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:  TREEDATA.TABLE: A WRAPPER FOR DATA.TABLE THAT ENABLES FAST MANIPULATION OF LARGE PHYLOGENETIC TREES MATCHED TO DATA
Review round: 1
Reviewer: 1

Output:
Basic reporting: • I commend the authors for presenting a professional structure of the manuscript, figures and tables.
• Similarly, a clear and unambiguous, professional English has been used throughout the manuscript and
package website.
• Moreover, supplementary material has been very well prepared, and has been made available from
the journal and from the software package website at https://ropensci.github.io/treedata.table/.
Supplemental files are written in English and Spanish. This is a very good step towards inclusivity. It
will also facilitate broader adoption of the package.
• Data to run all the example code has been shared as part of the software package. However, data from
the microbenchmarking of the package that were used to generate Figure 2 and 3, and raw data used
to generate Figure 1 have not been shared. For the sake of reproducibility, please share a link to the
repositories containing these data.
• The paper is self-contained, and results presented are relevant to the research goal: developing an R
package that facilitates fast and reproducible comparative phylogenetics analysis in large trees. I think
specifying in the title that the package is designed to deal with large trees (“fast manipulation of large
trees” instead of “trees” in general), will help readers immediately get a sense of the advantage of
learning and eventually using the package.
• The biological background of the package, as well as references supporting its importance are adequate.
The biological introduction is clear and relevant for the target audience. I would try to be more
specific in L42, stating that you are talking about the workflow for comparative phylogenetics and not
phylogenetics in general.
• The computational background can be improved by mentioning other packages that also deal with data
tables, maybe talk about the R tidyverse principles and argumenting why that structure is or is not
adopted for this package. I would consider making the section “A short description of data.table” part
of the introduction. In my opinion, this would round up the intro nicely.
Experimental design: • The authors present a research goal that is well defined, relevant and meaningful for the fields
of evolutionary biology and ecology. Authors successfully identify an analysis gap in these fields:
automatically creating comparative phylogenetics datasets with large trees in a fast and reproducible
way. The authors clearly state and demonstrate throughout the manuscript how the package they
present fills this gap.
• In this sense, the authors have developed original primary research that is within the Aims and Scope
of the journal.
• To achieve their goal, the authors develop a package written in the R language, that expands on the
fastest R package available for manipulation of data tables, the data.table package, to allow tree
manipulation. The authors appropriately justify the use of the R language, and present a rigorous
research of the available R packages for data table manipulation, and appropriately chose the one that
would give the fastest results for large tree manipulation. This should be made more explicit in the
introduction. For example in L52, they should mention the dplyr package and the data.frame workflow
from base.
• The authors described the package functions and usage very clearly and thoroughly. To test the speed
of the package they use the microbenchmark workflow, which is a current good standard for R package
benchmarking. However, the exact code used was not shared. This as well as the R data objects that
resulted from the microbenchmark runs should be shared as raw data.
• Statistical analysis used to analyze the temporal trend in phylogenetic tree size over time is adequate.
Code and raw data should also be shared.
Validity of the findings: • Following PeerJ recommendations, I am not leading the review with my perception on how impactful
and novel the research is. Nevertheless, I do believe this package will have a positive impact on the
field. I was able to successfully install the package from CRAN, and to run all example code in the ms
and supplementary data, and to reproduce the reported results exactly.
• Whenever possible, underlying data and code used to generate all figures should be provided.
• Conclusions are clearly stated, linked to original research question and limited to supporting results,
demonstrating that the treedata.table package effectively extends the functionality and syntax of
data.table for evolutionary research. However, the benchmark results they present seem to contradict
their conclusion that “treedata.table extends the speed of data.table”, in L230-231. Unless they mean
in comparison to treeplyr. But from the text I was unable to infer if treeplyr is also an extension of
data.table. If so, then treedata.table is not the first package to do this? This should be stated more
clearly.
Additional comments: To the manuscript:
• Adding a link to the website of the package (in the intro and abstract) would help readers know right
away that the package has been already published and is available for download. It would also prompt
readers to go explore it right away.
• I would highly recommend formatting function names with backticks (``) or quoting them. It would
help with readibility of the paper, especially for functions such as [ or operators such as $.
• L24: increasing -> increased
• L44-45: This language -> The R language
• L53-54: https://h2oai.github.io/db-benchmark, this should be a proper citation of the website with
date accessed. See an example of citation guidelines for websites here.
• L101: matrixes -> matrices
• L120-124: As I stated in general comments section, I think the output of these functions (especially
summary) should be described more, and/or refer readers to Table 1.
• L127: I suggest being more descriptive. Instead of saying “using the following line”, explicitly state the
function that is going to be used, e.g., “using the $ operator and the[ function, as follows:”
• L149: “furthermore, can create” -> “furthermore, we can create”
• L150: anoles. -> anoles:
• L171: Add a link to the vignette and specify which one of the 5 vignettes available.
• L211: “relative to” is repeated twice.
• L214: a comma is missing after “manipulation only”. Also, because you only tested speed of performance
among packages, “higher performance” should be “higher speed performance”.
• Figure 1:
– Legend: When using the rotl package, the Open Tree of Life project should also be cited, as
mentioned here https://cran.r-project.org/web/packages/rotl/citation.html, as Open Tree of
Life, B. Redelings, L.L. Sanchez Reyes, K.A. Cranston, J. Allman, M.T. Holder, & E.J. McTavish.
(2019). Open Tree of Life Synthetic Tree (Version 12.3). Zenodo. doi: 10.5281/zenodo.3937741
– When using log scale for a variable that is better understood in a non-logarithmic form, it is
best to replace the log numbers for their non-log equivalents. In this case the y axis showing the
number of tips can be modified to make the figure more readable, by replacing 1.6 for ~5, 3.2 by
~25, . . . , 8 by ~3k.
• Table 1: In the description column, I would remove “Function to . . . ”, it would save space and make it
less repetitive. You can start right away with the action that the function is performing. I would also
homogenize the verbs to present tense instead of using “ing” verbs sometimes.
• Figures in treedata.tables website preprint vignette appear broken, here: https://ropensci.github.io
/treedata.table/articles/E_Preprint.html.

To the README.md:
• add library(treedata.table) after installation instructions.
• in Line 54, explain that you are talking about function tdt and extractVector before using them in
line 57. It seems like you are only talking about functions from the geiger package, so it was not fully
intuitive for me to figure out the example in L57.
• add a license and citation
• section tl;dr seems out of place? It seems to me like it would go better as part of the first paragraph of
the README intro, maybe?
• L52: intutive -> intuitive
• L68: tipcs -> tips
• throughout the text: taxa names -> taxon names

To the AA_treedata.table_intro_english.Rmd file:
• L37 and L40: Some users might want to use your package without knowing about data.table. Try to
explain the main difference between data.table and data.frame. Maybe printing the class of the object
before and after would help readers visualize how the data object changes in structure.
• I like the named vectors!
• L78: Explain partial match and non-standard evaluation. Why are they relevant to be implemented in
your package? Linking partial matching to the corresponding vignette would be good.
• L80: consider a more straightforward wording for “all the tips not in the resultant data.table ”, to
maybe “all the tips absent from the resultant data.table ”
• Consider breaking down the Manipulating data section in subtopics. I would suggest at least making a
subtitle for Coindexing, so you can highlight this powerful functionality of your package.
• L101: again consider that readers might not be familiar with data.table, so a comparison with it is not
direct for all (most) users. It would make adoption of the package easier if the descriptions were a bit
more general, sort of speak.
• L107: the same goes for the concept of a “tidy approach”. Old school R users might not be familiar
with it, and new users might not have learned it yet. Try to think of a way to describe it in the simplest,
most general words.
• L129: Not sure I have it right, but maybe replace “out” for “the”?
To the file AB_treedata.table_intro_spanish.Rmd:
• It would be good to make the same editions I suggested for the English version of this vignette, i.e.,
making more subtitles, reducing the size of printed tables, elaborating more on “evaluación no estándar”,
“correspondencia o emparejado parcial”, “sintaxis del tidyverse”. And trying to explain things from
the point of view of someone that has little experience using the tidyverse, data.table and even R,
elaborating more on the comparisons with data.table.
• I am a native Spanish speaker, so I went ahead and checked typos and I made some suggestions to
change some of the wording to make it clearer. Feel free to disregard them or double check with other
Spanish speakers!
– L22:
* Anolis should be in italics
* arboles -> árboles
* están en formato -> estar en formato
– L18, 20 and 50: “Manipulando” should be “Manejando”. “Manipular” has a somewhat negative
connotation in Spanish, meaning that the data have been altered to change the results, or to
mislead.
– L65:
* “ser logrado” -> “ser obtenido”
* “El producto de extractVector como los brackets dobles es un vector con nombres” -> “Al
igual que con los brackets dobles, el resultado de la función extractVector es un vector con
nombres.”
– L78:
* I would remove the “Sin embargo”, it is not needed.
* match parcial -> correspondencia parcial
* múltiples columna -> múltiples columnas
* no standard -> no estándar
– L102:
* “pueden ser también operadas usando sintaxis en data.table” -> “pueden ser operadas
usando la misma sintaxis de data.table”
* arboles -> árboles
* simultanea -> simultánea
* y resumir -> y reducir
* numero -> número
– L108:
* “También podemos usar treedata.table para correr funciones en nuestros datos.” ->
“LA”treedata.table permite aplicar funciones directamente en nuestros datos de interés"
– L 116: “Los tips en el árbol también pueden ser removidos” -> “Las puntas del árbol también
pueden ser removidas”
– L135:
* ultimo -> último
* arbol -> árbol
* extraidos -> extraídos

