGH WINDOWS: AN EASY-TO-INSTALL PACKAGE TO ANALYZE MARKER GENE DATA
Review round: 3
Reviewer: 2

Output:
Basic reporting: see below
Experimental design: see below
Validity of the findings: see below
Additional comments: As previously stated, the authors made all suggested changes to the manuscript. Thus I can now recommend it for publication.


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

Input:
Title: BTW—BIOINFORMATICS THROUGH WINDOWS: AN EASY-TO-INSTALL PACKAGE TO ANALYZE MARKER GENE DATA
Review round: 3
Reviewer: 3

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: I am satisfied with the revised manuscript and response to my first comments.  I congratulate them on a valuable contribution.
I just need to point out to the authors a minor comment, to correct the typo on line 139 ("... supporting it at any time., On ...").


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

Input:
Title: THE VOLUNTARY CONTROL OF PILOERECTION
Review round: 1
Reviewer: 1

Output:
Basic reporting: This is a manuscript on voluntarily generated piloerection (VGP) and its self-reprted physiological and psychological correlates. It is the first broad study to investigate this phenomenon. The draft is clearly written and well structured. Please find corrections and suggestions for change below.

1) Although clearly written, the text contains several unexpected turns which are not easy to follow. For instance, after describing introspective abilities and physical self-regulation (lines 113-116) the authors write:  “In this tradition, the presence of piloerection during intense experiences has been shown to correlate strongly with reported emotional intensity […] (Benedek & Kaernbach, 2011, Wassiliwizky, Wagner, Jacobsen, & Menninghaus, 2015)…” The cited studies represent work on emotional induction using aesthetic stimuli. It is not clear why this research is in the tradition of the aforementioned work. Similarly, after reviewing a study on drug administration (lines 151-154) the authors proceed: “No hypotheses were formulated for other personality domains”, which doesn’t seem to be related to the previous.

2) Several relevant studies on piloerection are not mentioned or discussed in the manuscript. The authors state “there is an autonomic mechanism that to our knowledge has never been systematically investigated – piloerection”. However, several studies have done that, using objective approaches to measures piloerection: Craig (2005); Benedek & Kaernbach (2011); Kim, Seo, Cho (2014); Sumpf, Jentschke, Koelsch (2015); Wassiliwizky, Koelsch, Wagner, Jacobsen, Menninghaus (2017); Wassiliwizky, Jacobsen, Heinrich, Schneiderbauer, Menninghaus (2017). See also Schubert, Zickfeld, Seibt, Fiske (2016) for the link between goosebumps, tears and being moved and Nishida (1997) for animal piloerection in courtship behavior (missing in lines 419 et sqq: “Animals display piloerection as a functional social signal in both aggression and defence”).

3) In lines 148-151 the authors discuss the similarity between subjectively felt chills and objectively measured piloerection and conclude: “we may assume they are strongly related”. Two studies have looked into this issue empirically and come to different conclusions: Sumpf, Jentschke, Koelsch (2015) and Wassiliwizky, Koelsch, Wagner, Jacobsen, Menninghaus (2017).

4) Could the authors specify every time what kind of piloerection they are referring to? E.g. in line 116: “the presence of piloerection during intense experiences” should read “the presence of involuntary piloerection during intense experiences” or line 128-130 “he […] experienced [involuntary?] piloerection most of the time while watching football.”
Experimental design: no comment
Validity of the findings: 5) I find it particularly problematic that the student sample was excluded from further analyses because the participants did not comply with the VGP-criteria that were deduced from the online sample. Why are the answers from the online sample given more weight? One could argue that the online sample does not fulfill the criteria set by the student sample.

6) The authors do seemingly not correct for multiple comparisons (see lines 349-352 or line 367). This can be problematic in terms of alpha inflation. Either the p values have to be corrected for multiple testing or the authors could run a multivariate omnibus test instead of several t-tests.

7) The results show that people capable of VGP mostly associate it with psychological states of absorption (53.13%) and least with “being touched” and “like crying”.  In line 326 the authors state “VGP is associated with feelings congruent to experiences of involuntary piloerection”. However, I am not aware of any previous empirical work that links piloerection with absorption. On the contrary, several papers show a link to “being moved/touched” (Konecni, 2005/2007/2008; Benedek & Kaernbach, 2011, Wassiliwizky, Wagner, Jacobsen, Menninghaus, 2015; Seibt, Schubert, Zickfeld, Fiske, 2017). Taken together, this actually points in the direction of some differences between VGP and involuntary piloerection (rather than congruency).

8) The authors claim several times that VGP is an extremely rare phenomenon. However, in their study (pool 2) 120 out of 682 participants indicated to be able to self-induce VGP, which is about 18% of the sample!
Additional comments: minor points:

- Conceptual issue: One aspect in the introductory illustration (starting line 54) is not intuitive. The authors write “The second would most likely have normal reflexes […] and then subsequently a normal fear reaction […], but […] their heart remains steady, palms dry, and so on.” In most psychological accounts, the bodily component represents an essential part of the emotion. How can someone have a “normal fear reaction” without any physiological arousal? It is also surprising that the authors call the Schachter & Singer conceptualization of emotions (from 1962) a “modern account” (line 111).

- line 72: “nomos” is a singular form, please change the translation to “law”

- please explain what you mean by “sample of any size” (line 164) or change the wording.

- line 335: “we tested whether participants used their VGP abilities”; the more appropriate wording would be “we asked whether”


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

Input:
Title: THE VOLUNTARY CONTROL OF PILOERECTION
Review round: 1
Reviewer: 2

Output:
Basic reporting: Generally the reporting is fine except for a few issues raised below.
Experimental design: Generally the methods are fine except for a few issues raised below. The primary concern is that the authors overstate the meaning and implications of self-report results.
Validity of the findings: My main concern is the use of self-report measures. The authors need to reduce the strength of the claims they make.
Additional comments: 1. Tables
Table 1: Resp. time is unclear – I recommend abbreviating RT or spelling out Response.

Table 2: populations should be population, “and domain” is confusing here, consider rewriting for greater clarity. Confidence interval should be intervals. Define LL and UL – it seems these aren’t necessary.

2. Abstract
“physiological study in scientific history” seems a bit grandiose. I recommend just writing that only 3 previous cases have been reported to the best of your knowledge (this saves you from embarrassment in case some other cases are found …)

Rather than referring them as being “capable of VGP”, perhaps “report being capable of VGP”. It might also be valuable to state that their ability for VGP was formally documented/observed.

