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: DETECTION AND BENCHMARKING OF SOMATIC MUTATIONS IN CANCER GENOMES USING RNA-SEQ DATA
Review round: 1
Reviewer: 2

Output:
Basic reporting: The paper is well written and the hypotheses of this work are well defined. However, in my opinion the introduction should contain the background knowledge behind this work. From line 57 to 77, the authors explain the method but this is not the methods section.
Experimental design: This work fits very well the aims of the journal and it's potentially very interesting and useful for the community. The experimental design is well described however, several points need to be clarified.

1) First of all, in lines 63-67 the authors reported "First, we estimated the proportion of germline/somatic mutations by comparison of identified variants to the dbSNP database (Kitts et al. 2013) which catalogs known germline variants, and the Catalogue Of Somatic Mutations in Cancer or COSMIC database (Forbes et al. 2015)". These two database were used to differentiate between germline and somatic mutations. The key point here is that based on the dbSNP/NCBI handbook, dbSNP includes both germline and somatic mutations: "dbSNP accepts submissions for all classes of simple sequence variation, and provides access to variations of germline or somatic origin that are clinically significant"
https://www.ncbi.nlm.nih.gov/books/NBK174586/

Since this is a crucial step in the pipeline, the authors should comment on that. If the percentage of somatic mutations in dbSNP is low, the authors should show that and evaluate how this bias can affect the rest of the analysis. All the downstream analyses could be affected by this crucial step.

2) The number of samples is very low. You need a validation set to verify your discoveries.

3) The authors don't mention if the data are deposited in some repositories already or not (the internal dataset).

4) In line 129 and 130 the authors don't motivate why they used GATK, since 30 normal samples is recommended by GATK, and you only used 12 normal samples.

5) Related to my comment 1, the statement in line 392 can be taken into account at the beginning of the pipeline by considering only the germline mutations in dbSNP.
Validity of the findings: The findings depend on the answer to my previous comments, especially comments 1 and 2.
Additional comments: In general, this pipeline is well described, implemented and potentially useful in order to use RNA-seq to identify somatic mutations. However, the authors have to address several points (see 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: DETECTION AND BENCHMARKING OF SOMATIC MUTATIONS IN CANCER GENOMES USING RNA-SEQ DATA
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have addressed all the issues I  pointed out in the first review.
Experimental design: The authors have addressed all the issues I  pointed out in the first review.
Validity of the findings: The authors have addressed all the issues I  pointed out in the first review.
Additional comments: The authors have addressed all the issues I  pointed out in the first review. Therefore the article meets the PeerJ criteria and should be accepted as is.


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

Input:
Title: DETECTION AND BENCHMARKING OF SOMATIC MUTATIONS IN CANCER GENOMES USING RNA-SEQ DATA
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors have answered to all my comments.
Experimental design: The authors have answered to all my comments.
Validity of the findings: The authors have answered to all my comments.
Additional comments: All my comments/questions have been answered. I'm satisfied with the revision and therefore I suggest the publication of the work in its current version.


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

Input:
Title: VENOMIX: A SIMPLE BIOINFORMATIC PIPELINE FOR IDENTIFYING AND CHARACTERIZING TOXIN GENE CANDIDATES FROM TRANSCRIPTOMIC DATA
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript is mostly well written even though some sentences were occasionally confusing, I would comment careful reading of the whole manuscript. Some literature references could also be improved and updated. Some important considerations:

Comment 1: The annotation pipeline by Pineda et al. 2018 should be mentioned.

Comment 2: The authors only site Trinity for assembly and RSEM for quantification (lines 640-641). Please expand to include other softwares.

Comment 3: Figure 1, the authors write "Pulls expression information for each from toxin-like transcript.". This statement is confusing
Experimental design: Extracting useful information from transcriptomes is an important research question. The authors seemed to have fairly analysed their software but some additional analyses are required to fully understand if this pipeline could be useful at identifying toxins.

Comment 1: The authors should  include a discussion on the inclusion of proteomics data on the identification of toxins. Transcriptomics data are stained with a range of sequencing errors and combining a transcriptome analysis with mass-spectrometry outputs has become a main stream method for identifying toxins in venoms. The authors do not seem to have considered this point in their pipeline and some comments on that point or how to integrate mass-spectrometry data with existing tools (protein pilot?) would be useful.

Comment 2: The authors should mention signalP as necessary programs in Figure 1.

Comment 3: It should also be mentioned how are partial sequences treated? Are they flagged or not as partial? Do they receive partial treatments?

Comment 4: The authors compared the number of toxins identified using their pipeline with the number of toxins identified in the literature for the analysis of the same transcriptomes. Could the author also analyse what is the overlap between their identified toxins and the one reported in the literature? ie are the toxins the same or novel?

Comment 5: What about when the reads do not need to be assembled? Cone snail toxin transcripts are small enough to be included in the raw reads of a 454 sequences, potentially removing problems with spurious assemblies created by Trinity and similar softwares. This should be mentioned also by the authors
Validity of the findings: In this research article the authors describe a new pipeline to analyse the toxin contents of a venom by analysing transcriptomics next generation sequencing data. Their pipeline combined the outputs of several programs that quantify expression level, identify open reading frames, signal sequences, closest sequences from a dataset (ToxProt) and create phylogenetic trees. They used their pipeline to analyse the transcriptomes of four next-generation sequencing datasets extracted from public repositories. The authors made a number of claims, which unfortunately do not seem to be fully supported by the data presented in the manuscript.

Claim 1: The authors claim that their method identified more candidate toxins than reported in the original analyses. The data presented by the authors do not support that clain. Indeed, Table 3 highlights that the pipeline developped by the authors has missed a large number of gene superfamilies in the cone snail transcriptome. Have the authors tried to identify what part of the pipeline was causing problems? For example, the con-ikot-ikot proteins are quite large and should be easy to identify. Why did the pipeline only returned 3 toxins from the T-superfamily whereas Phuong et al. 2016 discovered 56 toxins?

Claim 2: The authors claim that "Venomix is the first pipeline to provide all these outputs in an easy to use search strategy" but I cannot see any evidence of this claim, what is the format of the output that makes it so practical? I cannot see any summary tables being produced by the pipeline neither easy to understand.

Claim 3: The authors state that they provided a pipeline that would help researcher with little bioinformatics skills to analyse transcriptome data but they require the users to provide: assembled reads into transcriptome, gene expression information in a tab delimited output and a tab delimited blast output against the ToxProt database. This cannot be considered as user friendly unless the authors provide a protocle with their package to help non-bioinformatic researchers produce these files.
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: VENOMIX: A SIMPLE BIOINFORMATIC PIPELINE FOR IDENTIFYING AND CHARACTERIZING TOXIN GENE CANDIDATES FROM TRANSCRIPTOMIC DATA
Review round: 1
Reviewer: 2

Output:
Basic reporting: Macrander and colleagues propose a very interesting tool designed for researchers who are not specifcally into bioinformatic and want to isolate toxin candidates in high-troughput sequencing data. Such tool is of great interest for the scientific community and the way the pipeline has been designed perfectly fills the gap between a very basic similarity search protocole and a refined machine-learning (or other cutting-edge) based method.
While the protocole is well designed, the way it is presented through this article does not allow to understand the advantages of this tool. The text itself is ok, sometimes going into too much details though, but the major issues are figures and tables. The principale figure presenting the pipeline itself should be simplified to allow authors to catch in a glance what are the keys steps of the analysis. Tables which summarize the comparative analysis should be represented as graphs and all other figures should go into the supplementary material. The discussion is ok but the material and methods as well as the results parts have to be shorten.
I thus suggest authors to rewrite the manuscript focusing their attention first on how the protocole and results are illustrated and second on giving a more synthetic view of the results.
Experimental design: The pipeline is well design. These are juste suggestions to improve it :

1)	If Venomix is designed to help non bioinformaticians in their search for new toxin candidates, I would like to see how it performs on a standard laptop, especially how long it takes for the similarity search. If the computing btime is reasonnable, it could be a big plus to mention it in the revised manuscript. Some people don’t have access to large cluster of calculation and it could encourage them to use Venomix
2)	For the similarity search process you could probably improve the sensity using an hmm approach . Hmms profiles are easy to build.
3)	You mentioned the influence of assembly steps. Something you could think about to adress this question is simpy mapping back the reads on toxin candidates (DNA sequences obviously)  or even predicting ORF on short reads ( what people use in metagenomic studies).
Validity of the findings: Venomix seems to allow the identification of more toxin candidates which makes it a valuable tool for the scientific community. I would like to see some basic statisctics as T-tests to see more clearly if the pipeleine allows to retrieve more toxin candidates compared to what have been found in the original publications. I
Additional comments: Specific points to adressto be  :