To the B_multiphylo_treedata.table.Rmd file:
• You do a great job describing the output for the multiphylo objects. By reading the vignette, it was
not clear to me what the dataset output would be using a multiphylo object. Knowing that all trees
must have the same tip labels, I think I was able to infer what it would be, but it is always good to
have confirmation from the authors. It would be good to clarify in the text that:
– there is only one output dataset and not one for each phy in the multiphylo object
– the output dataset contains only the overlapping taxa between the multiphylo objects and the
input dataset
• L33: Use of “Nevertheless” here was confusing for me. As I understand it, “nevertheless” is an adverb
used to contrast a first point with a second one. I do not easily see that the two points contrast with
each other (as they both indicate a restriction?): “all trees must have the same tip labels. Nevertheless,
both the provided multiPhylo and data.frame should partially overlap.” Maybe it is just a matter of
replacing “should” by “can”. If the trees and datasets have to partially overlap, then I would replace
“nevertheless” by “also” or “and”, just to make it straightforward for readers to understand your point.

To the C_PartialMatching.Rmd file:
• L31, 47, and 56: ahi should be ahli.

To the D_AdditionalFunctions_treedata.table.Rmd file:
• L18: character -> characters
To the vignettes in general:
• when dealing with large tables, consider doing head and tail instead, or printing an interactive table,
otherwise the text and explanations get a bit lost among the long printed outputs of the example code.

To the code in general:
• I really like the messages you print to screen. They are short, sweet, and helpful.
• Try to always keep messages affirmative, as far as possible. Somehow negative messages always feel to
me like an error and leave me feeling that I did not run the function correctly. For example, instead of
No tips were dropped from the original tree/dataset you can print All tips from original
tree/dataset were preserved. This will be kinder for your users.
• When trying the examples with the modified tip ahli to NAA, I noticed that the output message is
Tip labels detected in column: X
Phylo object detected
1 tip(s) dropped from the original tree
1 tip(s) dropped from the original dataset"
In the last line, instead of "tip(s)" should be "line(s)" or "row(s)", or maybe change "tip(s)" to
"taxon(taxa)" in both cases? Not sure about that, maybe the first suggestion is better. I leave that to you
:)


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:  TREEDATA.TABLE: A WRAPPER FOR DATA.TABLE THAT ENABLES FAST MANIPULATION OF LARGE PHYLOGENETIC TREES MATCHED TO DATA
Review round: 1
Reviewer: 2

Output:
Basic reporting: The package is quite simple and the manuscript is easy to follow. However, the review of relative works is not sufficient. The authors claimed in the manuscript that only treeplyr can combine tree with data. This is not true, there are several packages support this feature, such as phylobase, treeio and tidytree. The tidytree package is the fundamental package that the famous ggtree relies on. It can convert tree with data (parsed by treeio) into tidy data frame and supports dplyr verbs to manipulate tree with data. More packages should be included in the "Alternatives to treedata.table" session.
Experimental design: I have no doubt that treedata.table will have higher performance compare to other packages. However, as a user, I would like to see more comparisons on functionalities. Which packages can support the operation I needed is something more important. A table for detailed comparison is wanted.
Validity of the findings: In addition to speed comparison, more results on functionality comparison should also be included.
Additional comments: The limitation of this package is quite obvious that it only supports matching taxa with labels. This will restrict its application on internal nodes and on trees with identical tip labels.


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 INFLUENCE OF STORAGE TIME ON PONAZURIL CONCENTRATIONS IN FELINE PLASMA
Review round: 1
Reviewer: 1

Output:
Basic reporting: This is a straight-forward and readable article that contributes to the literature on measuring drug concentrations over time in individual animals. The importance of the selected drug, ponazuril, is highlighted, and the rationale for sample storage is included. It might be helpful to also include how important accurate drug concentrations can be to the estimation of accurate pharmacokinetic parameters, which may impact drug regimen design. Other context might also be provided in the introduction or methods by referencing the FDA guidelines there, in addition to the already included reference in the discussion.

The included figure is somewhat helpful, and it might be more helpful to use linear graphs, rather than bar charts, given the expectation that concentrations over time will decay in a linear or log linear manner.

Reference to literature on other drug decay over time after storage might be helpful to provide context to these findings, either by relevant chemical class, or drugs in this species of animals. If there is no relevant literature, noting this might also provide context.
Experimental design: The design is for the most part clearly explained. It might be more clear if the following were addressed:

1. Consider providing a brief explanation of why plasma was collected from healthy animals (i.e., as part of the description of this step in the study).
2. Re-word the description of plasma spiking and aliquoting into vials. It was not initially clear to me that 3 different concentrations would be followed throughout the study.
3. Considering explaining why duplicate or triplicate spiked plasma samples were not created for each day, and why duplicate or triplicate sampling of each vial was not performed - this would have provided a bit more confidence in the estimated concentrations.
4. Include a brief overview of the analytical method herein, even though it is a previously described method, perhaps at least the instrumentation and basic outline.
5. Include at what temperature the vials were thawed - was it room temperature? How long did it typically take for thawing before samples were analyzed?
6. Include perhaps parenthetically that the diclazuril was added as the internal standard.
7. Consider re-wording the description of the numeric results, or perhaps consider a table with the actual concentrations along with the percent losses.
Validity of the findings: The conclusions are valid based on the data, and clear interpretation of what these data mean is provided. As previously mentioned, it would be helpful to put these data and the conclusions in the context of similar drugs in other species, or other drugs in cats, in order to highlight the importance of knowing how much storage impacts drug concentrations and how that impact affects pharmacokinetic parameter estimates (and therefore drug dosing recommendations). In addition, comments on whether or not there is a contribution of this study to the expectation of linearity of drug degradation during storage could be interesting.
Additional comments: I appreciate the authors making the effort to evaluate and publish these kind of storage data - this is an understudied (or at least under-reported) area in clinical pharmacokinetics in veterinary medicine.


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 INFLUENCE OF STORAGE TIME ON PONAZURIL CONCENTRATIONS IN FELINE PLASMA
Review round: 1
Reviewer: 2

Output:
Basic reporting: see comments below
Experimental design: See comments below
Validity of the findings: See comments below
Additional comments: Comments for Authors:

The Introduction is a little long.  There is no need to review past pharmacokinetic studies or what it is used for.  Try and shorten the introduction and focus on just why this study was performed.

On line 52 is says “possible drug loss”.  Try another term here.  Perhaps “degradation of the drug” or “loss of stability”.

Line 58-59:  Be more specific here.  What animal species?  What concentration range?  Storage time?  What was the duration and conditions of storage to be examined?

Line 69:  It says “Ponazuril and diclazuril were purchased from Cayman Chemical”.  For a study like this you really should be using a USP analytical reference standard.  (obtained from www.USP.org)  If you didn’t use that, then there has to be some assurance that this source was equivalent to a USP standard, or met acceptance criteria from the USP.  The acceptance criteria for each of these standards is listed in the USP monograph.

Line 73-78:   This section describes the HPLC method.  For a study such as this, which focuses on the integrity of the method, you need a validated HPLC method.  Therefore, you need to consult with reputable sources (ICH, USP, FDA, etc.) and perform the necessary validation steps.  At the least, you must establish LOQ and LOD (and specify the criteria), repeatability of the assay, and performance of the calibration curve.  How many points in the calibration curve were used?  Were all points on the linear calibration curve?

I realize that on line 109 you said that a validation was performed.  But you should have stated that earlier (in the Introduction).  During the validation study that was cited on this line, the stability tests and effects of storage should have been performed as part of the validation.  Thus this study may have been unnecessary.  Usually authors are discouraged from splitting a single study into two publications.
Line 78:  You must specify the anticoagulant used.  The same anticoagulant must be used for all samples throughout the study.

Line 86:  Ideally in a study like this, at least 3 replicates per sample should be used.  You need this to calculate statistical parameters.  This is an important shortcoming of the study.

Line 87:  Were freeze-thaw effects measured?  What was the extraction efficiency?

Line 104-108:  Put all your specific results in a table and just refer to the table.  It is difficult to read when presented in the text.

Line 125:  Here you describe the loss of drug and amounts.  But what was your acceptance criteria?  At the beginning of the study (planning stages) you should have had some acceptance criteria listed (eg, loss of 5%, 10%, 15%, etc.).  What is acceptable before you reject the samples at these storage times?  This must be stated.  It is preferable that you use some published and accepted guidelines for this decision (ie, validation guidelines published by ICH, FDA, etc.)  This information doesn’t appear until the last line on line 139.  This should have been an objective stated at the beginning of the paper.

Figure 1:  Title says “percent recovery”.  This is not the correct term to use here.  Percent strength of solution or something similar is preferred.   Us the full axis for presentation.  (0% to 110%)


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 INFLUENCE OF STORAGE TIME ON PONAZURIL CONCENTRATIONS IN FELINE PLASMA
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: I am satisfied with revisions the authors have made.