3. Introduction
Generally, I think the introduction could benefit from being more concise and having a clearer linear structure. “Non-invasive imaging …” – this sentence seems unnecessary. The second paragraph doesn’t seem to me valuable in the context of the introduction. The third paragraph seems unnecessarily long and there is repetition (eg. control of HR). The structure of the 4th paragraph needs to be edited to improve the flow. It seems strange to state that VGP provides insights into personality when you have yet to report any personality differences in those reporting VGP.

4. Method
“exclusion criterial was based” should be “exclusion criteria were based”

“Those who endorsed the question (n=120) were invited to participate in the study, twenty-two completed the survey”
What survey? This is unclear.

Qualtrix is misspelled.

The fact that you have 2 samples that were administered questionnaires is confusing. Please make this clearer in terms of what was done with each sample. How was the online sample contacted? How many participants started the survey and how many completed it? This should be clearer and an attrition rate should be clearly stated.

The low internal consistency for every subscale (the cut-off is typically .7) is a huge concern and thus I think it probably makes the most sense to ignore these data.

Please report the number of items and scoring format for all scales.

Results
It seems “Online sample” would be a more suitable label than “Pool 1”.

P18: willfully is a word and thus [sic] isn’t required.

Please report ranges and/or CIs for %s in the text.

The descriptions of these different features of VGP should include details regarding the range of the scale. Otherwise, it’s unclear (e.g. the question about effort).

How was was practice defined here? Participants who did the act regularly (because it’s interesting/enjoyable) may be classified as practicing even though they weren’t actually “practicing” per se. Please clarify.

The section at the beginning of Pool 2 (see my comment above), shouldn’t include bullet points – put this in a paragraph. Also, this looks more like a discussion so why not put it in the Discussion?

How do you know it’s not involuntary and they just incorrectly perceive it as voluntary? The fact that they don’t have complete control over it (eg side of body) and that it occurs under similar states as involuntary piloerection lends plausibility to this.

The absence of a control sample is a concern and should be addressed. The same goes for the self-selected nature of this sample.

Did you ask about frequency of involuntary piloerection? A comparison with a control group would be very valuable here.

Please report descriptive statistics associated with inferential statistics (eg Aesthetic experiences questionnaire).

I recommend having subsections in the text.

Please report effect sizes for all effects (eg Fig 1).

Have the authors corrected for multiple analyses? There are many analyses reported in Table 1; accordingly, I’m nervous about the “calm” effect (p=.049). If no correction was applied, I suggest applying one, such as false discovery rate correction.

Discussion
“sizable sample ever characterised who have a voluntary ability to produce goosebumps.”
“Having established that VGP exists as previously described …”

Insofar as none of this was physiologically corroborated and it’s all self-report, I think these and other sentences absolutely need to be greatly tempered. For the record, I believe this is a real phenomenon but I don’t find the evidence presented (all self-reports, questionnaires with poor reliability, all online participants, small sample size) to be particularly compelling.

“Third, if the conditioning hypothesis were true, it would result in a remarkably consistent phenomenon which centres around a deliberative point on the back of the head or neck.”
Please explain this – I don’t understand the logic.

“However, in humans, we assume the ability is vestigial and observe little interindividual variation in site (except in the case of specific medical conditions such as pilomotor seizures; Loddenkemper et al., 2004).”
Awkward phrasing, especially since the research team didn’t seem to author that paper. I don’t understand what you mean about variation in site here.

“This suggests that VGP-capable individuals may be able to voluntary elicit these emotional states.”
This is tricky as the authors (to my knowledge) didn’t probe how it was done. Were these individuals directly producing piloerection or were they producing affective states (eg awe) that trigger piloerection?

Absorption isn’t an emotion.

I would highlight that absorption is a predictor for a range of phenomena from anomalous experiences, drug-induced synaesthesia, hypnosis, mental health worries, etc.
https://www.ncbi.nlm.nih.gov/pubmed/29452127
https://www.ncbi.nlm.nih.gov/pubmed/28174078
https://www.ncbi.nlm.nih.gov/pubmed/28758796
https://www.ncbi.nlm.nih.gov/pubmed/24146659
https://www.ncbi.nlm.nih.gov/pubmed/15849873
It may be that individuals high in absorption have liminal boundaries for anomalous experiences and increased liminality confers greater control (as is the case with highly suggestible individuals in the context of hypnosis, for example) or at least the perception of control.

I would qualify the openness findings on the basis of the poor internal consistency of the measure.

Future directions
I think the first goal is to confirm this phenomenon in the lab under controlled conditions. I’m surprised that’s not listed first. Frankly, I think the authors are jumping a bit ahead.

Conclusion
“This study is the first to outline the ability to produce voluntary piloerection in a sample of more than a single individual.”
This is overstated – you don’t demonstrate this ability. You identify a small group of individuals who claim to have this ability. The same goes for all the characteristics (e.g., that it’s effortless) – it should be changed to clearly indicate that these individuals report these characteristics rather than that they are characteristics of this phenomenon. The same goes for the term “VGP-capable participants “


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

Input:
Title: THE VOLUNTARY CONTROL OF PILOERECTION
Review round: 1
Reviewer: 3

Output:
Basic reporting: This is an exploratory study investigating voluntarily generated piloerection. This is a rare and under-investigated phenomenon and this paper makes an important contribution toward better understanding both the characteristics of the phenomenon itself and traits that distinguish individuals capable of VGP. The article is clear and well written. The introduction successfully establishes the potential value of research in this area, and the findings and limitations of this work are fairly evaluated.
Experimental design: This is very much an exploratory study that focuses mainly on describing the features of VGP. The main formal analysis is comparison of a self identified sample of individuals capable of VGP with previously reported samples from past literature.
My main concern with this paper is the comparison of personality measures between pool 1 and previous data. I do not think it is appropriate to compare this data at both the level of domains and also individual items. In particular, conducting separate analyses for each individual item without correcting for multiple comparisons seems problematic. I note that line 439 acknowledges that reported differences at the item level would not survive correction. With this in mind I suggest omitting the item level analysis completely.
Validity of the findings: This study investigates a research area where almost nothing is known. As such the findings here, although primarily descriptive in nature are a valuable and important contribution to the literature.
Additional comments: There are no other major issues that need to be addressed in this paper. Below are some general comments and requests for further detail that the authors may optionally like to respond to.

- The first paragraph provides some examples of famous case studies that have progressed human neuroscience. There is also mention of two case studies (BG and AM) using non-invasive techniques, but there is no information provided as to the details of why these particular cases were important. I wonder if it would be possible to provide some very brief comments along these lines to more fully illustrate the specific ways that studying exceptional individuals can increase scientific understanding.