1)	Key word : I think that transcriptome, toxins and bioinformatic are enough.
2)	The term TMP has to be clearly define in the manuscript.
3)	Figure 1 has to be modified a lot, mainly sinplified. Just draw some boxes with the major steps and remove all the remainig text. It was really hard forme to understand what the program was doing and how the decision to clasify a sequence as a toxin candidate was done. There are also some typos.
4)	Table 1 has to go to supplementary and the commentaried about assembly parameters etc etc has to be shorten.
5)	Line 760 : Do you mean toxins from distantly related taxonomic groups ?
6)	Table 2 has to be drawn as a graphic. It would simply help the readers to understand how Venomix perform.
7)	Line 765 : you just have to specify one time that the less stringent approach corresponds to the use of a higher e-value for the blast. A more general commentary about writing is that many concepts are named differently all along the manuscript.  Sometines toxin candidates is quoted (line 791 and after), sometimes not. Sometimes the number of toxin is designed by N=x, sometimes the number is written before. This seems to be small details, but it would really help the readers to have allthis small little things cleaned.
8)	Line 781 : too many o in Conodipin
9)	Line 784 : something wrong with tha sentence ?
10)	The description of each Venomix output is a little bit heavy, escpecially because they are not described systematicaly in the same order. You should make one big paragraph to summarize the results ans all the small details should go into the supplementary.
11)	Line 805 ; ‘Have no known function’. Could you say based on what ?
12)	Line 835 : Naive users have to know that rsem is a read mapping based approach as well as BWA. We should learn that at the begining of the manuscript, not at the end.
13)	Table 3 and Table 4 should use the same exact same notation. Toxin family / super family ?
14)	Line 846 and after : this is way too detailed.
15)	Line 862 :  what you named ‘no input’ are default parameters.


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: VENOMIX: A SIMPLE BIOINFORMATIC PIPELINE FOR IDENTIFYING AND CHARACTERIZING TOXIN GENE CANDIDATES FROM TRANSCRIPTOMIC DATA
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript appears to adhere to PeerJ policies and follows the standard PeerJ article template. It is written in English with clear unambiguous text.
In this manuscript, is presented Venomix as a new pipeline to characterize the toxin-like diversity from transcriptomes in phylogenetically distinct organisms. Venomix incoporates widely used programs into its pipeline and provides the user with several output files that can be used for different purposes. To evaluate the perfomance of Venomix, in this study were used four transcriptomes of different species. Venomix recovered numerically more candidate toxin transcripts for three of the four transcriptomes than the original analyses.

Undoubtedly this new tool is quite relevant in the area of "venomics" since can quickly identify toxin gene candidates from transcriptomes of a great variety of species. Although the authors note that Venomix is not designed to be used to as definitive validation pipeline fo toxin genes their outputs are very informative and can be used to further evaluate these candidates about several aspects.

Also, as the pipelines are versatile, mainly two parts of the workflow (sequence alignment and tree reconstruction) could easily be modified with the preferred programs which could give a comparative perspective within of this pipeline.
Experimental design: The methods are described in sufficient detail to be replicated. Research question is well defined and the knowledge gap is easily identifiable. Venomix succeeds to solve three problems found in similar approaches (Reproducibility, not be a toxic gene identifier and to accommodate a wide variety of taxa).
.
Validity of the findings: Overall, this study is clear from its original research until your conclusions being supported by your results. The authors meet the objetive to show that the Venomix package is a useful tool to identifiy and characterize the diversity of toxin-like transcripts.They also open a lot of possibilities in the development of pipelines centered around this goal,  the identification of components of venoms in poorly studied lineages.
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: VENOMIX: A SIMPLE BIOINFORMATIC PIPELINE FOR IDENTIFYING AND CHARACTERIZING TOXIN GENE CANDIDATES FROM TRANSCRIPTOMIC DATA
Review round: 2
Reviewer: 1

Output:
Basic reporting: The answers given to the other reviewers are adequate and the changes made to the manuscript have improved it. It is excellent to have this tool for research on venoms.
Experimental design: Experimental design is clear. The pipeline is well design. The modifications suggested in this part by the other reviewers were carried out appropriately.
Validity of the findings: The authors respond appropriately to the suggestions of the other reviewers. The authors make very clear the strengths and scope of the pipeline as well as its weaknesses and also where the pipeline could be optimized.
Additional comments: Congratulations for the work.


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

Input:
Title: A COMPARATIVE ANALYSIS OF THE COMPLETE CHLOROPLAST GENOME SEQUENCES OF FOUR PEANUT BOTANICAL VARIETIES
Review round: 1
Reviewer: 1

Output:
Basic reporting: The structure of the article conforms with PeerJ standards, the article has many minor grammatical inconsistencies.  In figure 2, the y- axis title has a typographical error.
Experimental design: The mentioned methodology is sound and backs the results aptly,  however I would suggest to include the tool used for genome assembly in lines 101-102  and line 145-153.
Validity of the findings: The authors have provided enough rational explanation for backing their study and it is commendable.
Additional comments: The outcome of the work is novel and I think it would attract researchers to look into this aspect of chloroplast genome variation among the variants of the same species. I would recommend the article for publications after necessary revisions have been implemented.


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: A COMPARATIVE ANALYSIS OF THE COMPLETE CHLOROPLAST GENOME SEQUENCES OF FOUR PEANUT BOTANICAL VARIETIES
Review round: 1
Reviewer: 2

Output:
Basic reporting: A lot of good data have been generated, but the results need to be revised thoroughly to derive the complete meaning of the data.

Language needs a major revision
Experimental design: Appropriate
Validity of the findings: Good
Additional comments: Major concerns:
I strongly recommend to check the language before publishing this work. Better to get a review from native English speakers or Professionals.
Comments:
Page 1: Background: The first sentence is not meaningful. Hence need to be clearly written.
Method: The sentence “According to the alignment sequences, the genome-wide genetic variations (SNPs and InDels) were developed”

Discrepancy in usage of terms “cultivars vs botanical types”;  “Genome vs cp Genome”; “America vs American” need to be checked in the entire manuscript. Need to maintain  uniformity.

Line#17: Abstract is not written properly. It is copied from the Background, method, discussion part as such.

Line#39: Keywords can be more specific.
Line#40: Short title can be changed.
Specific doubts:
Line#78: Are the nomenclatures AHP, AHL, AHD and AHZ are globally accepted?

Line#82: “genetic relationships based on four peanut cultivars” Are they of different botanical types? Need to specify.
Line#88: What are the genotype names of these botanical types?
Line#114: What are IR and SC region. Explain the details
Line#115: How many relative species- indicate the plant species used in Phylogenetic analysis.
Line#129: Cannot trace accession numbers at NCBI. Will it be accessible to public after publication of this MS?
Line#132: “Size and gene content of the peanut genome” Is it done for whole genome or cp genome? In the paragraph need to explain the IR, LSC and SSC region. The abbreviations should be expanded at the first instance.
Line#138-140: The sentence is not clear.
Line#145: Explain about DNA flexibility. Mention the methods to calculate this in Methods section
Line#162: Need to explain about the criteria used to identify InDels.
Line#173: Is this six or four cp genome sequences?
In discussions, the relation between different botanical types has not been discussed but it is indicated in the title of the Manuscript.


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

Input:
Title: A COMPARATIVE ANALYSIS OF THE COMPLETE CHLOROPLAST GENOME SEQUENCES OF FOUR PEANUT BOTANICAL VARIETIES
Review round: 1
Reviewer: 3

Output:
Basic reporting: While this article is written in passable English, it is riddled with grammatical errors. I don't think that it should be the job of a peer reviewer to copy-edit the manuscript, but I would highly recommend that the authors seek the services of a copy editor to correct the grammar and English usage before resubmitting the manuscript.

Technically speaking, this article is thorough in providing background and context for the study, but possibly it has too much background information. While some basic information about the cultivation history of the species is useful, perhaps it could be limited to a single paragraph. Similarly, the background information about chloroplast sequencing and phylogenetics could be simplified to perhaps a single paragraph.

The data presented is fairly standard for a paper on chloroplast sequencing and phylogenetics. I don't understand the meaning of the analysis of DNA flexibility in this context; I've never seen this issue addressed in a phylogenetics paper and the authors provide no citations for what this means and why it's relevant to the study.