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 INFLUENCE OF STORAGE TIME ON PONAZURIL CONCENTRATIONS IN FELINE PLASMA
Review round: 2
Reviewer: 2

Output:
Basic reporting: I did not fill in each box in this review.  That would be rather tedious and redundant.  Instead just scroll down to my comments below.
Experimental design: I did not fill in each box in this review.  That would be rather tedious and redundant.  Instead just scroll down to my comments below.
Validity of the findings: I did not fill in each box in this review.  That would be rather tedious and redundant.  Instead just scroll down to my comments below.
Additional comments: The authors have submitted a revision and addressed most, but not all of the concerns expressed by the reviewers.
This paper is acceptable as is, but has some significant limitations and short-comings that need to be stated in the final version.  They did not perform a full validation, and although we understand the limitations, many readers might assume that this paper constitutes a validation of their method.  The lack of replicates, among other criteria results in shortcomings.

The main criticism that still remains is that the authors performed a study without prior acceptance criteria to meet and a hypothesis to fulfill.  They should have stated at the beginning what their goals were.  They should have acceptance criteria for storage of the samples.  Do stored samples meet these criteria?
 It is a simple question.  Ordinarily +/- 10% would be an acceptable goal.  (This is common for many USP standards and guidelines.)  You met a goal in this range, so why not state this range as your acceptance?


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: NUTRITIONAL PROPERTIES OF SELECTED SUPERFOOD EXTRACTS AND THEIR POTENTIAL HEALTH BENEFITS
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment. understandable
Experimental design: understandable
Validity of the findings: need deeper scientific explanation
Additional comments: Thank you for your manuscript. its a nice result and bring a new "human and environment friendly".

here are some comments, I hope this will help to improve the quality of the manuscript.

General comments
It is mentioned that: there is a lack of explanation about the health benefits of those superfoods from the perspective of scientific study (in the abstract, and line 61-62). But, there are already several nutrition values of those mentioned materials in line 74-84. I  would suggest rewriting the sentence in the abstract and line 61-62 to avoid misunderstanding.

The term “superfoods” is  a term for food products available in the market. Are those materials claimed as superfoods, in the market where authors buy those products?

Abstract.
Methods: I would  suggest to mention the analysis; chemical constituent for mineral, etc etc and biological activity such as antioxidant and cell viability”
Future studies, isn’t this too specific  to mention against inflammation and oxidative stress? Because it can be studied in other non-communicable disease markers. I would suggest just “investigating their rules in regulating non-communicable diseases”. Or, it is mentioned  in the previous sentence that extract  composition and antioxidant composition are not directly related. So, this could be a  suggestion for further study to explore the specific compounds which are related to the antioxidant properties and other biological properties.

Introduction
Line 61: the citation is not in the list of references. As far as I understand, superfood is not allowed to be used in several countries (at least in EU countries) unless they are proven to have health value. So, I am curious about this paper and I can't find it in the reference list.

Line 66: these superfoods are claimed to have a large range of health benefits. Who claimed this? Please provide more information about this. For example from paper, from market survey, or from authors observation?

Line  86-90: this part  seems  trying to argue with previous study to support the methods used in this study. I would suggest  providing the citation in this statement. Or probably, it might be better just to mention that the mild aqueous extraction has never been  applied in those materials, without making any negative speculation from the previous method. In addition to that, the positive  value of  mild aqueous extraction can be added  to support this statement with citation.

Materials and methods
Line 103-105: the name of the company or  the producers  were mentioned. I am not sure what the regulation is if  the authors need permission to  put this name in the manuscript. Please check again and clarify.

Line 113: please provide the citation.

Line 117: please convert the rpm to g unit.

Line  170:  the “.” after the citation.

Line  178: what about the replication in the analysis?it is not mentioned in the manuscript.

Results
I would suggest to provide the level of significant difference according to the post-hoc test, whether 0.05 or 0.01.

Discussion
Line 280-284: the previous technique is efficient, but it may have an impact on health promoting activities. Please provide the citation and more detailed explanation. How much was the yield from the  previous extraction compared to this study.
	I  would suggest, to provide several techniques which have been applied as comparison to the novel technique.

Line  304-313: could  authors please provide some literature for the toxicity of solvent-based extracts to make a better understanding of this novel method?

Line 314-335: please identify or provide the literature, about the specific biological compounds in those materials which might be responsible for the antioxidant activity and other biological activity.

Conclusions
Line 334-343:  the citation is not important in conclusion
Line 334-343: please remove this part at the end of the conclusion
I would suggest, the novel extraction technique must be stressed in conclusion and what are the benefits from this finding, followed by the possible study to improve this technique in near future.

References: please check again to include all the citations in the text to the references list.


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: NUTRITIONAL PROPERTIES OF SELECTED SUPERFOOD EXTRACTS AND THEIR POTENTIAL HEALTH BENEFITS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The author(s) should show if there is clear difference(s) between super-food and functional food as shown in LINE 52 - 58. Or can the names be used interchangeably?
Experimental design: The fatty acid composition protocol is missing (LINE 189).

while LINE 261 should read In vitro......
Validity of the findings: No comment
Additional comments: The manuscript is well written in a simple and constructive language, with minor modification as shown in the individual sections.


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: NUTRITIONAL PROPERTIES OF SELECTED SUPERFOOD EXTRACTS AND THEIR POTENTIAL HEALTH BENEFITS
Review round: 1
Reviewer: 3

Output:
Basic reporting: Authors report a simple aqueous extraction technique of specific superfoods and the treatment of gastrointestinal cells in vitro, trying to provide simple methods to provide evidence of their clinical benefits.
Since other compounds are higher than compounds studied it would be interesting to focus more on antioxidant compounds present in these superfoods. Results are not clearly support hypothesis of study.

Please check my comments below on experimental work, result expression and literature cited.
Experimental design: L 11, for Lipid content and fatty acid profiles of flours please give a brief experimental description
For crude aqueous body temperature extracts, authors could have used acid since in the stomach acid conditions occur. Is the method used in house? if not please add reference/s and explain why specific amounts of flour and water were used (4g flour to 200ml water)..
L. 121-121 did authors prepare 5mg/ml in RO water for for compositional analysis as well. What RO stands for? why not use RO instead of deionized water for extraction? At L. 136-137 authors say that Extracts (5 mg/mL) were diluted in DMEM but at L. 120 in PBS, there is a conflict here, please explain.
Please add reference/s for IEC-6 and Caco-2 Cell Culture section experiment.
monosaccharides analysis is not clearly presented, how 1-phenyl-3-methyl-5-pyrazoline (PMP) derivatives were prepared?  Quantification of results how it was expressed? a typical HPLC chromatogram should be added.
A brief description of ICP-MS should be presented. Validation of method is not presented, what are the LOD and LOQ of each mineral?
Validity of the findings: Results of monosaccharides analysis are presented as D- and L- however at M&M thay are not mentioned the same way, also results are %w/w why not in concentrations of initial amount of flour used, please revise
For mineral content std deviation should be added to results.
Fig 2 and 3: error bars are not visible to check significant differences, also there are a and b for statistical analysis for flaxseed extract, why only for one value?
Significant digits should be checked for results at figure 3 for 1mg/ml flax seed extract
Additional comments: Concerning references they need to be rechecked for example, Arai 2002, Proestos 2018, Goyal et al., 2014; Ixtaina et al., 2011, Callaway, 2004, Mohamed et al.,2020 and many more are missing from Reference list even though they are present within text, please revise.
why authors used Ferric Reducing Antioxidant Power (FRAP) Assay? this is usually for biological samples and when antioxidants like phenolics are studied, please explain.


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: NUTRITIONAL PROPERTIES OF SELECTED SUPERFOOD EXTRACTS AND THEIR POTENTIAL HEALTH BENEFITS
Review round: 2
Reviewer: 1

Output:
Basic reporting: Well described
Experimental design: Well described
Validity of the findings: Well described
Additional comments: All comments have been addressed well, EXCEPT 1 more in conclusion: the citation is unnecessary in conclusion, which has not removed yet. even thought its mentioned in the rebuttal letter the revision has been made, in fact no revision is made in conclusion part.


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: NUTRITIONAL PROPERTIES OF SELECTED SUPERFOOD EXTRACTS AND THEIR POTENTIAL HEALTH BENEFITS
Review round: 2
Reviewer: 2

Output:
Basic reporting: The report is well written in simple language that would enable even non experts to understand the work. Though, not without some common errors as highlighted.

LINE 27 help with or  should be deleted

LINE 63 'both'  and 'a'  should be deleted
Experimental design: The experimental designs are appropriate.
Validity of the findings: The findings are valid within the concept design.
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: NUTRITIONAL PROPERTIES OF SELECTED SUPERFOOD EXTRACTS AND THEIR POTENTIAL HEALTH BENEFITS
Review round: 2
Reviewer: 3

Output:
Basic reporting: Article is written in clear, understandable English language
Authors added references that were missing.
The structure, the figures and Tables are corrected according to my comments and suggestions.
Experimental design: All queries concerning experimental procedures, materials and methods and statistical analysis were adequately replied. Response is accepted.
Validity of the findings: Results are clear and well presented. Response from authors regarding result expression is satisfactory and accepted. Conclusions are supported by results, well stated.  Authors provide all data replying to my comments.
Additional comments: Article is written in clear, understandable English language
Authors added references that were missing.
The structure, the figures and Tables are corrected according to my comments and suggestions.