- Many of the references for studies showing control of processes that are typically considered autonomic are quite dated (lines 68-87). Is there a reason that recent research has not addressed these phenomena? Is this worthy of comment?

- Regarding lines 101-103, what does it mean to say that the arrector pilli are ‘smooth muscles’ without somatic control? For someone with limited knowledge of physiology this seems confusing.  What is the property of these specific muscles that implies that conscious control should not be possible? More detail here would help.

- Line 176: what are the congruent phenomena that were targeted as part of recruitment?

-Line 199: Qualtrics


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

Input:
Title: THE VOLUNTARY CONTROL OF PILOERECTION
Review round: 2
Reviewer: 1

Output:
Basic reporting: Due to the changes the manuscript greatly improved in clarity. I have no amendments to make.
Experimental design: The authors now deal explicitly with the problem of alpha inflation and other issues I brought up in my last review.
Validity of the findings: All standards of the journal are met.
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: THE VOLUNTARY CONTROL OF PILOERECTION
Review round: 2
Reviewer: 2

Output:
Basic reporting: I have no further issues here.
Experimental design: I have highlighted my main concerns in my previous review.
Validity of the findings: As long as the authors highlight the central limitations (self-report measures, small sample sizes, online samples, some measures with poor consistency, multiple comparisons problem) and temper any strong conclusions, I think the results are fine to present as an exploratory study.
Additional comments: I have no further 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: THE VOLUNTARY CONTROL OF PILOERECTION
Review round: 2
Reviewer: 3

Output:
Basic reporting: In my view the original submission already fulfilled the requirements of basic reporting, and this revision has further increased the clarity and readability of the manuscript.
Experimental design: I previously expressed some concerns about potential duplication of analyses but I find the authors justification reasonable and accept that they have taken a sensible exploratory approach.
Validity of the findings: The findings are valid, interesting and well explained.
Additional comments: This is an impressive revision that appears to address all of the concerns raised. The added details, particularly in response to comments from Reviewer #1, have provided helpful additional context and situated these findings nicely within previously literature.
I congratulate the authors on a creative and engaging treatment of a puzzling and most interesting phenomenon.


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: ATTENDANCE, ENGAGEMENT AND PERFORMANCE IN A MEDICAL SCHOOL CURRICULUM: EARLY FINDINGS FROM COMPETENCY-BASED PROGRESS TESTING IN A NEW MEDICAL SCHOOL CURRICULUM
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript is clear and professional in its use of English and structure. The background provides sufficient depth to evaluate how the manuscript fits into the broader field of knowledge. Although increasingly used in health professions schools, more research is needed into the best practices of flipped-classroom implementation. This paper contributes to this field in addressing the effects of student attendance with respect to performance.

Specific suggestions, in order of importance, the authors should consider in this section of the review include the following.

1.	Lines 12-13: It would be helpful to provide more detail with respect to the other parts of the ECE. For example, how long are the large group sessions? How long are the problem-solving sessions? When do the problem-solving sessions happen relative to the weekly large group sessions? What is the relationship between the preparatory materials, and the activities in the problem-solving session and in the weekly large group sessions? How do the problem-solving sessions differ from the large group sessions in terms of the student activities? What is the attendance requirement for the problem-solving sessions? In the large group sessions, are students assigned to fixed groups or do they self-select?
2.	Line 7 and elsewhere: According to instructions to author, “PeerJ uses the "Name. Year" style with an alphabetized reference list.” Thus, the superscripted numbers for the reference should be revised as per the instructions and the reference list alphabetized.
3.	Line 7: The abbreviation PBL in parentheses is provided but the abbreviation is not used in the text subsequently. It can be deleted.
Experimental design: The experimental design, the assignment of students to cohorts, and the statistical analyses were all appropriate.

Specific comments, in order of importance:
1.	Line 112: “Tables” should read “Table”
2.	Lines 115-117: The indicated p value, p<0.01, should read p>0.01 since there was no statistically significant interaction.
3.	Line 38: Did the IRAT count toward their grade in the ECE or was it just based on the progress test? How did the students who attended the sessions perform on the IRAT?
4.	Lines 45-46: The authors hypothesize on why students missed class? Did students complete an evaluation of the ECE at the end? If so, were there any comments which can provide insight into why students did not attend class? It may be interesting to conduct focus groups with students who were absent.
5.	Line 58: The authors state the locally developed exams were “roughly equivalent.” Some testing programs (e.g., ExamSoft) provide an overall difficulty for an exam based on past performance of the questions. It may be interesting to know whether the difficulty of each exam was comparable. This will not affect the overall results and conclusions but would offer a more declarative statement with respect to equivalency of the exams.
6.	Lines 89-90: The authors state, “students who had two or less absences.” Just to be clear, does this mean that the data for the students without any absences were grouped with those who had one or two absences?
Validity of the findings: The raw data were provided and the results of the analyses were logically presented. The discussion aligned well with the results presented.
The discussion section could be enhanced, however, by some additional discussion of the effects of flipped-classroom on student performance with speculation on why absence from these sessions could lead to poorer student performance. For example, Street, et al. (Med Sci Educ 2015; 25:35) provides evidence in support of enhanced learning in the flipped classroom. Results from other health professions may be germane including Physical Therapy 2001; 896, Acad Med. 2014; 89:236, Am J Pharmaceut Educ. 2014; 78, and Adv Physiol Educ 32013; 7:316. Additionally, since the journal allows for speculation as long as it is identified as such, it may be useful to the reader to briefly mention the socio-constructivist view of learning which argues that knowledge is socially constructed through interaction, engagement and participation and that, pedagogically, the flipped classroom creates these social learning opportunities. It could be speculated that students who decide not to come to class are missing out on this social learning opportunity. The work of Vygotsky is relevant here.
Additional comments: Curriculum reform is increasingly occurring in medical schools. As a result, there are consequences, some intended, some not, of such reform. Thus, it is critical that schools evaluate the effects of the curriculum change on student performance to examine the consequences. This paper is timely in that the authors summarize briefly their new curriculum, the Shared Discovery Curriculum (SDC), and the consequences of allowing students to self-determine whether or not to attend class. The authors provided substantiating evidence that attending class enhances student performance. Accordingly, the school has elected to make student attendance a requirement. This is a welcome contribution to the field and will help the faculty at other medical schools provide evidence-based support when requiring student attendance. It will be interesting to follow how this change affects performance and the students’ reaction to the requirement.