I could not find the sequences on Genbank, so I can't comment on the quality of the annotations.
Experimental design: I am somewhat confused as to what the purpose of this study was: was it to clarify the phylogenetic positions of the four landraces, or was it merely to provide evidence about the plastome sequences of them?

If the purpose was the first, I don't think that it is sufficient to use a single sample of each landrace and use only one linkage group (the maternally-inherited plastome). I'd like to see a study that uses these reference plastomes and resequences many samples of each landrace and uses more rigorous phylogenetic analyses to at least determine with confidence the plastome's phylogenetic history of the landraces.

If the purpose was the second, I guess I can't really argue with the methods used. I'd still remove the phylogenetic results and discussion, for the reasons I stated above, but I don't have any particular complaints about the methods used to sequence four chloroplast genomes. I have never seen a DNA flexibility analysis in a paper before (and the authors provided no citations for it), so I cannot judge how well that particular component was performed.
Validity of the findings: While it is always useful to add more reference sequences to Genbank, I can't tell if I think that that feat alone is enough to justify a paper. If the authors want to use this data to write a paper about the phylogenetics of landraces in Arachis, I think that the data presented here is a good start towards that analysis, but I don't think that the research and analysis presented here suffices for that purpose. I wouldn't trust a phylogenetic analysis using only a single sample each for an intraspecies study.

If the standard for PeerJ is merely that the information presented is technically accurate and provides some new benefit to the literature, a stripped-down version of this paper without the phylogenetic analyses would be sufficient, but I think the paper would be greatly improved by doing more robust phylogenetic analyses with more sampling.
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: A COMPARATIVE ANALYSIS OF THE COMPLETE CHLOROPLAST GENOME SEQUENCES OF FOUR PEANUT BOTANICAL VARIETIES
Review round: 1
Reviewer: 4

Output:
Basic reporting: The manuscript provides new data on the chloroplast complete sequences of four peanut varieties (six accessions). The results are original and add structural knowledge to the huge genomic resources that are being provided for peanut and its wild progenitors. However, there are several items to be improved before the manuscript can be considered for publication.

1. The English language should be improved in all the extension of the manuscript to ensure that an international audience can clearly understand the text. Some examples where already marked.

2. REFERENCES
Should be corrected and complete. Examples are:

Titles of the papers, with and withot capitals in all the words.
266 Gepts P. 1993. The Use of Molecular and Biochemical Markers in Crop Evolution Studies.
267 Evolutionary Biology-new York 27:51-94.
268 Gibbons RW, Bunting AH, and Smartt J. 1972. The classification of varieties of groundnut
269 (Arachis hypogaea L.). Euphytica 21:78-85.

Incomplete citations
289 Krapovickas et al. 1960. revista de investigaciones agricolas 14(2): 197-228.
Krapovickas, A. & Rigoni, V. A., 1960. La nomenclatura de las subespecies y variedades de Arachis hypogaea L. Revta Invest. agric. B. Aires. 14: 198–228.
305 Martin M. 2011. Cutadapt removes adapter sequences from high-throughput sequencing reads.
306 2011 17. 10.14806/ej.17.1.200
307 pp. 10-12

3. The title is so ambitious because the variability and phylogenetic data is inferred only from six accessions considering only four taxonomic varieties. Indeed, to understand the genetic variation in cultivated peanut, with more than 10.000 accessions in international gene banks, and to make a confident phylogenetic analysis a larger sample is needed.
Experimental design: .The sample used to infer the variability and to make a phylogenetic analysis is not appropriate, because of the huge number of peanut  local races distributed worldwide, and kept in th einternational gene banks.
Validity of the findings: 4. The phylogenetic results are discussed citing statements that are false. For instance,
218 AHL is the most similar to wild species morphologically (Krapovickas et al. 1960).
This statement is not true. In the world monograph of Arachis (Krapovickas & Gregory 1994, English translation edited in 2006), as well as in the reference cited by the authors it is clearly established that A. hypogaea hypogaea var hypogaea presents the characters most similar to those found in wild species. Simpson 1996, also stated that the var. hypogaea presents the most ancestral characters.
219 importantly, AHL is regarded as the earliest peanut cultivar that was domesticated in the South
220 American based on the historical record.
This sentence is very confusing and the statement  is also not true. A. hypogaea var. hirsuta, is the variety found in the most ancient archeological remains, in the Pacific coast of Perú (Bonavia). However there is no evidence that this variety is the earliest domesticated. It is probably not, since the remains were found very far from where the ancestral wild species are distributed (Simpson et al 2001, Stalker et al. 2017).
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: A COMPARATIVE ANALYSIS OF THE COMPLETE CHLOROPLAST GENOME SEQUENCES OF FOUR PEANUT BOTANICAL VARIETIES
Review round: 2
Reviewer: 1

Output:
Basic reporting: The English in the manuscript is actually worse than it was in the last revision. I strongly recommend that the authors find a copy editor who is fluent in English to revise the manuscript: not only are there problems with the phrasing, but there are instances throughout where words are run together ("minimumevolutionary" on line 103, "Fabaceaeserved" on line 102), places where there are multiple spaces for no reason, repeated words (line 218), extra punctuation, etc. These sorts of mistakes are not related to the scientific merit of the paper, but demonstrate a carelessness in the revision process.

The discussion is improved from the previous version: I think that the authors clearly articulate why their limited phylogenetic analysis is still useful in supporting a previous hypothesized relationship within the varieties. There is no mention at all in the discussion of the rationale and conclusion drawn from the DNA flexibility analysis; either it should be discussed and justified in the discussion or it should be cut out entirely.
Experimental design: It seems like the authors merely mean to present some new plastome sequences within botanical varieties of A. hypogaea; if this is their purpose, it is well-explained and the paper does address this topic. The methods are clear enough to replicate the study.
Validity of the findings: I still don't understand the point of the flexibility analysis; while the authors address it in the rebuttal, none of that is present in the revised manuscript.
Additional comments: No additional comments


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

Input:
Title: A COMPARATIVE ANALYSIS OF THE COMPLETE CHLOROPLAST GENOME SEQUENCES OF FOUR PEANUT BOTANICAL VARIETIES
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors attended all the major points requeted.
Experimental design: The authors attended all the major points requeted.
Validity of the findings: The authors attended all the major points requeted.
Additional comments: The authors attended all the major points requeted.


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: CAPRELLID AMPHIPODS (CAPRELLA SPP.) ARE VULNERABLE TO BOTH PHYSIOLOGICAL AND HABITAT-MEDIATED EFFECTS OF OCEAN ACIDIFICATION
Review round: 1
Reviewer: 1

Output:
Basic reporting: This paper is well written and a nice little preliminary study on interactive biotic and physiological effects. One could argue that it might need a little more analysis on the heart-rate side of things. That being said it is of note that the authors have worked on a lesser known group of crustaceans for which very little work on heart rate has been performed.

Overall I prefer papers when they are not written in the first person.

Minor points
L70 ..and more (Rossoll,..  more what?
L97 for sentence ending quality

L116 onwards - These are really predictions rather than hypotheses. I would tend to omit these and give a shorter (1-2 sentence aims). Something like the aim was to determine the potential effects of reduced pH on metabolic rate and if and how this may interact with habitat complexity to determine the future distribution of caprellid amphipods

L131 Field studies were conducted, and organisms were collected...

L189 - McGaw et al is 2018, also check - one of the authors last names is incorrect!

Results
Very brief, can you give a bit more detail and guide the reader through the results. For example, in biogenic habitat preference how many more caprellids were found on Obelia - give some details rather than general trends

Figures
Can you indicate significance on the graphs. Also could you include more description in all the legends. I do not understand the C and D figure 1- can you explain what these are and what they mean please. Same for figure 3 can you explain what this is, what the line represents and the shaded area
Experimental design: It is interesting that the authors appear to have used a novel method of a cell phone to record heart rate. But it is only briefly described, this is novel and while would not be great for most applications, it might be very useful for undergraduate projects given the low cost and ease of use. Some more detail would be beneficial and even some screen shots included in the paper as figures.

I do have some concern as to how accurate/useful the rates will be. I would assume the animals would be stressed only being in a small amount of water, but more importantly it would likely become hypoxic, and crustaceans show a strong bradycardia response under such circumstances. The stress response could be reduced, but not abolished, by allowing them to settle in the petri dish for several hours and flow through water would reduce oxygen depletion. The common way to hold these animals is to put a spot of superglue on the carapace and then stick a Q-tip or similar structure to the carapace - keeps the animal in place and they can be manipulated for seeing the heart. I know this may not be feasible at this stage, but keep this in mind for future studies. In addition HR of crustaceans tends to be quite variable between individuals, but also within an individual over even short time periods. Therefore having an N=4, and a short trace of 20sec may not be that reliable if one wants to draw definite conclusions - but it is a nice start and this phone method should be explored further.