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: APPLICATION OF ULTRASOUND GUIDANCE IN THE ORAL AND MAXILLOFACIAL NERVE BLOCK
Review round: 1
Reviewer: 1

Output:
Basic reporting: Well done to the authors for an interesting review. Overall it is well written and supplemented with comprehensive summary tables, clear figures and relevant references.

Proposed improvements:
1. The results and discussion in the abstract is too general. Authors may consider to specifically state the important findings from the literature review.
2. The result section can be improved with a descriptive summary of selected article types  and parameters of interest (i.e nerve, approach, etc.) at the beginning of this section.
Experimental design: The design of this study is acceptable for a literature review.

For improvement:
1. In view of an almost similar literature search process according to PRISMA protocol, authors may add a flow chart (diagram) to show the process of literature selection.
Validity of the findings: The findings from the literature review are well summarized and reported. Excellent summary tables.
Additional comments: Additional comments:
1. Additional diagram on ultra sound application at the oral and maxillofacial region can be considered.
2. Brief discussion on study limitation is also valuable.  This can be supplemented with a summary table of level of evidence for each selected article. Another alternative is to add column for 'Level of Evidence' next to 'Authors' column in all 3 tables.
3. Minor typographic error: 'In palne' in table 1 (first row).


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: APPLICATION OF ULTRASOUND GUIDANCE IN THE ORAL AND MAXILLOFACIAL NERVE BLOCK
Review round: 1
Reviewer: 2

Output:
Basic reporting: Please correct the grammar. Commas are used injudiciously throughout the text.
Experimental design: The search strategy for the articles is not very clear. "Most of the papers were published after 2010" the statement is very subjective. Please rewrite it objectively.
Validity of the findings: Intra oral approach – inferior alveolar nerve. The authors have relied on insufficient evidence to conclude that there is limited utility in using ultrasound guided nerve block of inferior alveolar nerve. It is better to stress on the paucity of clinical studies while concluding the same.
Additional comments: There is insufficient information regarding the type of ultrasound probe and the frequency used in these studies


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

Input:
Title: APPLICATION OF ULTRASOUND GUIDANCE IN THE ORAL AND MAXILLOFACIAL NERVE BLOCK
Review round: 1
Reviewer: 3

Output:
Basic reporting: The article is well written and in professional English.
The review have covered a wide range of the area and does not need any amendments or changes.
Experimental design: The article have reviewed the relevant papers and succeed to come up with a solid coherent article.
Validity of the findings: Findings and conclusions are well stated.
Additional comments: I highly recommend this article 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: APPLICATION OF ULTRASOUND GUIDANCE IN THE ORAL AND MAXILLOFACIAL NERVE BLOCK
Review round: 2
Reviewer: 1

Output:
Basic reporting: The article meets the standards of the journal
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The article can be published in its current form


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

Input:
Title: EVALUATING WHOLE-GENOME SEQUENCING QUALITY METRICS FOR ENTERIC PATHOGEN OUTBREAKS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The article overall is well written, offers a good list of references that are used in the context of the results observed by the authors. Figures, tables and data are well made.
Experimental design: The methods are well described and in detail. The research question, although overall significant, looses a bit of impact due to the choices of dataset and tools selected by the authors. Specifically, the authors selected four datasets, representing (i) one E. coli outbreak, (ii) two Salmonella outbreaks and (iii) one Shigella outbreak. These three species are some what related (in comparison to other species also relevant to food safety such as Listeria monocytogenes and Campylobacter) and share many characteristics not shared by these other foodborne pathogens, such as a large genome (>= 5 MB) and ~50% GC content (in comparison to smaller genome sizes and low %GC of L. monocytogenes and Campylobacter). Therefore, it seems inappropriate to generalize the results obtained in this study to  these other important foodborne pathogens. This problem could have been easily "fixed" by inclusion of other species representing a more diverse genomic context. In addition, the authors selected lyveSET as a tool for assessing the impact of raw read quality in SNP discovery. Although this tool has shown its usefulness in SNP discovery applied to foodborne pathogens, it does not seem to be currently used by any public health or regulatory agency for real-life investigations using WGS data. It could have been more useful, for example, to know how raw read quality impact the SNP discovery using the CFSAN SNP pipeline, which was developed by FDA-CFSAN, and is currently used by this agency in outbreak/traceback investigations. Also, public health agencies in the US, currently rely on cgMLST for outbreak investigation and, therefore, learning the impact of raw read quality on cgMLST results would have been more useful.

A second, unrelated concern: did the authors check the raw sequence data (and/or assemblies) for contamination? Can you please describe?
Validity of the findings: The conclusions are well stated. However, I missed a comment on why and how the raw read quality impacts on assemblies and SNP discoveries. In the case of SNP discovery, is it because of read mapping not being successful or because of not passing the selected cutoffs (minimum number of reads mapping, % concordance, etc), or both? For assemblies,  do reads with low quality generate misassemblies or just prevent assembly of larger contigs? Providing the readers with some explanation on this would be very helpful.
Additional comments: Line 92: fix last reference (Hannon 2010)
Lines 109-110: The causal-effect relationship presented in this sentence does not make sense to me. Why isolates collected over the span of several months necessarily would need to have their genomes sequenced using 3 different Illumina cycles?
Line 114: "...such as high..."
Lines 170 (in comparison to line 167): Why using a different notation here? I suggest using:
FPi = | Si - (Sj U Sk U Sl)|


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: EVALUATING WHOLE-GENOME SEQUENCING QUALITY METRICS FOR ENTERIC PATHOGEN OUTBREAKS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The language is clear throughout. However, should the sentence in line 99 begin with "This study" instead of "The study", as the previous sentence refers to "the current study"?

The authors provide adequate explanations for the filtering and trimming read-healing strategies (lines 87 to 93), but the explanation of how error correction strategies work is lacking. How do kmer and alignment techniques correct miscalls?

The figure caption for Figure 2 appears to be incorrect, it appears to be a repeat of the caption for Figure 1.

Figures S1 and S3 are not referred to in the text.

Line 368 to 369 - Figure 3 is referenced, but this text is referring to Figure S3.

There were some errors in references cited in the text (e.g. line 92, is there supposed to be a semi-colon after the Schmieder & Edwards 2011 reference?; line 531, the dates are missing).
Experimental design: Methods are described in detail, including locations of scripts and flags used during analyses. The Benjamani-Hochberg correction is mentioned in the caption for Table 2, but not described in the methods section.
Validity of the findings: The authors describe in detail their findings and conclusions based on the data.

Although the authors explain that SPAdes is demonstrated to be among the most accurate assemblers, it would be interesting to see the impact of an assembler such as SKESA (Souvorov et al., 2018) which is another popular assembler that is more scrupulous than SPAdes and has been shown to produce higher quality assemblies.
Additional comments: Overall well written. As a note, line 53 states that genome assembly "enables" characterisation of genes involved in resistance and virulence, however assembly is not required for gene detection (e.g. SRST2 by Inouye et al., 2014; and the KMA tool by Clausen et al., 2018).

The article may also benefit from a description of how widespread SNP analyses are, and what they are commonly used for.


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: EVALUATING WHOLE-GENOME SEQUENCING QUALITY METRICS FOR ENTERIC PATHOGEN OUTBREAKS
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment. I thank the authors for clarifying and responding to my previous comments.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: No comment.


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

Input:
Title: IDENTIFICATION AND FUNCTIONAL ANALYSIS OF BACTERIA IN SCLEROTIA OF CORDYCEPS MILITARIS
Review round: 1
Reviewer: 1

Output:
Basic reporting: 1. The language of this manuscript needs to be polished.
2.The expression of research results needs to use the past tense. For example, "shows" in the “abstract” should be changed to "showed".
3. The sentences in lines 33-36 need to be language polished and relevant references need to be added.
4. The format of Table S1 needs to be modified. For example, please delete the blank rows in the Table S1.
5.The sentences on lines 237-239 need grammatical corrections.
6.On lines 265-266, "indicate" should be changed to "indicated", and "is" should be changed to "was".
Experimental design: 1. The authors need to explain in detail what is the significance of this research.
2. In line 70 "The 12 C. militaris...", what does this "12" mean? Please explain in the manuscript.
3.Is the "102.92 W" on line 228 correct? I think it is wrong.
Validity of the findings: no comment
Additional comments: 1. "Potato-dextrose-agar (PDA)" appears in line 202, but "potato-dextrose-agar" also appears in line 80. Therefore, "potato-dextrose-agar" in line 80 should be changed to "potato-dextrose-agar(PDA)", and "potato-dextrose-agar (PDA)" in line 202 should be changed to "PDA".
2. The “P” on line 209 needs to be italicized.


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

Input:
Title: IDENTIFICATION AND FUNCTIONAL ANALYSIS OF BACTERIA IN SCLEROTIA OF CORDYCEPS MILITARIS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The English language and grammar in this manuscript should be greatly improved throughout. Please also see Detailed corrections and comments.

Line 15: “the corresponding ecological functions” should be “their corresponding ecological functions”.

Line 21: “shows” is changed to “showed”.