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: ATTENDANCE, ENGAGEMENT AND PERFORMANCE IN A MEDICAL SCHOOL CURRICULUM: EARLY FINDINGS FROM COMPETENCY-BASED PROGRESS TESTING IN A NEW MEDICAL SCHOOL CURRICULUM
Review round: 1
Reviewer: 2

Output:
Basic reporting: Backgroud:
Although authors describe about Early Clinical Experience (ECE), large group sessions and others about Shared Discovery Curriculum (SDC) in the text fairly in detail, it will be lot easier for readers to comprehend by showing the curriculum map.
Experimental design: Methods:
Description at lines 63-65 about the Progress Clinical Skills Examination (PCSE) needs more detail. Although they cite URL of the article by Gold et al., readers would like to know at least the number of the questions in the post encounter components.
Validity of the findings: Discussion:
	Please discuss about the conflicting results of the relationship between attendance and grades in the field of medical education in the previous researches.
	Authors address the limitations only about that this is not the experimental study. The data is the only one year is also the limitation of this study, although authors will not be able to accumulate the data because medical school’s decision to start requiring students attend the large group sessions.

Conclusions:
I believe that the conclusion to suggest causal relationship between large group sessions and students’ mastery of clinical applications of basic science knowledge is inappropriate.
Additional comments: The authors are to be congratulated on this piece of excellent work seeking the evidence for the newly introduced curriculum. This paper would be of interest for many medical educators. The methods are appropriate for this type of study. The paper is also clearly written and presented. However there are several minor points to be corrected.


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: ATTENDANCE, ENGAGEMENT AND PERFORMANCE IN A MEDICAL SCHOOL CURRICULUM: EARLY FINDINGS FROM COMPETENCY-BASED PROGRESS TESTING IN A NEW MEDICAL SCHOOL CURRICULUM
Review round: 2
Reviewer: 1

Output:
Basic reporting: No Comment
Experimental design: No Comment
Validity of the findings: No Comment
Additional comments: Thank you for the revisions to the manuscript. I am pleased you found the comments useful.


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: ATTENDANCE, ENGAGEMENT AND PERFORMANCE IN A MEDICAL SCHOOL CURRICULUM: EARLY FINDINGS FROM COMPETENCY-BASED PROGRESS TESTING IN A NEW MEDICAL SCHOOL CURRICULUM
Review round: 2
Reviewer: 2

Output:
Basic reporting: 'no comment'
Experimental design: 'no comment'
Validity of the findings: 'no comment'
Additional comments: Authors’ revisions are well accepted. This paper would be of interest for the readers in the field of health professional education.


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

Input:
Title: THE DIVERGENCE HISTORY OF EUROPEAN BLUE MUSSEL SPECIES RECONSTRUCTED FROM APPROXIMATE BAYESIAN COMPUTATION: THE EFFECTS OF SEQUENCING TECHNIQUES AND SAMPLING STRATEGIES
Review round: 1
Reviewer: 1

Output:
Basic reporting: Overall, the manuscript is clearly written in professional, unambiguous language. The manuscript provide sufficient introduction and background with relevant prior literature.
The manuscript provides an important evaluation of the demographic inference of mussels when employing different sequencing techniques, sampling method, number of loci and methods of binning jSFS. However, this is not clearly reflected in the title and other parts of the text.
For example, the title is a bit misleading. You do not look at the impact of model complexity on ABC-inferred demographic history. You are inferring the demographic history of mussels among a set of competing models. To evaluate the impact of model complexity I would expect different sets of competing models in which you increase or decrease the level of complexity. In addition, to evaluate different data sets in which the “true” model would change complexity (not only mussels, in this case)
What do you mean with high-throughput sequencing strategy? To the sequencing techniques? The type of jSFS? It is very ambiguous. In the abstract, you mention sampling strategy. Are you referring to that? Then it is not sequencing strategy.
You could write a title on this line: Inferring demographic history of mussels employing different sequencing techniques (or specify the techniques here) and sampling methods (or configurations of data) as you write on line 106.

Line 106-111 You are somehow mixing the terms throughout the manuscript sampling strategy, configuration of the data, sequencing strategy. Be consistent on what do you mean with each term.  You can do it here at the Introduction for example, and keep this throughout the text
Experimental design: The research is original, the knowledge gaps are identified, and the methods are well described. However, I am mainly concerned of one fundamental issue: the true or correct model is unknown.
Although the authors mentioned that the history of the species is well known, the exact model is unknown. In this sense, it is not possible to conclude that the model selected is the correct model. It is only possible to describe consistency of the results among different sample size, sequencing techniques, etc.
In addition, if the model is unknown. How can you reject the possibility of a systematic error on the estimate, if you are not contrasting the results with a “correct” and an “incorrect” model? This can be assessed by simulating data under different migration conditions, perform the ABC analysis with this data as you did with the mussels and see if you are able to recover the correct model among those models. Otherwise, you are simply inferring the demographic history of the species and not assessing the ABC method itself.
Validity of the findings: The authors show the results of the model with the highest posterior probability (Table 3) and the Bayes factor. However, I could not find the results of the other models. Results of the other models should be presented at least on the supplementary material. What does the values of Bayes factor means? Most models show Bayes factors close to 1-2. These values are usually consider low. How different are the values from the other models?

The goodness-off-fit is only shown from the most likely model. How different was compare to the goodness-off-fit from the results of the other models? You should present some examples in the supplementary material as well, or describe it in the results.

Conclusions do not emphasize clearly the main findings of the research. Conclusions should be focused on the consistency of the results and not on obtaining the correct model or making inference about the complexity of the models. For example:

Line 35. "We show that these models consistently outperform the simpler alternatives." This statement should be reformulated. Obtaining a more complex model as the most likely model does not necessarily mean that complex models outperform simpler models. They simply were selected as the most probably model in this case. You can write something like: We show that these model were consistently selected as the most probable model". You still don't know the real model of the mussels, and you don't know that if there is a species with a "true" simple migration model, the simple migration model will be selected  more accurately than if there would be a species with a "true" complex models, as you assume in this case.

Line 36. "This argues that methods that are restricted to simpler models may fail to reconstruct the true speciation history." This is actually true for all models, including the "complex" models that you are evaluating.