The authors state that HR in crustaceans can be used as a proxy for metabolism, but they also will have access to a whole other set of data - stroke volume! By modelling changes in shape of the heart one can calculate the stroke volume of the heart, and thus cardiac output. This is important because HR and SV vary independently of one another to determine how much blood is pumped from the heart. See papers by Harper and Reiber on grass shrimp for methods. It can be measured from a short video clip.

The authors state why they used 17C, but why were animals acclimated for 15h after capture and then 72h to the test pH are these times particularly relevant to anything?
Validity of the findings: This paper makes a nice connection between the biotic (environment) and physiological changes. The heart rate looks like it does change, the authors shouldn't be too surprised by this, whether it is a stress or a metabolic effect/cost is unclear. The authors have used 2 embryonic papers that found no difference in other crustaceans - but this is not to say that there may be an increase in this adult species. There are a lot of physiologically different mechanisms between juveniles and adults. I would try and improve the discussion by strengthening the links, for example if cover disappears maybe the animals would be more stressed or food deprived, how would this affect them given they now also have a higher metabolic rate? At present the two effects are discussed somewhat separately.
It would also be interesting to see what effect long term pH had on growth and feeding ability to draw definite conclusions. Maybe this is something for another study.

Discussion - Ok I would try and tie in the ecological an physiological changes a little more. For example it does appear that metabolic rate is higher in low pH. If then they cover and possible food source is lower what might this do to an animal that is already stressed by low pH.

Overall I enjoyed reading this paper - I liked the way the authors used both physiological and habitat changes to determine possible effects of OA.
Additional comments: A couple of questions that I always pose to this type of predicted environmental data and climate change scare mongering
1. You subjected the animals to a sharp decrease in pH, when in reality pH is going to change gradually over the next 80 years. I would assume that these organisms are reaching maturity quite quickly and there could be tens of generations (if not more) in that time. Given both the hydroids and caprellids may show developmental plasticity, or even short-term evolution, how realistic do you think these experiments are in general? Do you think that most organisms would adapt over several generations to any aspect of climate change?
2. If these organisms do decrease, or even disappear altogether why should we care? Are they important in the big picture of things, what feeds upon them, are they key players in the ecosystem?
IF all these climate changes come to fruition, (and given the previous media hype during the last 50 years about nuclear war, the ozone hole, various flu pandemics, acid rain, meteorites, the Aztec calendar, over population and Uncle Tom Cobbly), I doubt whether they will have any noticeable effect. However, should they come true the actual global social disruption that will occur will mean than no one gives two hoots about some little animals, or even any other animals. They will all be buying guns and running for the hills!
So I am with President Donald Trump on the whole climate change thing!


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: CAPRELLID AMPHIPODS (CAPRELLA SPP.) ARE VULNERABLE TO BOTH PHYSIOLOGICAL AND HABITAT-MEDIATED EFFECTS OF OCEAN ACIDIFICATION
Review round: 1
Reviewer: 2

Output:
Basic reporting: The paper by Lim and Harley, is a relatively simple and somehow indirect experiment to estimate the synergistic effects of physiological and habitat-mediated aspects of Ocean Acidification (OA) on amphipods caprellids....
This is a quite challanging and interesting topic and the paper, although a bit simplistic, deserve publication. However, before final acceptance it is necessary to consider and expand in the discussion and in the comparisons the following points:
a) discuss and add literature on physiology (respiration and metabolic rate) with particular focus on crustaceans and OA (e.g. Turner et al., 2016  Marine Biology 163, 211)
b) expand  and discuss more paper related to abundance pattern of amphipods to OA and their relationships with the habitat (e.g., the macroalgae) (e.g. see es. Scipione, M.B. 2013. On the presence of the Mediterranean endemic Microdeutopus sporadhi Myers, 1969 (Crustacea: Amphipoda: Aoridae) in the Gulf of Naples (Italy) with a review on its distribution and ecology. Mediterranean Marine Science 4, 56–63.
Kroeker, K.J., Micheli, F., Gambi, M.C. & Martz, T.R. 2011. Divergent ecosystem responses within a benthic marine community to ocean acidification. Proceedings of the National Academy of Sciences USA108, 14515–14520.
SCIPIONE M.B., KROEKER K.J, RICEVUTO E., GAMBI M.C. 2017. Amphipod assemblages along shallow water natural pH gradients: data from artificial substrata (Island of Ischia, Italy). Biodoversity Journal, 8(2): 469-470.
Ricevuto, E., Lorenti, M., Patti, F.P., Scipione, M.B. & Gambi, M.C. 2012. Temporal trends of benthic invertebrate settlement along a gradient of ocean acidification at natural CO2 vents (Tyrrhenian Sea). Biologia Marina Mediterranea19, 49–52.)
Poore et al., 2013 Oecologia https://www.ncbi.nlm.nih.gov/pubmed/23673470)

I found in fact that respect to the interest and complexity of the topic more literature could be considered.
this is the only point I warmly suggest, for the rest the paper is well written.
Experimental design: Quite simplistic but correct
Validity of the findings: The results should be better discussed and compared at the light of various papers whihc have not considered and whihc rae related to bot capreilli amphipods and other crustaces, related with OA
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: EFFECTIVENESS AND ADEQUACY OF BLINDING IN THE MODERATION OF PAIN OUTCOMES: SYSTEMATIC REVIEW AND META-ANALYSES OF DRY NEEDLING TRIALS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Well done
Experimental design: Well done
Validity of the findings: Reasonably well done given available evidence and validated/acceptable methods
Additional comments: Authors performed a very thorough and rigorous review.  Specific, mostly minor, comments are:
1.	What is inverse-var random effect model? DL model?
2.	Blinding adequacy is 1/0?
3.	Some parts are repetitive. I think shorter text is more read by readers.
4.	Blinding is relevant in n-of-1?
5.	Line 328: how to determine intervention credibility?
6.	Baethge et al. (Psychother Psyochsom2013) may be cited; a similar research was done but without BI.
7.	Effect estimate is Effect size? Please clarify how it was defined. Change score or difference in change scores or something else?
8.	Line 378: P-value or CI may be added?
9.	Line 437 and 479: randomization is important in meta reg and subgroup?
10.	Line 481: Clinical significance is addressed?

Editorial:
1.	Line 123: The vs. the.
2.	Line 325: What is DN?
3.	Line 414: broken letter.
4.	Line 524-5: N and n is correct?
5.	Line 551: pearled?
6.	Table 2: 0.2.0.


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: EFFECTIVENESS AND ADEQUACY OF BLINDING IN THE MODERATION OF PAIN OUTCOMES: SYSTEMATIC REVIEW AND META-ANALYSES OF DRY NEEDLING TRIALS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The English grammar and spelling were correct with adequate and appropiate scientific terms. The literature references are update and according to the methods description. The background and context are correct and justify the research question and hypothesis, which are very appropiate and necessary. The article structure, figures, and tables showed a well-planned study which provides important considerations in a correct way.
Experimental design: The experimental research design of this manuscript shows a high quality research in a rigorous way. Methods are described in a clear style with enough detail. Nevertheless, I only suggest adding the Ethics committee approval code in the methods section, which is an important item in order to add this information to the PRISMA and PROSPERO data.
Validity of the findings: The validity of the findings is appropiate. The conclusion are established according to the objectives. Therefore, the finddings are useful and necessary according to the described literature.
Additional comments: Congratulations for the authors. This manuscript is well-planned study and provides important information which is necessary in the dry needling field. The placebo and sham treatments mechanisms needs to be investigated and this research presents the necessity of futher investigations regarding the effects of these interventions in order to establish the adequate effectiveness in blinded randomized clinical trials. I only suggests that the Ethics committee approval code should be added in order to clarify this key point in the methods section. In addition, adding the MeSH term "Myofascial pain syndromes" may be useful.


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

Input:
Title: EFFECTIVENESS AND ADEQUACY OF BLINDING IN THE MODERATION OF PAIN OUTCOMES: SYSTEMATIC REVIEW AND META-ANALYSES OF DRY NEEDLING TRIALS
Review round: 1
Reviewer: 3

Output:
Basic reporting: All satisfactory
Experimental design: All satisfactory
Validity of the findings: All satisfactory
Additional comments: The most fascinating part of this report, for me, was the revelation that out of 4894 individual publications only 24 were good enough to be included in the analysis.  And that, of these, 19 trials showed risk of methodological bias that was high or unclear. Five trials were adequately blinded, and blinding was assessed and sufficiently reported to compute the blinding index in 10 trials.