Line 22: “Serratia could reduce nitrate” should be “and Serratia could reduce nitrate”.

Lines 23-25: “Two strains of bacteria, N-2 and N-26, were isolated from the sclerotia of the C. militaris, cultured, biochemically characterized, and identified as Stenotrophomonas maltophilia and Pseudomonas” can be improved into “Two strains (N-2 and N-26) of bacteria isolated from the sclerotia of C. militaris were identified respectively as Stenotrophomonas maltophilia and Pseudomonas, based on the cultural and biochemical characters” .

Lines 37-38: “Much attention has been paid to the development of C. militaris (Jin et al., 2018; Lee et al., 2020).” should be “Much attention has been paid to the cultivation and ultilization of C. militaris (Jin et al., 2018; Lee et al., 2020).”.

Line 39: This statement is not accurate enough. In fact, Cordyceps militaris can parasitize insect larvae, pupae and adults.

Lines 39-41: These three sentences could be combined into one sentence.

Line 58: “The effect of microorganisms on the sclerotia of C. militaris needs further research.” could be replaced by “The effect of microorganisms in the sclerotia on the growth and metabolite production of C. militaris needs further research.”

Lines 59-63: The whole paragraph should be rewritten to make sense.

Line 65: “Sample source and treatment” should be “Sample preparation”.

Line 66: “Sample:” should be omitted.

Line 67: The latitude and longitude information should be followed “Tieling City”.

Line 69: (GZUIFR) (Strain SYCM1910).

Line 71: “In other words, each sample was a mixed sample.” Should be omitted.

Line 72: Sclerotia samples were prepared by the method of Zeng (Zeng et al., 2019) with slight modifications.

Line 73: “C. militaris was rinsed” or “The sclerotia formed by C. militaris were rinsed”? C. militaris is a fungus.

Line 75-76: “After removal of the body wall of the insect, the specimen was sclerotia.” is changed into “After removal of the body surface of the insect, the sclerotia was used for analysis”.

Line 78: “Culture media and isolation of bacterial strains” should be “Bacterial community by cultural method”. I would suggest that “Identification of bacteria isolated from sclerotia” section is combined into “Bacterial community by cultural method”, to make the description more clearly.

Line 84: “Total bacterial DNA extraction, PCR amplification, and high-throughput sequencing” into “Bacterial community by non-cultural method”.

Line 97: “Isolated bacteria were observed” into “Isolated bacteria by the cultural method were observed…”.

Line 115 and 120: The subtitles should be omitted.

Lines 116-118: The bacteria grew well on PDA? Usually PDA is not a medium for bacteria.

Line 121: A piece of mycelia of C. militaris from a 21-day PDA plate was inoculated…?

Line 122: on a magnetic stirrer at ? rpm? You should indicate the speed of the stirrer.

Line 125: “Measurement of physical indicators:” should be omitted.

Line 129: “Biochemical determination:” should be omitted.

Line 135: “data processing” section should be combined into “Bacterial community by non-cultural method” section in Line 84.

Line 147: “Prediction of bacterial function” section should be also combined into “Bacterial community by non-cultural method” section in Line 84.

Lines 148-150: “FAPROTAX (http://www.zoology.ubc.ca/louca/FAPROTAX/) is a tool that can predict ecological functions of bacterial and archaea taxa from 16S rRNA amplicon sequencing (Sansupa, et al., 2021). We used it to identify ecological functions of OTUs.” may be replaced by “FAPROTAX (http://www.zoology.ubc.ca/louca/FAPROTAX/), a tool that can predict ecological functions of bacterial and archaea taxa from 16S rRNA amplicon sequencing (Sansupa, et al., 2021), was used to identify ecological functions of OTUs.”

Line 154: “Data analysis and presentation” should be “Data analysis”.

Line 157: “Results and Analysis” should be “Results”.

Line 158: In the “Methods and materials” section, you descripted “Bacterial community by cultural method” first, so it is better that you introduced the results according to this order. So I would suggest that you put the “Isolation and identification of bacteria” section (in Line 179) first, then “Bacterial community composition and ecological function” section.

Line 159: “A total of 204,067 effective sequences were detected in three samples of C. militaris” is changed into “A total of 204,067 valid sequences were detected in three sclerotia samples of C. militaris”.

Line 190: “By Basic Local Alignment Search Tool (BLAST) analysis” should be “By BLAST analysis”.

Line 202: “On potato-dextrose-agar (PDA) plates” should be “On PDA plates”.

Line 209: F and P should be italic.

Lines 237-239: These microorganisms may help to maintain plant hormone balance, control root development, promote nutrient acquisition, prevent disease, improve plant growth and maintain plant health?

Lines 239-241: The microorganisms are also ingested by root-feeding insects. When Cordyceps spp. invade insects, the microorganisms in the insect gut interact with the fungus (references?).

In your supplemental files, why the dry weight of the three replicates in the group N-26 showed significant difference (Raw_data_of_Figure_5)? Please this problem with your data.

Line 292: the references should be well formatted. They are not acceptable in the present format.
Experimental design: Line 70: “The 12 C. militaris” meat what? 12 insect larvae or pupae infected by C. militaris? You should indicate the insect stage (larva or pupa). And which species of the insects were infected by C. militaris?

Line 82-83: You should indicate the temperature you used to culture the bacteria. For bacterial isolation, why you also used PDA medium which is usually used for fungal isolation?

Line 129: You should provide more details about the sample preparation of cordycepin analysis. You used the liquid medium co-cultured with C. militaris and a bacterial strain for cordycepin analysis by HPLC?

Line 180: Only two bacterial strains were isolated from the sclerotia of C. militaris by cultural method?

Lines 212-214: You stated that “The addition of bacteria decreased the cordycepin content of the culture medium from 44.04 μg/mL in the control to 6.55 μg/mL with strain N-2 or μg/mL with strain N-26 (N-2: F=342.421, P < 0.01; N-26: F=406.635, P < 0.001) (Fig. 5C)”. You should make sure that the cordycepin decrease in the culture medium was not caused by the decreased biomass of C. militaris mycelia. From the “Methods and Materials” section, I do not know clearly how you prepared the samples for HPLC analysis.

In the Results, the results on the physiological and biochemical description of two bacterial species from “Isolation and identification of bacteria” section are not so interesting, because you can identify the bacterial species only by molecular marker and the identified bacterial species are common in insect community.

Although the isolated N2 and N26 showed significant inhibitory effect on the growth of C. militaris on PDA plates and liquid Sabouraud’s medium, and the cordycepin content in the liquid medium decreased sharply, it is uncertain that the cordycepin decrease in the culture medium was not caused by the decreased biomass of C. militaris mycelia. I suggest you express the cordycepin content in the culture medium by μg per g dry weight of mycelium pellets, to evaluate the influence of the bacterial strains on the cordycepin content produced by C. militaris mycelia.
Validity of the findings: Lines 174-178: It seems to me that Stenotrophomonas (OTU1490), Sphingobacterium (OTU2342), Pseudomonas (OTU1448, OTU2330, and OTU2314) and Rhodococcus (OTU1539 and OTU1423) are big groups with many species in each genus, so the predicted functions unlikely cover different species in the genus. Please verify these statements.

Lines 279-280: You indicated that “Our data indicate that some symbiotic bacteria may inhibit the production of cordycepin.”. You have to express the cordycepin content in the culture medium by μg per g dry weight of mycelium pellets, to evaluate the influence of the bacterial strains on the cordycepin content produced by C. militaris mycelia.

Line 284: Inclusion should be rewritten according to the new analysis of the the cordycepin content in the culture medium by μg per g dry weight of mycelium pellets.
Additional comments: No additional comments


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

Input:
Title: IDENTIFICATION AND FUNCTIONAL ANALYSIS OF BACTERIA IN SCLEROTIA OF CORDYCEPS MILITARIS
Review round: 1
Reviewer: 3

Output:
Basic reporting: The authors of the manuscript are interested in  Identification and functional analysis of bacteria in sclerotia of Cordyceps militaris. However, Zhang et al (2021) have compared the diversity and composition of the bacterial and fungal communities associated with naturally
occurring C. militaris, including sclerotia, stromata and the habitat soil and also the bacterial functions. The difference is the sample from different locations. In this Ms. the samples from Liaoning province have been used, and  only bacteria in sclerotia was studied. There was little new results.
On the other hand, I think the research question is not meaningful. The infection of C. millitaris on insect is easily. The bacteria in sclerotia should be studied in  other entomogenous fungus e.g.Beauveria bassiana and Metarhizium anisopliae which were been used in biocontrol.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: No additional comments


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

Input:
Title: IDENTIFICATION AND FUNCTIONAL ANALYSIS OF BACTERIA IN SCLEROTIA OF CORDYCEPS MILITARIS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The manuscript has been greatly improved, especially in the language, method description and result experession.

Some minor errors:

1.	Lines 159-160: Each C. militaris sclerotia sample weighed about 3.5 g. However, in Line 293, you indicated that “Each C. militaris sclerotia sample (0.5 g) was ground”. It seems that 3.5g is quite different from 0.5g.