The conclusion paragraph. Line 491-506. Reads more like an introduction. There are not clear concluding remark of the study per se.
Additional comments: The main focus of the manuscript should be modified or simulated data should be added.
You can focus on the  demographic history of mussels and the consistency of the results when employing sequencing methods, sampling, etc. However, in order to make inferences about the impact of these conditions (i.e., sequencing methods, sampling, classes of jSFS) in the accuracy of the ABC inferences, you need to base your results on simulated data in which you can have control of the true model.
You can also combine simulated data with the mussels as an example, but you can not talk about the accuracy of detecting a correct model with only the mussels 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: THE DIVERGENCE HISTORY OF EUROPEAN BLUE MUSSEL SPECIES RECONSTRUCTED FROM APPROXIMATE BAYESIAN COMPUTATION: THE EFFECTS OF SEQUENCING TECHNIQUES AND SAMPLING STRATEGIES
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript by Fraisse et al. is written well, with the appropriate literature cited. The figures are of high quality and overall it's a nice contribution. My only suggestion has to to do with the conclusion; the real value I see of this study was the ABC models with novel summary statistics, small sample size, and common genomic data sets. However, the conclusion is about speciation and the current genomic islands debate; I appreciate the authors interest in the subject, but I'm not sure the conclusion is the most reflective of this ms' contribution.
Experimental design: The paper and models are appropriately designed.  I was slightly interested in the use of RNAseq (i.e. coding) as I have not really seen these data used in an ABC-coalescent framework. If your model assumes random mating (as the W-F model does), then selection at these loci means the assumption of random mating is violated - would this not be the case to some extent for RNAseq data? Stronger selection yields shorter the coalescent times (assuming directional and not balancing selection) and estimates of historical Ne will be downwardly biased. As I see the main value of this paper in the idea of using RNAseq on a few samples to learn something about the population history, I just wanted to make sure it was indeed an appropriate use.
Validity of the findings: Data and models are robust - nice contribution to the genomics and demography. Again, as more of these genome ABC studies are produced, the more applications and limitations we are discovering. This is a very important contribution in that regard.
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: THE DIVERGENCE HISTORY OF EUROPEAN BLUE MUSSEL SPECIES RECONSTRUCTED FROM APPROXIMATE BAYESIAN COMPUTATION: THE EFFECTS OF SEQUENCING TECHNIQUES AND SAMPLING STRATEGIES
Review round: 2
Reviewer: 1

Output:
Basic reporting: The manuscript has improved significantly; the suggestions were taken into account.
Experimental design: No comments
Validity of the findings: The aim of the study and conclusions are now well explain and consistent with the results.
Additional comments: I only have minor suggestions:
Be consistent with the use of abbreviations and only introduce the terms once. For example, the term:  joint site frequency spectrum (jSFS) is introduced together with the abbreviation in line 26 of the abstract, in line 30 or the same abstract, the entire term is used again.
Similarly, you introduced joint site frequency spectrum (jSFS) in line 56 of the introduction but on the rest of the text, you mix the use of the term and the abbreviation, e.g., see lines 443, 454.

The word “our” is used on several occasions. Avoid the use of that and instead, specify what it is such as the method, the specific results, etc. For example, line 283. “We checked our ability to…” It seems that it is the ability of the authors and not the ability of the ABC method. The same in line 363. Line 416. “our capacity…”, the authors capacity or is it the capacity of the method?

Line 459. The word “confirming” is probably not the most appropriate since Robinson et al., 2014 evaluated few and large number of individuals (2-50 individuals) but this study only few (2-8). Maybe you can use other word like “consistent with”, or you can write it in a different way in which you use the results from Robinson et al., 2014 to support the results from your data.

The inferences based on the simulated data during the model checking and the inferences based on the empirical (mussel data) are now clear. I acknowledge the use of the word “simulated” to clarify. However, it is not necessary to completely remove the term pseudo-observed datasets (since it is a common term when referring to the data sets simulated for the model checking).

Line 489. Which simulation results? Clarify. Is it simulated data generated as part of the model checking ABC framework or is it additional simulated data that was independently analyzed in ABC, such as the paper you are citing in line 495 which employs simulated data generated externally using Hudson’ ms?


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

Input:
Title: GENOME-WIDE ANALYSIS REVEALS FOUR KEY TRANSCRIPTION FACTORS ASSOCIATED WITH CADMIUM STRESS IN CREEPING BENTGRASS (AGROSTIS STOLONIFERA L.)
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript provides a thorough and motivating view of existing literature to support the importance and relevance of the present study. The language is clear, and all but figures 1-3 are self-contained and provide sufficient information. Figures 1-3 could use more information in caption to describe them adequately.
Experimental design: Overall, the discussion and results of the studies is well written, very clear, and provides sufficient information. Materials and methods are also described with sufficient information.
Validity of the findings: Impact, novelty, and description of results is well stated, and relevance to answering the central research question of the manuscript is clearly determined.
Additional comments: The manuscript by Yuan et al. studies and analyzes the role played by four transcription factor families associated with Cadmium stress in creeping bentgrass. The manuscript provides a thorough and motivating view of existing literature to support the importance and relevance of the present study. The discussion and results of the studies is well written, very clear, and provides sufficient information. The manuscript can be accepted for publication after addressing the following minor comments:
1.	The authors can mention briefly in the text their rationale for using KEGG database for analysis
2.	Figures 1-3 need more information in caption to describe them adequately


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

Input:
Title: GENOME-WIDE ANALYSIS REVEALS FOUR KEY TRANSCRIPTION FACTORS ASSOCIATED WITH CADMIUM STRESS IN CREEPING BENTGRASS (AGROSTIS STOLONIFERA L.)
Review round: 1
Reviewer: 2

Output:
Basic reporting: It is highly recommended that the authors rewrite the manuscript, ideally, with some help from a native English speaker. English may not be the first language for the authors, but in the current form, it is very difficult to understand the points authors are trying to convey. Sentences are incomplete and grammatically incorrect.  There is excessive use of colloquial language instead of scientific terms – which makes it hard to interpret the context and research findings.

Examples:
Abstract:
“Studying the uptake, translocation, and accumulation of Cd by plants is crucial for cultivation of pollution-tolerant plant species.”

How the findings in this study could be applied towards cultivation of pollution-tolerant plant species is not discussed anywhere. Without any conclusive results, making such overreaching claims is hasty.

Repetitive sentences:
Abstract:
“The main objective of this study was to reveal differentially expressed genes (DEGs) under lower (BT2_5) and higher (BT43) Cd
concentration treatments in creeping bentgrass. This study includes characterization of DEGs from creeping bentgrass leaves under Cd stress.”