This is a damning comment on the quality of acupuncture research, and well worth publishing.
The only bit that I would change is the conclusion:

"Future trials should be adequately blinded and should collect robust blinding data to allow quantification of moderating effects of blinding effectiveness on pain outcomes; "

My conclusion, from the same data would be that it isn't worth spending any more time and money on acupuncture research.  After over 4000 trials there is still room for doubt about whether it works to any useful extent. and the best quality trials show that either it doesn't work at all, or produces a benefit that's too small to be useful to the patient, my conclusion would be that no further research is needed. Another 4000 trials are unlikely to change the conclusions.


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: EFFECTIVENESS AND ADEQUACY OF BLINDING IN THE MODERATION OF PAIN OUTCOMES: SYSTEMATIC REVIEW AND META-ANALYSES OF DRY NEEDLING TRIALS
Review round: 2
Reviewer: 1

Output:
Basic reporting: None
Experimental design: None
Validity of the findings: None
Additional comments: One small comment:

I think "REML model was used.." is an incorrect sentence. Authors have statistical collaborators or coauthors. So they should consult with statistician and fix the sentence if they agree.


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: EFFECTIVENESS AND ADEQUACY OF BLINDING IN THE MODERATION OF PAIN OUTCOMES: SYSTEMATIC REVIEW AND META-ANALYSES OF DRY NEEDLING TRIALS
Review round: 2
Reviewer: 2

Output:
Basic reporting: Thanks for addressing the review suggestions
Experimental design: Thanks for addressing the review suggestions
Validity of the findings: Thanks for addressing the review suggestions
Additional comments: Thanks for addressing the review 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: EFFECTIVENESS AND ADEQUACY OF BLINDING IN THE MODERATION OF PAIN OUTCOMES: SYSTEMATIC REVIEW AND META-ANALYSES OF DRY NEEDLING TRIALS
Review round: 2
Reviewer: 3

Output:
Basic reporting: OK
Experimental design: OK
Validity of the findings: The findings are OK. The conclusions are totally wrong, in my opinion.
Additional comments: I made only one suggestion but the authors declined to implement it. Therefore I think that this paper should be rejected.
I said
"My conclusion, from the same data would be that it isn't worth spending any more time and money on acupuncture research. After over 4000 trials there is still room for doubt about whether it works to any useful extent. and the best quality trials show that either it doesn't work at all, or produces a benefit that's too small to be useful to the patient, my conclusion would be that no further research is needed. Another 4000 trials are unlikely to change the conclusions.’"

The authors' response was
"We agree that further research is unlikely to change the conclusions of acupuncture research."
They seem to agree but then declime to change their conclusions on the follwoing grounds.
"However, the current review investigated dry needling specifically and excluded acupuncture trials. The philosophy and the proposed mechanisms for dry needling are considered different from those of acupuncture"

Since one thing that has emerged from the 4000+ trials of acupuncture is that it makes no difference to the outcome whether needles are inserted in the traditional acupuncture points or anywhere else, these grounds seem to me to be invalid.  It's already known that the position of the needles makes no difference.That's how we know that traditional acupuncture points are figments of imagination,, dating from a prescientific age.

If we accepted the authors argument, we'd have to accept another 4000 trials from every subtype of acupuncture -auricullar, electro etc etc etc.


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: EFFECTIVENESS AND ADEQUACY OF BLINDING IN THE MODERATION OF PAIN OUTCOMES: SYSTEMATIC REVIEW AND META-ANALYSES OF DRY NEEDLING TRIALS
Review round: 3
Reviewer: 1

Output:
Basic reporting: All the authors have done in response to my comments is to replace

"Future trials should be adequately blinded and should collect robust blinding data to allow quantification of moderating effects of blinding effectiveness on pain outcomes; these will be important steps towards determining the true mechanisms of dry needling."

with

"Any future trials in this or related areas should be adequately blinded and collect robust blinding data to allow quantification of moderating effects of blinding effectiveness on pain outcomes’ .

Although this is slightly better, it still implies that the authors are unwilling to offer advice on future trials.  The authors said after the first review that "We agree that further research is unlikely to change the conclusions of acupuncture research."  Yet they have still not included this important statement in the paper. In my opinion, this is not scientific objectivity but an implicit condonement of pseudo-scientific medicine,  Some very respectable journals like Anesthesia and Analgesia have stopped accepting papers on things like acupuncture altogether. One reason for that is that such papers tend to keep alive the idea that there is controversy. That is standard practice for alternative medicine advocates. It is exactly the same tactic as was used by the tobacco industry: manufactured doubt.

In my view the paper should be rejected if the authors are not willing to include something like
"Further research is unlikely to change the conclusions of acupuncture research, and to that extent no further research can be justified.  If more trials are done in this or related areas should be adequately blinded and collect robust blinding data ..."
Experimental design: No comment
Validity of the findings: No comment
Additional comments: All the authors have done in response to my comments is to replace

"Future trials should be adequately blinded and should collect robust blinding data to allow quantification of moderating effects of blinding effectiveness on pain outcomes; these will be important steps towards determining the true mechanisms of dry needling."

with

"Any future trials in this or related areas should be adequately blinded and collect robust blinding data to allow quantification of moderating effects of blinding effectiveness on pain outcomes’ .

Although this is slightly better, it still implies that the authors are unwilling to offer advice on future trials.  The authors said after the first review that "We agree that further research is unlikely to change the conclusions of acupuncture research."  Yet they have still not included this important statement in the paper. In my opinion, this is not scientific objectivity but an implicit condonement of pseudo-scientific medicine,  Some very respectable journals like Anesthesia and Analgesia have stopped accepting papers on things like acupuncture altogether. One reason for that is that such papers tend to keep alive the idea that there is controversy. That is standard practice for alternative medicine advocates. It is exactly the same tactic as was used by the tobacco industry: manufactured doubt.

In my view the paper should be rejected if the authors are not willing to include something like
"Further research is unlikely to change the conclusions of acupuncture research, and to that extent no further research can be justified.  If more trials are done in this or related areas should be adequately blinded and collect robust blinding data ..."


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

Input:
Title: CYTOGENETIC ANALYSES IN TRINOMYS (ECHIMYIDAE, RODENTIA), WITH DESCRIPTION OF NEW KARYOTYPES
Review round: 1
Reviewer: 1

Output:
Basic reporting: Major comments
This is a simple and short communication article, which provides a description of a new karyotype for the genus Trinomys. Molecular analyses were performed to check taxonomically the specimens. The manuscript highlights that Neotropical biodiversity is still poorly known. Methods described are important and with sufficient detail and information to be replicated, however morphological information was not investigated and it should be done to give more support to the discussion.

The title and abstract highlight a cytogenetic study, but the item Material and Methods starts inverted - in opposition to the main idea of the manuscript. The authors should consider the possibility of describing firstly the cytogenetic information and secondly the molecular information. It would be easier to describe karyotypes and then correlate them to the names - the trees should carry diploid numbers and the species names.
A map should be included in the manuscript with all the localities from where the sequences are and the distribution of the species as well.
The authors should remove Table 3 from the manuscript because all the information is in the Figure 4. In this figure, by the way, they should also check the G-banding pattern comparison of the triplet chromosomes 4; 7, 5, 5; 9; 12, 7, 7; 13; 15.
Experimental design: The cytogenetic and molecular methods used are adequate. To really improve the manuscript, as mentioned in the previous item, morphological studies should be included to improve the article. Additionally, FISH using specific probes could clarify what G-bands do not reveal (synteny).
Validity of the findings: According to the discussion (lines 179-181), the authors mentioned that the differences in the morphology of pairs 26 and/or 27 may be real or may reflect different chromosome condensation. This is an important point because if this is not real, the FN is the same for all individuals and some points are supposed to be reviewed.

Regarding the sex chromosome variation, other rodent families/subfamilies/tribes show X and Y polymorphisms. The discussion should be improved, for example, evaluating the several mechanisms evolved in the evolution of these chromosomes - addition/deletion of constitutive heterochromatin, centromere repositioning, for instance, and the cytogenetic alterations it could (or not) generate. Would it lead to a speciation process?
Additional comments: Minor comments
The authors should mention that Minas Gerais represents one state of Brazil and the number of metaphases counted per individual as well.
Figure 2 is indicated with letter A and B. Why do not use the same criteria in the Figure 3?
It is worth to insert the collecting localities in the legend of the figures.
The legend of Figure 3 should be corrected: Karyotypes of Trinomys setosus: a female with 2n=56, FN=106 (on the left) from Serro, Minas Gerais State, and of a male with 2n=56, FN=108 (on the right) from (locality) after GTG- (on the top) and CBG-banding (below). Scale bar = 10 μm.
The individual MCN-M 3296 (Table 2) is a male or a female?