2.	Line 393: (Guo, et al., 2016) should be formatted.
Experimental design: No comments
Validity of the findings: No comments
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: REMOVAL OF HEAVY OIL FROM CONTAMINATED SURFACES WITH A DETERGENT FORMULATION CONTAINING BIOSURFACTANTS PRODUCED BY PSEUDOMONAS SPP.
Review round: 1
Reviewer: 1

Output:
Basic reporting: Basic data has been represented well by the authors.
Experimental design: Data on chemical characterization is missing
Interpretation of CMC in the form of plots are missing
Panel diagrams can b created to represent the emulsification of oils, carbon sources etc. It would help for the better comparison
Validity of the findings: It has good scientific data. However, just washing  or dipping the oil coated material not sufficient to prove removal of oil. Some quantitative data is required which could be proved with other supporting experiments
Additional comments: Need to incorporate suggested data to improve the quality of the manuscript. It has been written well.


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: REMOVAL OF HEAVY OIL FROM CONTAMINATED SURFACES WITH A DETERGENT FORMULATION CONTAINING BIOSURFACTANTS PRODUCED BY PSEUDOMONAS SPP.
Review round: 1
Reviewer: 2

Output:
Basic reporting: The language is mostly fine and clear.
Background is not sufficient and hypothesis is not stated.
Experimental design: The study reports original data and the topic falls within the scope of the journal. However, the research question and knowledge gap  are not clearly identified. The adopted methodology is standard for preliminary biosurfactants studies. Some experiments are not sufficiently presented with the necessary information. Replicates are included and statistical analysis was performed. Proper controls are lacking for some experiments.
Validity of the findings: Rationale of the study and benefit of the work to the literature are not clearly stated. Controls are lacking in some experiments.
Conclusions have some overstatements.
Additional comments: General Comments
This study deals with the production of biosurfactants by some Pseudomonas strains and investigates the potential of biosurfactants as components in a detergent formulation for removal of heavy oil from artificially contaminated metal, glass, and plastic surfaces. The manuscript is written in a clear and mostly correct language.
However, the study did not provide tangible insights into, or advancement of, our current understanding of the field. No dought, biosurfactants are highly interesting microbial products with tremendous applications in the environment and industry. Nonetheless, large-scale commercialization is still suffering from economic obstacles due to high production and purification costs. There is a major knowledge gap that needs further investigations towards enhancing the commercial viability of biosurfactants. The literature is full of reports on biosurfactants produced by Pseudomonas spp., and their physicochemical characteristics are very much similar to those reported in this study.
Perhaps the most interesting part of the study is the detergent formulation part. However, it is very truncated and many proper controls are lacking. I propose that the authors remove the initial experiments on the production of biosurfactants and dedicate a large-scale study with proper controls
to further explore the potential of biosurfactants-containing detergents.

Specific Comments
Title:
The title is too generic and can be modified to better reflect the performed work. For instance: “Removal of heavy oil from contaminated surfaces with a detergent formulation containing biosurfactants produced by Pseudomonas spp.”
Introduction:
This is one of the weak aspects of the study. The introduction presented general information on biosurfactants. The knowledge gap, rationale, and research questions, are not clear. The lack of several studies on biosurfactants-containing detergents does not necessarily make it a significant knowledge gap in the field. It is very important to elaborate on detergent formulations by answering the following questions.
-How detergent formulations are formed?
-What components do they have?
-What is the role of each component?
- Are they more efficient than using the surfactants alone? Why?
- Are there earlier studies on biosurfactants-containing detergents? Give examples.
- How they perform compared to conventional (synthetic detergents)?
- What are the knowledge gaps or questions in the topic?
- Which questions are addressed in this study and what is the approach?
It is fine to start the introduction with some general information in one or two paragraphs, albeit this should be followed by more specific presentation of the research problem and rationale of the study.
Materials and Methods:
Overall, the adopted methods are standard for biosurfactants studies and mostly described with sufficient detail. Replicates are included and statistical analysis is relevant. The main issue is the part describing the detergent formulations.
Line 116: What was the volumetric ratio of ethyl acetate to the culture broth?
How the product was concentrated?
Treatment with the base and acetone needs some details.
Line 128: Specify which liquid and which conc. of the biosurfactants.
Line 132: Which concentrations were used for CMC measurement?
Line 136: Why the emulsification activity was not measured for the extracted biosurfactants?
Line 150: The negative control should be uninoculated culture medium because the biosurfactants were in cell-free broth.
Line 159: The techno-economic feasibility was not investigated in this study.
Line 188: The designation “industrial detergent” is irrelevant.
Line 191-192: Was the biodegradability of the component assessed? The mentioned previous studies should be cited.
Line 193: The identity (chemical names and sources) of the natural organic solvent and the thickening agent should be revealed.
Line 198: Heating at which temp?
Line 202: This is not clear.
Line 203-206: This part needs some details on how the efficiency of the different formulations was evaluated? Which industrial field applications were investigated?
Line 244: Some of the physicochemical characteristic should be included.
Line 244-255: For feasible industrial application larger surfaces and higher amount of oil contamination should be tested. The glass slide and metal nuts with 100 μl oil do not reflect the application potential at industrial scale. What was the form of the biosurfactants preparation used in the detergent? Cell-free culture broth or crude extract. Was it added as solid or dissolved in a solvent?

Since the detergent formulation has several components, it is interesting to test them separately and in different combinations for oil removal. These controls should be included to reveal their effect (if any) on the oil removal efficiency of the biosurfactants.
Obviously, the biosurfactants should be tested separately for the oil removal. For all these controls, the different components should be in comparable concentrations. This is important to show whether the detergent has any benefit. If it does not have, then it makes no sense to use the biosurfactants in a detergent formulation to avoid further unnecessary costs.

Results:
Interpretations of the results should be moved to the discussion. For instance
Lines 317-321, Lines 393-404, Lines 334-343.
The quality of the figures should be improved in terms of resolution. For the bar charts, significance of data should be indicated by letters.
Line 400-401: Abrasion and lubrication were not investigated.
Line 402: Technical and environmental feasibility were not investigated.

Discussion:
The discussion is lengthy and, for the major part, cited literature without meaningful interpretation of the results.
The authors should elaborate on the observed variations in the properties of biosurfactants produced by different bacteria. Also, the differences in the performance of the detergent formulations need some explanation.
Line 132: The CMC is reported as mg/L

Conclusions:
 The conclusion was presented more like a summary and focused only on the detergent formulation, although it represents a smaller part of the study. Moreover, some statements are not sufficiently supported with data. For instance, biodegradable (Line 565), industrial use (Line 566), organic pollutants (Line 569).
This is a lab study. Viability for industrial application needs large-scale or pilot studies.


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

Input:
Title: BIOGEOGRAPHIC EVIDENCE SUPPORTS THE OLD AMAZON HYPOTHESIS FOR THE FORMATION OF THE AMAZON FLUVIAL SYSTEM
Review round: 1
Reviewer: 1

Output:
Basic reporting: This manuscript uses the spatiotemporal pattern of six Metazoan clades in order to test the Old vs. Young Amazon hypothesis. This is to be commended, as this question has been hotly debated in the last decades.

In my opinion, one of the main issues is the relevance of such six clades for solving this decade-long controversy. I may be wrong, but I suspect that way more clades may follow all four criteria as summarized in the “Selection of taxa” subsection (see 101-109) among the myriad of Metazoan genera and families documented in the Amazon basin. Apart from these criteria, nothing is said about the significance of such sample for addressing the question, or the potential biases it may suffer from. Moreover, I see no clear consistency in this sample: the authors have studied a bee genus (volant and social), an amphibian (non-volant / semi-aquatic), as well as a bird family (volant) and three mammalian families (arboreal monkeys; rodents with a wide array of locomotory and habitat preferences; phyllostomid bats, volant and mostly fruit-eating). In other words, these groups have very distinct biogeographical histories, feeding habits, and dispersal/vicariant behaviors and capabilities when facing potential biogeographical barriers (especially overwater).

“Most studies support a “Young Amazon” model” (line 81). I do not agree, as the trend had considerably changed in the last decade. The literature survey as provided in the introduction fully ignores an entire range of recent references. Sedimentologists, structural geologists, palynologists, and vertebrate paleontologists have gathered a bunch of data supporting the decay of the PMWS and the onset of a transcontinental Amazon drainage by early late Miocene times in the last decade. See for instance Roddaz et al. (2010; Hoorn & Wesselingh book), Boonstra et al. (2015, Palaeo3), Salas-Gismondi et al. (2015, Proc Roy Soc B; 2016 PLoS ONE), Antoine et al. (2016; Gondwana Research), Jaramillo et al. (2017, Science Advances), Marivaux et al. (2020, J Hum Evol). Moreover, some studies have demonstrated that the Amazon basin had been split into two major subbasins (South vs. North) no earlier than in latest Miocene to Pliocene times (e.g., Espurt et al., 2007 Geology), which seems to match the biogeographical history of various plant and animal clades from Amazonia. In other words, the Amazonian geological history is much more complicated than the authors seem to depict it, in that events involving Amazon River and its tributaries have obviously occurred throughout the last 12 My.

Similarly, the authors write that they “agree with Shephard (2010), who suggested that the PMWS was greatly reduced in the middle-late Miocene.” This is fully at odds with more recent literature providing primary data on palynomorphs, marine microfossils, and aquatic communities (Boonstra et al., 2015, Palaeo3; Antoine et al., 2016, Gondwana Res; Jaramillo et al., 2017, Sci Adv; Feussom Tcheumeleu et al. (2019, Palaeodiv & Palaeoenv). In this scheme, an updated map is available for middle Miocene times (PMWS: see map in Marivaux et al., 2020, J Human Evol). It is adapted from that of Boonstra et al. (2015, Palaeo3).