Ambiguous pronouns:
Abstract:
“This study is the first report of the molecular characteristics of DEGs under Cd stress using transcriptomic analysis in
creeping bentgrass. These results provide new insight into the key roles played by these TFs under different Cd stress conditions and provide an important reference for breeders cultivating pollution-tolerant species.”

Which ‘these’ results are referred to here?

Sentence structures are very confusing:
Line 33-34
“As an increasing factor in environmental pollution, cadmium (Cd) pollution has once again attracted public attention.”

Write shorter sentences (Line 34-36):
“Cd is a non-essential element that is highly toxic and easily accumulates in plants, inhibiting plant development through its effects on physiological and metabolic processes (Nawrot et al., 2006; Hasan et al., 2009; DalCorso et al., 2010).”

Break up such sentence into 2-3 shorter sentences. Short and clear sentences will help convey point easily.

Unclear study objectives:
“Studying the uptake, translocation, and accumulation of Cd by plants is crucial for cultivation of pollution-tolerant plant species.”

What is the scope of the current study?

Manuscript structure:
Several references are missing.
Several studies have investigated differentially expressed genes in response to Cd stress in different plant species.
Yue R, Lu C, Qi J, et al. Transcriptome Analysis of Cadmium-Treated Roots in Maize (Zea mays L.). Frontiers in Plant Science. 2016;7:1298. doi:10.3389/fpls.2016.01298.
Oono Y, Yazawa T, Kanamori H, et al. Genome-Wide Transcriptome Analysis of Cadmium Stress in Rice. BioMed Research International. 2016;2016:9739505. doi:10.1155/2016/9739505.
Oono Y, Yazawa T, Kawahara Y, et al. Genome-Wide Transcriptome Analysis Reveals that Cadmium Stress Signaling Controls the Expression of Genes in Drought Stress Signal Pathways in Rice. Tran L-SP, ed. PLoS ONE. 2014;9(5):e96946. doi:10.1371/journal.pone.0096946.

Figures:
Please rewrite all the figure legends. (Once published) readers may (and most likely) try to understand the research primarily by looking at figures. Do the figure legends in their current format help readers interpret your results?

Please include in the introduction section, a brief summary of all the studies you discuss in the discussion section.
Results section needs to be more structured. In its current format, the results section reads like a materials and methods section.
Discussion reads like a results section.

Please look at the following article for manuscript structure and wiring:
Scientific reports: Transcriptome analysis of molecular mechanisms responsible for light-stress response in Mythimna separata (Walker)
Experimental design: No rationale for study design has been provided:
Why did you select 7-day treatment?
Why did you select 2.5 mM and 43 mM Cd concentrations? Concentrations in mM ranges are very high – are these environmentally relevant?
See the following studies – Cd concentrations in µM ranges:
Song J, Feng SJ, Chen J, Zhao WT, Yang ZM. A cadmium stress-responsive gene AtFC1 confers plant tolerance to cadmium toxicity. BMC Plant Biology. 2017;17:187. doi:10.1186/s12870-017-1141-0.
Oono Y, Yazawa T, Kawahara Y, et al. Genome-Wide Transcriptome Analysis Reveals that Cadmium Stress Signaling Controls the Expression of Genes in Drought Stress Signal Pathways in Rice. Tran L-SP, ed. PLoS ONE. 2014;9(5):e96946. doi:10.1371/journal.pone.0096946.

“Hoagland nutrient solution was used for irrigation”. Either cite a reference or include details.

After 3 months, mature plants were obtained and subjected to 0 mM (CK), 2.5 mM (BT2_5), and 43 mM (BT43) CdCl2 treatments.
How exactly was this ‘subjecting’ done? Provide details.
Validity of the findings: Methods section indicates that “After 3 months, mature plants were obtained and subjected to 0 mM (CK), 2.5 mM 88 (BT2_5), and 43 mM (BT43) CdCl2 treatments.” Whereas the results section indicates the effect of Cd stress on ‘development’. To study the effect on development, samples need to be collected at different time points after exposure to Cd.

Line 357:
No justification of how upregulation of certain genes is related to tolerance is provided.
MAPKKK12 may play an important role in activating tolerance to Cd stress in creeping bentgrass.
Line 372-373:
WRKY23 and WRKY75 were quickly upregulated from lower to higher Cd concentrations (Table S9), indicating that WRKY23 and WRKY75 could increase tolerance to Cd stress.

This study is the first report of the molecular characteristics of DEGs under Cd stress based on a transcriptomic analysis.
This is a false claim. Check the publications that you have not cited. Maybe in Agrostis stolonifera L, but that is not what the sentence states.
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: DOES ELEVATED ATMOSPHERIC CO2AFFECT SOIL CARBON BURIAL AND SOIL WEATHERING IN A FOREST ECOSYSTEM?
Review round: 1
Reviewer: 1

Output:
Basic reporting: English could be improved in places, literature references and explanation of previous work currently incomplete.
Experimental design: I believe that the experimental design is sound and the research question is well defined.
Validity of the findings: Generally sound-I disagree with the 100% radon loss model but their interpretations are reasonable. .  see comments below
Additional comments: Review of “Does forest growth at elevated CO2 affect bioturbation and soil carbon burial rate?” by Gonzalez-Meler et al.
In this manuscript, the authors show stable carbon isotope and radionuclide tracers of organic carbon transport in soil profiles measured at the Free Atmosphere Carbon Exchange (FACE) site which allows them to test the effect of high CO2 and ambient CO2 conditions on burial and bioturbation.  Overall I find the study to be interesting and novel and the dataset is sound.  The authors tackle a timely and significant problem: how rising atmospheric CO2 concentrations might affect soil processes, and possibly drive a feedback involving carbon burial.  The paper is written reasonably well and is easy to follow but there are some places where the English could be tightened.  Some of the background and context also need improvement.  Finally, the possibility that 137Cs transport is at least partially decoupled from organic matter transport should be more adequately addressed.
Below I give more specific comments/suggestions:
1)	Abstract: the first part of the first sentence is a bit awkward:  “studies of effects…” should read “studies measuring the effects”
2)	I don’t believe that “bioturbation” needs to be defined in the abstract- this is a standard term
3)	Introduction line 50: the words “accessibility of” should be removed, and so the statement should simply be “Soil organic carbon decomposition depends on….”
4)	The in-text referencing is inconsistent: sometimes the authors list multiple authors, and other times they use “et al”.  See lines 63, 73, and 94 for examples that need to be fixed.
5)	More background on previous research using fallout radionuclides as tracers of organic matter transport in soils are needed.  The authors reference some studies but don’t really explain the work that has been established.  For example, Dӧrr and Munnich (1989, 1991) were I believe the first scientists to use radionuclides (14C, 210Pb, 137Cs) to trace soil organic matter transport in soils yet they are not referenced here.  They found that 210Pb was a good tracer of soil carbon transport, but that Cs was too soluble and seemed to have an additional transport mode that was probably dissolved Cs+ cation. I believe that their results may be applicable to the current study.   Kaste et al. (2011) showed that 210Pb was a good tracer of soil organic matter burial and organic matter age in Podzols collected from both Norway and the northeastern United States.  I find the introduction and context of the manuscript to be somewhat incomplete with regards to describing previous work.
6)	Material and Methods: could be improved.  Describe the coring device a bit more- was it a plastic tube?
7)	The authors fit two different models to their 210Pb/226Ra data- one that assumes 25% of 222Rn loss from soils, and the other that assumes 100% loss of 222Rn from soils.  While I agree that the loss of 222Rn is probably depth-dependent, and that bioturbation/weathering might increase the loss at the surface, I do not think that 100% 222Rn loss is realistic.  Most studies show that the loss is closer to 10-25% (Graustein & Turekian).  While assuming 100% loss of 222Rn at the surface permits a stronger fit to the data in the upper 10cm, a more likely scenario is that the diffusion and advection rates are variable with depth.  I suggest that the authors address this possibility more thoroughly in the discussion.
8)	The reference list at the end of the manuscript needs to be checked carefully for formatting.
Ultimately I believe that this is a very novel and useful study.  I hope to see it published after some revisions.