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: CYTOGENETIC ANALYSES IN TRINOMYS (ECHIMYIDAE, RODENTIA), WITH DESCRIPTION OF NEW KARYOTYPES
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript is an important contribution to the study of the genus Trinomys. It is well written and with a complete bibliographic research. I have a few suggestions and considerations.

In-text citations need to be checked. Looking at the authors instructions it is stated that: "for three or fewer authors, list all author names (eg Smith, Jones & Johnson, 2004)." For example the citations in the introduction: Emmons et al. 2015 and Dunnum et al. 2001 have 3 authors. And when there are two authors, the "&" should be used instead of "Lara and Patton 2000", "Carvalho and Salles 2004", for example.

Tables and Figures:

In table two could have another column with the Genbank accession number.

In relation to the figures, I understand that the manuscript already has many but I think it would contribute a lot if it had a map with the collection localities. This would be interesting even to see where are the localities of T. setosus and compare with the distribution of the two subspecies.

It would be interesting to have in Figure 1 the corresponding karyotype for the specimens included in the study. Otherwise, we have to go into the supplementary files to see which karyotype corresponds to which sequence. And and the phylogenetic tree (although based on only 401 bp) seems to show a structure in T. setosus. Do karyotypes confirm this? or not? This part could be more discussed as well.

The figure 3 legend is confusing; first replace “Metaphases” with “Karyotypes”. The title legend says: “Metaphases of a female Trinomys setosus (2n=56, FN=106)” but the figure shows both karyotypes (2n=56, FN=106, FN=108) of the female and the male.
In Supplementary Figure S3, why are T. setosus specimens in different colors?
Experimental design: Regarding molecular sequencing I have some issues about sequencing only 401 bp. But I understand that the tool was used only for specimen identification, and the results were congruent with previous studies.
Three of the five specimens were sequenced in the study. But as I could see in Supp. Files, it seems that the other two (T. setosus KX655539.1 (UFMG 6024) and T. moojeni KX650080.1 (MCN-M2816)) had previously been sequenced and deposited in GenBank. I suggest that this information should be included in the Material and Methods section.
Validity of the findings: In the results section, lines 139-140 says: “…and composed of 26 pairs of biarmed (pairs 1-26) and one pair of acrocentric (pair 27) autosomes”
but in line 143: “… that differed from those described above in the morphology of one of the smallest autosomes (pair 26) that was biarmed instead of acrocentric in these three animals” I guess it is pair number 27 instead of 26?
But, again, in lines 148-150 we see: “… and to verify that the complements of T. setosus with FN=106 and FN=108 (Fig. 4, Table 3) differed in relation to pair 26, which was acrocentric or biarmed, in the animals with FN=106 and FN=108, respectively.”

Line 163: Replace (Corrêa et al. 2005; Fig. 2) with (Corrêa et al. 2005; Fig. 1).

Pessoa et al. (2015) report that there is an unpublished karyotype for T. setosus: "The karyotype of T. s. setosus from Almenara, Minas Gerais state, is 2n = 56 and FN = 108 (unpublished data)." Although it does not have the published karyotype, the information is present in the last revision of the genus. It is worth to include this information in the discussion, even because this karyotype was related to T. setosus setosus with a related locality, as the karyotype 2n = 56 and FN = 104 was related to T. s. elegans (Pessôa et al. 2015). I think this part of the discussion could be a bit more developed, taking into account the subspecies, the karyotypes localities and their 2n and NF.

I don’t understand line 191 – The constitutive heterochromatin evidenced in our specimens was mainly located on the smallest chromosomes and the sex chromosome pairs in the two species” – as we can see in Figures 2 and 3 the first pairs are marked with constitutive heterochromatin (also written in lines 151- 153).
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: CYTOGENETIC ANALYSES IN TRINOMYS (ECHIMYIDAE, RODENTIA), WITH DESCRIPTION OF NEW KARYOTYPES
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors improved the manuscript, however I still have some considerations to be made.
Figure 1 - Coordinates and a kilometer bar should be inserted in the maps.

Figure 2 - The collection sites of the individuals could be included in this figure - at least the five animals of this study.

Concerning the lines 145-146 -- "Morphological characters analyzed corroborated the phylogeny and allowed assigning the T. setosus specimens to T. setosus" -- and lines 196-198 -- "Although our phylogenetic analyzes do not distinguish between T. s. setosus and T. s. elegans (Fig. 2, Supplemental Fig. S1 and S2), the karyotype and the morphological analysis seem important to recognize these subspecific taxa. ": The morphological analysis indicates T. setosus as a single entity? Cytogenetically, do the authors considered the karyotype as a diagnosable character for species identification (even considering the variation in the pair 26 and the similarity with the diploid and fundamental numbers of T. moojeni? Is that similar to T. gratiosus which also has 2n = 56 and FN = 108? Phylogenetically (Figure 2), the clade referred to T. setosus is composed of several relatively well-supported subclades, one of them (support 1) includes the animals from Morro do Pilar and Serro, with 2n = 56 and FN = 106 and FN = 108, due to variation in the pair 26 of autosomes; other clade (support 0.98) recovered the specimen from São Gonçalo do Rio Abaixo, which has 2n = 56 and FN = 108, besides the sister clade, for which th 2n is not shown. What is the distance between/among these localities, vegetation, topology, altitude, etc.?
Experimental design: Have the authors checked the presence of stop codon sequences? This detail can be crucial for alignment and analysis.
Validity of the findings: Figure 4 - It is really difficult to see that the pairs 26 in the Figures 4B and 4D are morphologically different. Are the authors absolutely sure that this happens?
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: CYTOGENETIC ANALYSES IN TRINOMYS (ECHIMYIDAE, RODENTIA), WITH DESCRIPTION OF NEW KARYOTYPES
Review round: 2
Reviewer: 2

Output:
Basic reporting: All previous suggestions were accepted and improved
Experimental design: All previous suggestions were accepted and improved
Validity of the findings: All previous suggestions were accepted and improved
Additional comments: I would like to congratulate the authors. They accepted all previous suggestions and in my opinion the manuscript has been improved and should be accepted for publication. I have no further 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: TOUCH-SCREEN-GUIDED TASK REVEALS A PROSOCIAL CHOICE TENDENCY BY CHIMPANZEES (PAN TROGLODYTES)
Review round: 1
Reviewer: 1

Output:
Basic reporting: Context:
From my perspective it seems like all important background is covered. Not really sure why the section on understanding intentions is included in the intro, if you think it is very relevant, could you please clarify, otherwise, maybe this is an opportunity to shorten the MS.

Language:
Please proofread and double-check grammar throughout manuscript. Check lines 75, 91, 98, 99, 102-105, 127, 136, 153, 155, 238, 298, and Table 1, among others.

Tables and Figures:
Could you please add the information about mother-offspring relationships in the tables. I know that this is also explained in the text, but it might help readers understand the table more quickly, too.
Figure 4 the label and caption don’t match (Fig 4 / 6), at least in the PDF that I got.
Figures 4 and 5 include information on “Proportion of choice X above choice Y”, not only for Experiment 2 and 3, but also for Experiment 1. However, in Experiment 1, three choices were provided. So what does the proportion mean? Is “prosocial above altruistic” e.g. “proportion of prosocial choices among all of the choices that were either prosocial or altruistic, ignoring selfish choices”. If that’s what you did, it is difficult to compare to proportions in pure two option choices. At the very least the readers should know. So could you please explain, either in text or in figure caption. Sorry if I misunderstood!
Figures 3, 6 and 7 speak of fitted values but I could not find any information on the underlying models that were fitted. Could you please provide this information in supplementary.
Figures 4, 6 and 7: given that the main message of the MS is that prosocial was chosen over selfish very often, would it not make more sense to flip around the lines from “selfish above prosocial” to “prosocial over selfish” for more intuitive readability?
Figure 5, the caption speaks of error bars, but there are no error bars in the figure
Finally, what is the difference between Figure 4, bottom right, and Figure 5, other than that one seems to be the invert of the other?