To end with, a similar study focused on biogeographical history of Amazonian frogs was published by Vacher et al. (2020, J Biogeogr). It somewhat supports the current results, with a distinct geographical grid and pattern. It may be worth mentioning it.
Experimental design: As a paleontologist, I have not addressed molecular phylogeny-based methodology. Instead, I have focused my revision on geological premises, paleontological literature and taxonomic issues, notably related to the fossil record, in order to improve the manuscript.
Validity of the findings: Aside from the geological context, the authors seem to have missed some recent literature of primary interest regarding calibration points, at least for Echimyidae (Boivin et al., 2019 Geodiversitas; Courcelle et al., 2019, Mol Phyl Evol; Pérez et al., 2019, J Syst Palaeont) and Cebidae. For instance, the cebine Panamacebus is dated at c. 21 My (Bloch et al., 2016, Nature) and Cebus sp. was recognized in c. 11.5 My-old deposits (Marivaux et al., 2016 AJPA [cited for Cebuella, but seemingly not used for Cebus].
This may affect the ages of several dichotomies in the Fig. 4 and probably reinforce the authors’ conclusions. Accordingly, the Supplementary table with taxa and their ages needs to be widely enhanced, as some references are outdated, systematic assignment of several taxa is questionable, and more precision would be required. For instance, “Middle Miocene” is not precise enough as an age for cebids found at La Venta (see primary references in Kay, Madden, Cifelli, and Flynn, 1997; La Venta book). I would also recommend citing updated primary references (note that no reference section is available in the SI).

Moreover, the periods ranging the 10–6 My and 2–0.5 My time intervals coincide with drastic global climatic changes (e.g., Westerhold et al., 2020, Science), mainly affecting rainfall in tropical-equatorial areas (Martínez et al., 2020, Sci Adv). At a regional or a local scale, this has in turn affected plant community composition, with cascading consequences on animals feeding on plants and those feeding on insects (i.e., all clades studied here). In other words, the authors should be aware of the existence of confounding factors. I would suggest to clearly mention such caveats.
Additional comments: I have uploaded an annotated pdf of the main manuscript with some typos and minor issues, remarks, and suggestions. I could not upload the annotated version of the Supplementary data I had prepared (using track changes), as only one file can be uploaded on the review page but I keep it available if needed.


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: BIOGEOGRAPHIC EVIDENCE SUPPORTS THE OLD AMAZON HYPOTHESIS FOR THE FORMATION OF THE AMAZON FLUVIAL SYSTEM
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript uses clear English, but a few statements are somewhat ambiguous. I comment on these and other issues in my line-by-line. No specific hypotheses are put forward – I believe the authors have the necessary background information and are working on an appropriate system to do so
Experimental design: The manuscript uses clear English, but a few statements are somewhat ambiguous. I comment on these and other issues in my line-by-line. No specific hypotheses are put forward – I believe the authors have the necessary background information and are working on an appropriate system to do so
Validity of the findings: The results are very interesting for our understanding of Amazonian biogeography. However, I believe some assertions/conclusions should be alleviated, in particular the one that already appears in the abstract and is developed in the discussion – “we suggest that the temporal range for the beginning and complete formation of the Amazon fluvial system might be older than previously proposed”. The method the authors used to estimate phylogenies and associated diversification times is robust, however, there are theoretical principles that are not taken into account when such conclusions are put forward (please check my main concern #1 in the line-by-line review). Both fossils and trustworthy biogeographic information could be used to estimate divergence times. What the authors are implying is that the former would provide more realistic information on the actual divergence times and, hence, could be used to assess our current knowledge on the geologic dating of the formation of such geologic events (the formation of the rivers) and its importance to understand the biogeography of the region. The arguments on lines 312-314 highlight, again, my main concern #1: your analyses cannot be used to ‘assign’ dates to geologic events such as the subsidence of the Purus. They can only provide evidence that these dates might need to be reassessed, or that the particular taxa used in this study do not show biogeographic patterns that support the currently recognized dates (but others might).
Additional comments: Please find my complete line-by-line review below. I raise three points that I consider of particular relevance, and that should be addressed when preparing a revised version of the manuscript.

1)	Dated geologic events are the main information that evolutionary biologists can use to investigate the history of life on earth, and molecular dating evidence can be used to corroborate the geologic evidence. However, the opposite is not necessarily true unless we are talking about poorly understood system. Much more needs to be understood about the geologic history of the Amazon basin, but it is certainly not a poorly understood system (Wesselingh et al., 2011). The fact that in many instances there is an inversion of this interpretation should definitely be addressed by the authors (e.g., my comments on line 61–63).
2)	The lack of a quantitative comparative approach, instead of a visual comparison of results.
3)	The lack of a clearly-stated hypothesis-testing framework. One can infer the author’s working hypothesis, but clearly stating it would make the presentation of results easier to follow.

57: typo, should be “Plio” not “Pilo”.

59: Ribas et al 2011 should be 2012.

61-63: This assertion relates to my main concern on this study – the interpretation that using dated phylogenies can provide estimates for the dating of the geologic events, which seems to be an inversion of how the evidence should be used in biogeographic studies. Of course, biogeographic information can and should be used to corroborate the estimates made from geologic information. However, geologic dating is the main primary evidence for the history of the planet and not the opposite. Robust geologic dating cannot simply be refuted using molecular dating techniques, since those are known to have many caveats (Duchene et al., 2014; Bromham et al., 2017). Also, even assuming that a molecular dated phylogeny provides ‘perfect’ dates, the processes behind the actual dates might not be the geologic events in question. Many other evolutionary/biologic processes can be invoked to explain the evolution of particular clades in a particular biogeographic scenario. The null hypothesis that a simple vicariant biogeographic scenario is the best evolutionary scenario will certainly not hold true in many cases. The processes behind the divergence times must be interpreted in a population-based scenario, i.e., other evolutionary mechanisms that, for example, cause changes in population sizes, or introgression among the selected clades, might influence the estimation of branch lengths and, hence, dating results in a Bayesian framework (Ho & Duchene, 2014; Bromham et al., 2017; Dos Reis et al., 2018), and not only major biogeographic events. In summary, Alfaro et al 2015 do not, in any way, propose plausible dates for the formation of Amazonian rivers – they do provide evidence that the evolutionary history of Saimiri was influenced by the rivers, supporting a geologic model that was built based on geologic (and not molecular dating) evidence.

77: “either” implies that another reason would also be put forward, which was not the case.

81: Should be a comma after “general”, not a period.

81–83: Background knowledge on Amazonia biogeography is only briefly reported, although I understand the approach considering the vastness of the subject. However, I believe it would be interesting to develop a better argument on the possible reasons why some studies support a “young Amazon” model and others an “old amazon”. In the next paragraph, a few reasons are put forward (secondary calibrations, number of species, studies at the population level, etc), but they are only mentioned as possible problems. Considering the scope of this manuscript, I believe that a paragraph dedicated to highlighting the main differences between studies that corroborate young versus old models would be highly beneficial to the readers, and also to support the aims of the study.

85–89: Although I appreciate the brief reminder on historical biogeography and panbiogeography, it seems that appropriate references are missing (Croizat is actually cited in the reference list, but no publication date appears here).

89: You mean that there are no “biogeographical studies that incorporate different taxa” for the study of Amazonian diversification? This is a strong assertion, and I highly disagree with it. First, what do you mean by taxa here? You used different taxonomic ranks here, which is fine, but other studies have certainly incorporated much more dense sampling, covering dates that could certainly indicate both old and new diversification, and used many taxa as well, such as (Santos et al., 2009; Smith et al., 2014).

95–97: “the goal of the present study is to evaluate the fit of molecular phylogenetic and biogeographic data to the previously described models regarding the age of formation of the Amazon fluvial system”. The way that this is put forward is correct and contradicts the way other arguments are built in the study (see my main concern #1).

Material and Methods
I commend the authors for their clear and well-designed selection of taxa strategy.

I am also particularly happy with the phylogenetic reconstruction and divergence times estimation methods. The authors employed the best available methods for their case and used many strongly recommended error-checking strategies, and I can only congratulate them for that.

159–165: I am not convinced that the VIP analysis was suitable for this case. It seems to be a highly simplistic model and, more importantly, is not able to actually provide the information that is presented in Fig. 5. I understand one can assume that the highlighted river was the biogeographic barrier responsible for the vicariant event, but this is not actually tested (and only visually inferred as far as I can tell). There is a more modern version of this type of analysis, written and implemented by the same author (Arias, 2017). Nonetheless, I suggest the authors to use a model-based inference based on evolutionarily explicit models  - rase (Quintero et al., 2015), in line with their beautifully performed phylogenetic inferences.

The BioGeoBEARS analyses were also quite nicely performed, thank you.

Congruence between events (main concern #2): it seems to be a problem in the proposed “comparative” approach since no statistically explicit comparison is performed, but a simple interpretation of congruence based on visual estimates. There are a few available methods that could infer the concordance among the dates (Oaks, 2019) or, maybe more important in this case, estimate the congruence among the vicariant events (nodes on the trees) (Satler & Carstens, 2016).