Suggested References:

Using atmospheric fallout to date organic horizon layers and quantify metal dynamics during decomposition
By: Kaste, James M.; Bostick, Benjamin C.; Heimsath, Arjun M.; Steinnes E., and Friedland AJ
GEOCHIMICA ET COSMOCHIMICA ACTA   Volume: 75   Issue: 6   Pages: 1642-1661   Published: MAR 15 2011

LEAD AND CESIUM TRANSPORT IN EUROPEAN FOREST SOILS
By: DORR, H; MUNNICH, KO
WATER AIR AND SOIL POLLUTION   Volume: 57-8   Pages: 809-818   Published: AUG 1991

DOWNWARD MOVEMENT OF SOIL ORGANIC-MATTER AND ITS INFLUENCE ON TRACE-ELEMENT TRANSPORT (PB-210, CS-137) IN THE SOIL
By: DORR, H; MUNNICH, KO
Conference: 13TH INTERNATIONAL RADIOCARBON CONF Location: DUBROVNIK, YUGOSLAVIA Date: JUN 20-25, 1988
RADIOCARBON   Volume: 31   Issue: 3   Pages: 655-663   Published: 1989


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: DOES ELEVATED ATMOSPHERIC CO2AFFECT SOIL CARBON BURIAL AND SOIL WEATHERING IN A FOREST ECOSYSTEM?
Review round: 1
Reviewer: 2

Output:
Basic reporting: This article is well written and the English is of a high quality. For the most part it is clear and easy to follow. I have a few minor comments on clarity:
Line 53-55. I find this sentence rather long and difficult to process. I would suggest to cut it down to:
"Climate change forcing factors can directly and indirectly affect soil C stocks, altering the resilience of vegetation and human society to climate change."
Lines 75. Saying that previous studies do not link long term soil C movement to 'climate change forcing factors' implies that that will be done so here. To me that implies the impact of climate change, and I do not consider a CO2 change as a change in climate (in fact, a lot of model studies 'climate' and 'CO2' are considered as two different forcings). Perhaps you could say something like 'climate change or CO2 change' instead of 'climate change forcing factors', it might just be clearer. Hope that makes sense.

Figure 4 is missing and has been replaced with a copy of Figure 1. Please address this. So I cannot comment on Figure 4 but otherwise the figures appear to be appropriately presented.

The hypothesis is that elevated CO2 could enhance bioturbation and/or soil weathering, and the analysis addresses this, thus the study is nicely self contained. My one concern is that the title does not very clearly reflect the hypothesis. The study considers more than one potential impact of elevated CO2, including direct effects on weathering and therefore not only the impact of 'forest growth'. I would suggest removing 'forest growth' and perhaps something along the lines of:
'Does elevated CO2 affect soil carbon burial and soil weathering in a forest ecosystem?' (it wouldn't have to be exactly this!)
Experimental design: In general the work seems to be original, well-defined and relevant. I cannot comment too much on the methods since I don't have a large experience with experimental methods.

Method description:
Line 174. Perhaps this is commonly known but I had not heard of a 'feeding rate constant' so I would like to see a few words here to explain what this represents.

Regarding the experimental design, since the differences in some of the key variables were not statistically significant and yet could have been reasonably large (for example, the mean mixing rate, which directly addresses the hypothesis, was 0.63 cm^2/yr in elevated CO2 and 0.53 cm^2/yr in ambient CO2, but with error bars too large to distinguish them), taking a larger number of soil samples might allow the potential differences (or lack of difference) to be resolved. I would suggest adding some comments in the discussion on this and potentially a call for additional sampling in further studies.
Validity of the findings: My main concern with this analysis is that after fitting a model with both advection and diffusion terms, only the advection term is then considered. For example on line 288-290, the advection rate is considered but not the diffusion rate. With a diffusion rate of 0.5 cm2/year, that could certainly result in 13C-depleted C reaching a greater depth than from the advection term alone. The advection process relates more to soil accumulation and burial, whereas the diffusion term will relate to the bioturbation (e.g. worms) which will mix some of the surface carbon to depth (and deep C to the surface). Even considering both terms, it is still likely that the other processes mentioned on the following lines can also have an impact, but definitely the diffusion term needs to be also considered, and in a quantitative manner (i.e. how much of the deeper C might this account for). Then the abstract lines 37-39 will also need some modification.

Another concern is the conclusion that mixing may prevent detection of the additional carbon in the layer (for example 347-349). While this could be a factor, could it also be that both control and elevated CO2 plots have carbon addition at those depths, but in the elevated CO2 plot it is depleted C? Surely the presence of depleted C does not on its own imply that the C addition has been greater than in the control?

Finally just a question regarding the Pb/Ra profile shown on Figure 5 - the conclusion is that it decays too rapidly with depth to be consistent with the advection term implied from the 137Cs profile, and therefore there must be some additional losses. However, 137Cs is more prone to desorption than 210Pb, and if the 137Cs is to some extent desorbed and leached downwards, could this also be providing additional advection which would not be seen for the 210Pb?