Raw Data:
I really appreciate that the authors make available their data, thank you for this!
The data could be presented in a more accessible fashion, here are some suggestions.
The paper speaks of Experiments, but the data speaks of Conditions, please harmonize.
The three conditions (experiments) are currently saved in three different data sheets, but they could be in one big data set that simply has another variable “Experiment” that codes where the data comes from.
It is not clear to me why for the different conditions, some variables have to be repeated in multiple columns (e.g. Condition 1, why are there three columns each for the Individual and Trial variable, and why are there six empty columns). The same is true for Experiments 2 and 3. For all three sub datasets, there could simply be one simple structure “Experiment – Individual – Trial – Prosocial – Altruistic – Selfish”. For trials that provided only two options (in Experiments 2 and 3), the third option has to be a missing value of course.
Experimental design: Description of Methods:
I found it a bit difficult to follow the sections on the training phase (151 - 163). The way I understand the training phase, there was only ever one individual, the individuals did not touch anything, they just heard the three sounds: one for selfish (food to their feeder), one for altruistic (food to what would later become the recipient feeder) and one for prosocial (food to one of the feeders, randomly chosen). And the actor always had access to both feeders, including the recipient feeder. And in the learning phase the screen was active and there was only ever one symbol, the individual touched it and the appropriate feeder delivered a reward. Is this correct? It would be great if this section could clarify this.
Validity of the findings: I think many sensible precautions were taken to avoid that individuals may simply learn to prefer the prosocial option, which, as I understand it, may have been a problem with some earlier studies. The very quick development of clear preferences (Figure 3), and the consistency in the other Experiments speaks for a more “insightful” task understanding than gradual preference learning.

I would recommend to be a bit more careful in making general statements about chimpanzees in the discussion and conclusion (e.g. l 331 “a clear prosocial tendency on chimpanzees”). Your sample is too small for meaningful statistical inference to “a population” of chimpanzees, and even if it was larger, it is not representative of “the chimpanzee” because the PRI chimps are a captive group with many unique features. This is not to say that anything is wrong with these chimpanzees or that their behavior is “unnatural”, I would simply caution not too make too broad a generalization.

The only thing I found a bit confusing was your take on the social pressure / fear of harassment explanation of prosocial behavior. As you point out, one may argue that individuals may behave prosocially when in the same compartment only to avoid being pressured by their partner (l 267 to 268, see also Tennie, Jensen, & Call, 2017 for a recent example). You argue that this may not apply to your own study because of (1) the results by Claidiere et al. speak against it, and (2) you did not observe signs of frustration in your own study (L. 270 to 272).
I wonder if Claidiere et al (2015) is really a good counter-example, because in that study the chimps were trained by the experimenters to always accept the reward distributions, even if a lower ranking individual received more (“In the together condition [… we] removed the apparatus from reach if one or the other tried to reach for the reward on the other side.”).
Further, with regard to signs of frustration in your own study, do you mean you did not see signs of frustration during the early parts of Experiment 1 when individuals still chose selfishly sometimes? Because in later sessions most subjects chose prosocial most of the time (when it was an option) so there would be no reason for either chimp to get frustrated, because, one might argue, their partner had already “preemptively” provided them with food most of the time? Or do you mean that you did not observe frustration or attempts to steal food in the selfish vs. altruistic trials only, where you would most likely expect them to occur (l 224 to 226)?
Finally, when speculating about some of the deviations in your data (why adults choose selfish over altruistic most of the time but two of the offspring choose altruistically occasionally and one does not, l 312-314, 322/3), you say that social pressure may be part of the explanation after all. So why would the offspring choose so un-selfishly (for one individual in Experiment 3 even more often than not), if not because of fear of repercussions? And so if social pressure may play a role in motivating “altruistic” behavior in the altruistic vs. selfish condition, at least by lower ranking individuals, why would this logic not also apply in the prosocial vs selfish condition? I think the behavior still fits your own definition of prosocial in that it is intended to benefit one another, so I am not recommending to rewrite the whole discussion. But it would be good if you could clarify the logic a little bit here. I apologize if I misunderstood.
Additional comments: I think this is a very interesting study and a well written manuscript.


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

Input:
Title: TOUCH-SCREEN-GUIDED TASK REVEALS A PROSOCIAL CHOICE TENDENCY BY CHIMPANZEES (PAN TROGLODYTES)
Review round: 1
Reviewer: 2

Output:
Basic reporting: The use of the English language in this manuscript should be improved so that an international audience can clearly understand the text. I have annotated the manuscript to correct grammatical errors and typos. Also throughout the text, many of the numbers used should be spelled out according to Peer J's guidelines.
The authors provide a good introduction to the prosocial literature and context for their study. The literature referenced is relevant as well.
The figures are high quality but there are some issues with the descriptions (please see annotated manuscript).
One broad suggestion is to include an age class column in Tables 2-4 so that it’s easy to see who is an adult and who is juvenile while interpreting the results.
Experimental design: This research is interesting and novel in the field of prosociality with the addition of the touchscreen used in the experiment.
There are some ambiguities in the methodology which I have addressed in the annotated manuscript and below. This was mainly related to the description of experiment 3 and how it differed from experiment 2. To clarify this difference I would also suggest rewriting the description in Table 1 to something like this:
We ran 96 trials for each pair of choices (P-S, S-A, P-A). Each pair was presented in a block of 48 trials and the order of choices counterbalanced between individuals.

In the training phase, were the different sounds also paired with each reward location? The phrasing in lines 157-158 is unclear as it appears that the experimenter was just randomly choosing where to reward, was it a random presentation of sounds paired with the experimenter delivering food to the appropriate locations? Or just random reward locations?
Line 166- was it random whether the adult or juvenile began as actor?
Line 172- did the sound they were trained with also play when they made a choice along with the sound of the feeder?
Lines 174- Chimpanzees only changed positions (recipient vs actor) after an entire experiment was over? As I understand it there were 3 sessions per experiment so I suggest clarifying that after the completion of each experiment the roles were switched. And again in line 175- two experiments were run per day if I interpreted this correctly.
I also think that lines 204-213 should be in the methods section.
Validity of the findings: These findings are significant to the field and the data is analyzed well. I would like to see any data or descriptions about whether there was scrounging of rewards between the chimpanzees during the study. The text states that there weren't behaviors directed towards the partner, but the behavior related to the food could be explicitly noted.
Line 341- I would suggest adding that the touchscreen methodology helps control for the effect of visible food along with the ability to increase trial numbers could cite Cronin 2012 in Animal Behavior.
Additional comments: I think this is an interesting advancement in prosocial testing with the addition of a touchscreen apparatus. With some clarifications regarding methodology and improvements in the language it deserves to be published.


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

Input:
Title: TOUCH-SCREEN-GUIDED TASK REVEALS A PROSOCIAL CHOICE TENDENCY BY CHIMPANZEES (PAN TROGLODYTES)
Review round: 1
Reviewer: 3