Results
232–249: A few sections could be improved for clarity.

251–252: The fact that the analyses using only the priors resulted in unresolved topologies is not surprising. Running the analyses only using the priors should be used to assess how informative the data are, and also to check if the priors were correctly specified. This was already reported in the previous paragraphs, so the authors might consider removing this information.

Isolation barriers and ancestral reconstruction: it would be much easier to understand this section if the results were directly associated with the proposed models (young versus old). This relates to my main concern # 3: if a hypothesis-testing framework was put forward, the importance and understanding of each of these results would be largely improved.

Discussion
285: please use ‘proposes’ instead of ‘recreates’.

339: ‘than’ instead of ‘that’.

353: ‘less precise’ in relation to what?

I appreciate how some potential problems with the divergence times estimations are taken into consideration, but I suggest that these are presented in light of their implication; e.g., how would your interpretation of the whole system change considering that your ages might be older because no internal constraints were present for most taxa?

Figure 3: It seems that two distinct plots were used to visually accommodate the differences in the age scale (y-axis). I suggest the authors write this out in the legend.

Figure 4: the authors might consider depicting all trees using the same scale, so they can be directly compared.

References cited in the review:
Arias, J.S. (2017) An event model for phylogenetic biogeography using explicitly geographical ranges. Journal of Biogeography, 44, 2225-2235.
Bromham, L., Duchene, S., Hua, X., Ritchie, A.M., Duchene, D.A. & Ho, S.Y.W. (2017) Bayesian molecular dating: opening up the black box. Biol Rev Camb Philos Soc,
Dos Reis, M., Gunnell, G.F., Barba-Montoya, J., Wilkins, A., Yang, Z. & Yoder, A.D. (2018) Using Phylogenomic Data to Explore the Effects of Relaxed Clocks and Calibration Strategies on Divergence Time Estimation: Primates as a Test Case. Syst Biol,
Duchene, S., Lanfear, R. & Ho, S.Y. (2014) The impact of calibration and clock-model choice on molecular estimates of divergence times. Molecular Phylogenetics and Evolution, 78, 277-89.
Ho, S.Y. & Duchene, S. (2014) Molecular-clock methods for estimating evolutionary rates and timescales. Molecular Ecology, 23, 5947-65.
Oaks, J.R. (2019) Full Bayesian Comparative Phylogeography from Genomic Data. Syst Biol, 68, 371-395.
Quintero, I., Keil, P., Jetz, W. & Crawford, F.W. (2015) Historical Biogeography Using Species Geographical Ranges. Syst Biol, 64, 1059-73.
Santos, J.C., Coloma, L.A., Summers, K., Caldwell, J.P., Ree, R. & Cannatella, D.C. (2009) Amazonian Amphibian Diversity Is Primarily Derived from Late Miocene Andean Lineages. PLoS Biol, 7, 0448–0461.
Satler, J.D. & Carstens, B.C. (2016) Phylogeographic concordance factors quantify phylogeographic congruence among co-distributed species in the Sarracenia alata pitcher plant system. Evolution, 70, 1105-1119.
Smith, B.T., McCormack, J.E., Cuervo, A.M., Hickerson, M.J., Aleixo, A., Cadena, C.D., Perez-Eman, J., Burney, C.W., Xie, X., Harvey, M.G., Faircloth, B.C., Glenn, T.C., Derryberry, E.P., Prejean, J., Fields, S. & Brumfield, R.T. (2014) The drivers of tropical speciation. Nature, 515, 406–409.
Wesselingh, F.P., Hoorn, C., Kroonenberg, S.B., Antonelli, A., Lundberg, J.G., Vonhof, H.B. & Hooghiemstra, H. (2011) On the Origin of Amazonian Landscapes and Biodiversity: A Synthesis. Amazonia: Landscape and Species Evolution (ed. by C. Hoorn and F.P. Wesselingh). Blackwell Publishing Ltd.


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: BIOGEOGRAPHIC EVIDENCE SUPPORTS THE OLD AMAZON HYPOTHESIS FOR THE FORMATION OF THE AMAZON FLUVIAL SYSTEM
Review round: 2
Reviewer: 1

Output:
Basic reporting: This manuscript is the revised version of a work that I had the opportunity to evaluate. This new version is widely improved with respect to the original one, and most of my comments have been taken into account (most premises, rationale, and literature).
Experimental design: Geological premises, paleontological literature and taxonomic issues, notably related to the fossil record, are now correct.
Validity of the findings: The results appear to be robust; the discussion is clear and the conclusions are not oversold.
The only sticking point is the depiction of a fully modern Amazon River network 14 Mya (Fig. 6C), both lacking the Pebas Mega-Wetland System and running continuously between the Andes and the Atlantic Ocean. There are no geological grounds for this. On the contrary, several authors (Croft, 2007 – Palaeontology; Antoine et al., 2013 – op. cit.; Marivaux et al., 2020, J Human Evol) use the concept of “western corridor” to explain the relative uniformity of terrestrial vertebrate components along the central to northern Andes. This could be a good premise.
Additional comments: Some references listed in the text do not follow a chronological/alphabetical sequence (lines 51-53, 60-61, etc.).

Line 64 and throughout the text (e.g., 346, 434): “middle-late Miocene” seems to be used in a wrong way. From what I have understood, the authors mean ‘the middle part of late Miocene times’ (around 10-9 Mya), which should be “middle late Miocene” (i.e., without the minus sign). When using the minus sign, the meaning is fully distinct, in pointing to the complete time interval encompassing middle and late Miocene times. Alternatively, f the authors mean the short time between the middle Miocene and the late Miocene, then they should use the “middle-late Miocene transition”.

Lines 71 and 75: “changed”

Line 86: “Pliocene-Pleistocene”

Line 126: “Meldrum”

Line 227: “undersampling”

Lines 369 and 374: “middle Miocene” instead of “mid-Miocene’

Figure 6: The panel C fully ignores the comprehensive corpus of data documenting the Pebas Mega-Wetland System by 17-10 My (see paleomaps by Antoine et al., 2013 – op. cit.; Boonstra et al., 2015 – Palaeo3; Marivaux et al., 2020 – J Human Evol [about a cebid, 13 My-old!]). In other words, there should be a “mega-pantanal” in Western Amazonia instead of a modern-like Ucayali-Marañon River network. Perhaps, both could be superimposed graphically, to help the readers.
 The authors are also suggested to take into account the western Corridor hypothesis (Croft, 2007; Antoine et al., 2007 – 4th EMPSLA proceedings; Marivaux et al., 2020 – J Human Evol) for explaining the expansion of distributional ranges between Atlantic areas and Western Amazonia by that Pebasian-climax time.
Moreover, there is absolutely no argument in favor of an uninterrupted transcontinental drainage (Andes–Atlantic Ocean) at that time and their results do not allow the authors to pass through such geological/geographical grounds. In other words, I would recommend them not to draw a continuous line between Western Amazonia and the lower Paleo-Amazon Basin (the mouth of which was probably located more to the south).

Pierre-Olivier Antoine


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: BIOGEOGRAPHIC EVIDENCE SUPPORTS THE OLD AMAZON HYPOTHESIS FOR THE FORMATION OF THE AMAZON FLUVIAL SYSTEM
Review round: 2
Reviewer: 2

Output:
Basic reporting: The revised version of the manuscript is a much-improved version, and I congratulate the authors for their efforts in responding to all my concerns. The manuscript uses clear English, but a few section could be improved for stylistic reasons – I briefly comment on those below, but please use them at your discretion – they are not mandatory changes, but only suggestions.
Experimental design: The research questions are clear, and the phylogenetic and biogeographic methods are particularly robust in this revised version of the manuscript.
Validity of the findings: The results are very interesting and add important evidence to our understanding of Amazonian biogeography.
Additional comments: The first paragraph consists of only two short sentences, highly contrasting with the second one. The riverine barrier hypothesis was strongly developed since the publication of the seminal work of Haffer on the distribution of lowland vertebrate Areas of Endemism. I understand the authors intention on not particularly touching on this subject, but a few studies do provide the means to incorporate such novel information on the background they choose to develop, such as (Santorelli et al., 2018), and (Oliveira et al., 2017).

I reiterate that only using the priors resulting in unresolved topologies is not surprising – the analyses have no information to rely on in order to obtain branch lengths and bipartitions. These results should be used to compare the fit of the data to the prior (Nascimento et al., 2017). I do believe the authors used this information well, and no actions should be taken apart from, maybe, changing the text (nonetheless, it should be noted that it is expected that the posterior is narrower, and cases where it is not might indicate that the calibration points are not informative, although this should be interpreted with caution).

Nascimento, F.F., Reis, M.d. & Yang, Z. (2017) A biologist’s guide to Bayesian phylogenetic analysis. Nature Ecology & Evolution, 1, 1446-1454.
Oliveira, U., Vasconcelos, M.F. & Santos, A.J. (2017) Biogeography of Amazon birds: rivers limit species composition, but not areas of endemism. Scientific Reports, 7
Santorelli, S., Jr., Magnusson, W.E. & Deus, C.P. (2018) Most species are not limited by an Amazonian river postulated to be a border between endemism areas. Sci Rep, 8, 2294.