Line 367-368, "Our results indicate..." I think this sentence is too strong a claim given that the results did not show much of an impact.
Line 259 "do not show unequivocal evidence" I would change this to "do not show evidence", because not only is it not unequivocal, it simply doesn't appear to be there.
Additional comments: Line 201. I think this should say g/cm^3 and not kg/cm^3!
Line 259. Says 222Ra, should be 226Ra?


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: SKELETAL MUSCLE MITOCHONDRIAL VOLUME AND MYOZENIN-1 PROTEIN DIFFERENCES EXIST BETWEEN HIGH VERSUS LOW ANABOLIC RESPONDERS TO RESISTANCE TRAINING
Review round: 1
Reviewer: 1

Output:
Basic reporting: Please find my review within the enclosed document.
Experimental design: Please find my review within the enclosed document.
Validity of the findings: Please find my review within the enclosed document.
Additional comments: Please find my review within the enclosed document.


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: SKELETAL MUSCLE MITOCHONDRIAL VOLUME AND MYOZENIN-1 PROTEIN DIFFERENCES EXIST BETWEEN HIGH VERSUS LOW ANABOLIC RESPONDERS TO RESISTANCE TRAINING
Review round: 1
Reviewer: 2

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: This paper by Robert’s and co-workers investigated whether total myofibrillar and sarcoplasmic protein concentrations, myosin and actin protein content, myofibrillar protein levels of genes involved with myofibril formation, or markers of mitochondrial physiology present differences in expression between ‘high’ and ‘low’ responder clusters to a 12-week resistance training program. In contrast to their hypothesis, it was demonstrated that total myofibrillar and sarcoplasmic protein concentrations along with total myosin and actin content were not differentially altered between or within the ‘high’ and ‘low’ responder cohorts. The authors concluded from this finding that the higher increases in muscle fiber cross-sectional with ‘high’ responders is perhaps more likely due to factors other than myofibrillar or sarcoplasmic protein concentration increases. The authors have performed a very well-designed study and investigated a unique, but justified, hypothesis. They have adequate participant numbers for their analyses and a highlight of their methods were the three measures used to generate the high and low responder cohorts (which presents a sound basis as opposed to other publications on this topic that aren’t based on such stringent measures). The paper is very well written (consistent and appropriate use of language and grammar), and my comments/ concerns are only minor in nature.

1. Regardless of the sound hypothesis stated in the manuscript, wouldn’t an increase in mitochondrial content/ function/ capacity with resistance exercise work more against the ‘specificity of adaptation’ where the muscle is more phenotypically adapted to promote molecular events promoting muscle growth (i.e.: increases in translation initiation signalling, etc) than try to promote the opposite of this?

2. Substantial detail regarding the methodology used to measure myofibrillar and sarcoplasmic protein expression is provided, however I still wonder about the sensitivity of the measure. How well optimised are these blots- for instance, have different concentrations of loaded protein been used to determine the amount of protein that provides optimal signal while still keeping sensitivity to measure differences between samples? Also, how well are antibody specificity and associated dilutions optimised?


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: SKELETAL MUSCLE MITOCHONDRIAL VOLUME AND MYOZENIN-1 PROTEIN DIFFERENCES EXIST BETWEEN HIGH VERSUS LOW ANABOLIC RESPONDERS TO RESISTANCE TRAINING
Review round: 1
Reviewer: 3

Output:
Basic reporting: Well done
Experimental design: Well done overall. My one comment would be to include detailed description of the resistance training protocol. It is inconvenient for readers to have to locate another paper, and some may not have access to the paper. The RT protocol has relevance to the findings, so it should be explained.
Validity of the findings: Well done
Additional comments: Overall, this is very well-done paper with potentially important implications to the field. The writing was excellent, the methods sound, and the conclusions appropriate. I have a few suggestions for improvement:

First, the finding of greater mitochondrial capacity is interesting, and I would agree that the higher energy cost of MPS is a potential link. However, are high responders better able to upregulate mitochondrial proteins or is this simply a consequence of a greater increase in turnover and thus a byproduct? I realize that there is no way to know, but it would be worthwhile to provide speculation here.

Second, you hypothesize that intracellular fluid accumulation may explain differences in muscle size between high and low responders. What mechanism could be responsible for such a hypothesis? It would seem counterintuitive to me that this would be explanatory, given that fluid changes are temporary and can be altered in a very short time frame (thus, the hypertrophy conceivably would not be “lasting”). If the fluid changes were somehow chronic, then I don’t see how this would occur without accompanying increases in sarcoplasmic proteins, which would necessitate greater surrounding fluid to maintain a stable milieu. I would think there could be negative consequences on the function of the cell from higher intracellular fluids without accompanying protein accretion, as this would conceivably disturb the “spacing” of tissues required for cellular balance. Again, I realize that there is no way to know the answers to these questions, but I feel they should be addressed.


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: SKELETAL MUSCLE MITOCHONDRIAL VOLUME AND MYOZENIN-1 PROTEIN DIFFERENCES EXIST BETWEEN HIGH VERSUS LOW ANABOLIC RESPONDERS TO RESISTANCE TRAINING
Review round: 2
Reviewer: 1

Output:
Basic reporting: See my general comment(s)
Experimental design: See my general comment(s)
Validity of the findings: See my general comment(s)
Additional comments: Dear Dr Roberts and colleagues,

Thanks for the detailed responses to my comments and questions, and the other reviewers', including the provision of additional data for my own perusal.

I'm happy with the responses you've provided, and the subsequent edits made.

Llion


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: SKELETAL MUSCLE MITOCHONDRIAL VOLUME AND MYOZENIN-1 PROTEIN DIFFERENCES EXIST BETWEEN HIGH VERSUS LOW ANABOLIC RESPONDERS TO RESISTANCE TRAINING
Review round: 2
Reviewer: 2

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: The authors have satisfactorily addressed all my comments and are to be commended on their quality 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: SKELETAL MUSCLE MITOCHONDRIAL VOLUME AND MYOZENIN-1 PROTEIN DIFFERENCES EXIST BETWEEN HIGH VERSUS LOW ANABOLIC RESPONDERS TO RESISTANCE TRAINING
Review round: 2
Reviewer: 3

Output:
Basic reporting: See below
Experimental design: See below
Validity of the findings: See below
Additional comments: The authors have satisfactorily addressed my comments and I feel this will be an excellent addition to the literature.