Output:
Basic reporting: 1.	Language: The English is most of the time very good, easy to follow and appropriate for the context. There are some spelling mistakes (e.g. Line 102: to examined; Line 155:  so that the actor could easily ear the sound) or wrong wordings (e.g. Line 98: were modulated across different the conditions), which can be easily eliminated by thorough proof reading.
2.	Literature: The literature cited is appropriate and informative.
3.	Structure: The structure of the manuscript mainly conforms to PeerJs standards. However, no clear hypotheses and predictions are given. Furthermore, the results section includes too many information on methods, which needs to be moved to the Material and Method sections (Lines 189-190; 204-213; 224-228).
4.	Figures: There are too many figures and tables showing similar or even the same data (e.g. the right Panel of Figure 4 shows data included in the tables; same goes for Figure 5). Furthermore, the right and left panels of Figure 4 basically show the same thing, the error bars already indicate whether there are large or small differences between individuals. So the right Panel is not really necessary. And if I’d like to know the individual performances, I can look them up in the Tables. I would suggest to better summarize the results in fewer figures and tables, e.g. combine Table 3 and 4. To indicate Mother/Offspring you could just highlight which Initial refers to which category, e.g. typing the mothers in bold, or add a column indicating whether the subject is mother or offspring. And restrict yourself to one sort of diagram for presenting the data (not two as in Figure 4). Furthermore, I do not think that you need to show the cumulative means for all conditions. Figure 7 e.g. shows that not much is happening throughout the course of the experiment, so why do you show us this diagram?
5. Figures: Furthermore, the Figure captions are not sufficient to understand the Figures easily and do show quite some mistakes (spelling and form). E.g. in Figure 4 you should reference each part of the figure with an additional letter (a,b,c..), and explain accordingly. Don’t put the error bar explanation in the middle of the caption. And pay attention that the caption does not start with referencing another Figure (e.g. Figure 4: Figure 6 Mean proportion of choices for S-A trials…). The caption of Figure 3 says “Proportion of the cumulative mean […] as a function of time/trial (x-axis)”. There is no time referenced. Just the number of trials. So please clarify the description. Furthermore, rephrase the ‘bin’ explanations: “Trials are grouped in 8 bins of 18 trials”. You probably mean something like “trials are grouped in bins (each comprising 8 trials) for a total of 144 trials, i.e. 18 bins”.
6.	Especially the Figures (3,6,7) showing the cumulative means need more explanations and better referencing. It took me some time to understand the “altruistic above prosocial” (and similar) phrasing in the Figures and Tables. I would suggest rephrasing these descriptions to better understand which data are shown. You mean the results in trials where only the referenced two options had been shown, right (e.g. altruistic and prosocial)? You need to either better explain that in the captions or use a different term.
7.	Results: The results sections needs the most improvement. As there are too many Figures and Tables, the referencing is too complicated. After restricting the figures and tables, please rephrase the results section accordingly and explain better. Especially the results of Experiment 1 are difficult to understand. How can you report a “selfish over prosocial” choice (e.g. Line 188, 189), when there always have been 3 options, i.e. selfish, prosocial and altruistic? I do not get that at all. Same goes for reporting these kind of data in Figures 4 and 5. I do not understand how you calculated the “selfish over xy” for Experiment 1. That does not make any sense to me.
8.	Raw data are supplied
Experimental design: The research question is indeed interesting and meaningful. However, I think that the experimental design and analysis need some improvement.
1.	First, the authors state that they want to better understand the cognitive processes involved in the prosocial choice tasks. However, I do not see how the current experiments examined any cognitive processes? As stated in the discussion the experiments rather explored the influence of “nurturing history and rank-relationships”. So the initial aim of exploring ‘cognitive processes of PCT’ should be reconsidered.
2.	Furthermore, the authors state that experiment 2 served as a control to ensure that the chimpanzees understand the task. I do not see that. Why proves a consistent choice in Experiment 2 that the chimpanzees understood the meaning of the task? They could have just learned to choose a specific symbol in Experiment 1, without a deep understanding of all the consequences. Some subjects may have just learned to choose the circle, irrelevant of the second option. Or could have learned to avoid a specific symbol. Actually, Cl ALWAYS choose the prosocial symbol. She may have just learned that this specific symbol is rewarding her. How do you know that she understood the other two symbols? Especially, when forced to choose between the altruistic and selfish option, she chose randomly. Demonstrating that she did not know what the other two symbols meant. I know, you report a learning and training phase at the beginning, but what happened there exactly? No results of these phases are reported. So, the alternative explanations are just missing. I am not convinced that the subjects indeed understood the meaning of each symbol, at least not all of them. And this needs to be discussed.
3.	The authors try to explain in the discussion why they did not include a ‘ghost control’. However, I think this is a necessary control condition, rather than Experiment 2. Why can’t the authors just block the subjects access to the ‘partners’ reward, by building a barrier or something? This would solve the issue of over-rewarding the subject in prosocial choices.
Validity of the findings: As stated above, I am not entirely convinced that every chimpanzee understood the meaning of each symbol. Alternative explanations are possible and need to be discussed or even explored further. Therefore, I am not sure that the experiment indeed showed clear prosocial choices in chimpanzees. I feel the authors’ assumption is not valid and at least needs a lot more discussion.
Additional comments: The authors state in the discussion that there had not been any negative interactions between the subjects. Really? So when e.g. Pn always chose the selfish option, his mother just sat there and did nothing? That is hard to believe. Did the experimenters interact with the second chimpanzee somehow? More information on the behaviour of the recipients would be great.


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: TOUCH-SCREEN-GUIDED TASK REVEALS A PROSOCIAL CHOICE TENDENCY BY CHIMPANZEES (PAN TROGLODYTES)
Review round: 2
Reviewer: 1

Output:
Basic reporting: -
Experimental design: -
Validity of the findings: -
Additional comments: All my concerns from review round 1 have been addressed to my satisfaction. The English language in the manuscript still needs improvement. I strongly recommend to have a native speaker have a second look at it.


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: TOUCH-SCREEN-GUIDED TASK REVEALS A PROSOCIAL CHOICE TENDENCY BY CHIMPANZEES (PAN TROGLODYTES)
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors addressed all issues to my satisfaction.
Experimental design: The authors addressed all issues to my satisfaction.
Validity of the findings: The authors addressed all issues to my satisfaction.
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: TOUCH-SCREEN-GUIDED TASK REVEALS A PROSOCIAL CHOICE TENDENCY BY CHIMPANZEES (PAN TROGLODYTES)
Review round: 2
Reviewer: 3

Output:
Basic reporting: Thanks for the revision of your manuscript. I am happy with most of the changes, but have some minor points:

- Thanks for including predictions, however, I still think that the underlying hypotheses need also to be formulated, e.g.  why should the prosocial tendency increase over trials? Furthermore, the predictions sound a bit circular/obvious, the way you formulate them. I would suggest to rearrange a little, so it is rather something like: 1) If chimpanzees have a tendency to behave prosocial, they should choose the prosocial option more often than the selfish and altruistic options. In addition, there are again some spelling/grammar erros in this sections, e.g. ll. 113: this tendency increase(s) over trials. Please check again

ll. 174: “didn’t” should be “did not”
ll. 177: This sentence sounds strange/wrong. First, it should be “This way ‘we encouraged’ or ‘the subjects were encouraged’. But ‘encouraged to understand’ is also odd. I would suggest something like: “This procedure should ensure that the individuals understand that both…”
ll. 196: I guess you mean ‘rewarding the ACTOR” not the author
ll. 235: delete ‘the’
ll. 309-311: this sentence needs some rearrangement
ll. 312: it should be ‘have shown’
ll. 327: haven’t should be have not
ll. 330: delete ‘the’ before ‘experiment 3’
Figure 6: In the caption you refer to experiment 2, but Fig. 6 shows the results of experiment 3, right?

Raw Data: The authors copied the results of the experiments next to each other, but that was not what Reviewer 1 intended (at least I think so). Instead of putting the results next to each other, they should have put them underneath and added the column ‘Experiment’, filling in 1, 2 and 3 accordingly. So the results could be analysed much easier. The way it is presented now is not that appropriate.
Experimental design: s. above
Validity of the findings: s.above
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: ANXIOLYTIC AND ANTI-STRESS EFFECTS OF ACUTE ADMINISTRATION OF ACETYL-L-CARNITINE IN ZEBRAFISH
Review round: 1
Reviewer: 1

Output:
Basic reporting: This manuscript is written well with only a few grammatical/stylistic issues.

The introduction is lacking content and is too short.  There should be mention of the novel tank dive test and light/dark test and their use in zebrafish research.  Why are the authors using zebrafish to test ALC?  This must be clear early on before statements like those on line 204 that relate results to treating 'mental disorders' (line 204). More background is necessary linking the use of zebrafish as a medical model and details about why these tests are relevant. Has there been any research on ALC and fish?

The figures and tables were great.

Minor comments:
line 26 - "...and on excessive neuronal death."  This does not make sense with the start of the sentence.
line 52 - a growing body of "evidence"
line 86, 87, 89 - change 'submitted to' to 'placed in'
line 105 - 'indicator'
line 143 - 'was mixed'
line 170 - 'Pearson'
Experimental design: It is interesting that fish were kept for only 15 days before being experimented upon.  Why only 15 days?

Many facilities require a quarantine of 30 to 60 days (common duration of bacterial of parasitic illness) before experimentation.

The authors may want to consider this for future studies.

Were the experimental groups staggered with the control groups?  Why were there less control fish than experimental fish in the first round of the NTT and LDT?

Why was the NTT done for 6 minutes and the LDT for 5 minutes?  This should be explained in the manuscript.

Why was the ACS test done after the NTT and not the LDT?  This should be explained in the manuscript.

In the result section (lines 177-178) it is stated that ALC significantly increased the time spent in the top.  However, this is only true for 0.1 and 1.0 mg/L.  This statement should be revised to represent the results.  Moreover, why is there an opposite dose-response relationship here?  0.1 has a much greater effect than 1.0 and 10 mg/L.  This needs to be addressed in the discussion.

Furthermore, in the NTT the 0.1 and 10 mg/L were significant but not the 1.0 mg/L.  Why might this be?  The authors also need to address this in the discussion.
Validity of the findings: The data is statistically sound, in my opinion.  I liked the graphs (bars and individual data points) and diagrams.
Additional comments: No additional comments