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 ENERGETIC COST OF MOUNTING AN IMMUNE RESPONSE FOR PALLAS’S LONG-TONGUED BAT (GLOSSOPHAGA SORICINA)
Review round: 2
Reviewer: 3

Output:
Basic reporting: There are still a lot of grammatical error and awkward phrasing throughout. For example, the first 2 sentences of the abstract could be condensed to:

Mounting an immune response is thought to be energetically costly, however, direct measures of metabolic rate during immune challenges contradict this assumption.

Also, in the first paragraph of the introduction sentences 2, 3, 4, 5, 9 & 10 all start with a prepositional or introductory phrase (In particular, For example....). This is poor writing and should be revised.
Experimental design: The authors have met my original suggestions. Thank you.

166. Because you included random effects, you used generalised linear mixed effects models. Not just a GLM. Terminology matters here.
.
Validity of the findings: Looks good.
Additional comments: Thank you for taking the time to address my comments & those of the other reviewers. This will make a nice contribution to understanding the immune systems of bats and their overall variability.

Both Reviewer 2 & I raised issues with the author's discussion of injection dosages on 249-266 as one of the reason for not being 'strictly comparable with most of these studies for at least two reasons.' Especially with the laundry list after it, it minimizes that fact that species do vary incredibly in their response to LPS. It's just what happens and it's not just about body size as you present in your response to the reviewers. Without dosage trials or TK studies to find comparable physiological responses regardless of dosage level, this reasoning is not fair. I suggest minimizing this bit of discussion altogether or highlight _why_ this is true if you feel that a general audience won't intuitively understand it.

308-309. I believe that Brian McNab is the authority on this & shouldn't be ignored.


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: DIVERSITY, HOST-SPECIFICITY AND STABILITY OF SPONGE-ASSOCIATED FUNGAL COMMUNITIES OF CO-OCCURRING SPONGES
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript by Nguyen and Thomas explores the temporal stability of fungi associated to three different marine sponges and compares data with surrounding seawater communities and fungi recovered from the same samples through isolation. There are few similar papers with replicates on sponge-fungal communities. Therefore the manuscript adds substantially to the state of the art of the topic.
The study design is appropriate, as are the methods used, the paper is well-written and therefore I think it deserves publication in peerJ.

I do have minor comments specified below and a number of comments to be more cautious with the presentation and discussion of results (in general comments to the authors):
Experimental design: see comment in box 1.
Validity of the findings: see comments in box 1 and some specific remarks related to this in "general comments for the authors"
Additional comments: MATERIALS AND METHODS
l120: "pemit" = "permit"
l124: what are "fungal samples". At this stage, it is not clear where they came from or what they are. Do you mean the sponge samples harbouring the fungi?
l125: 200 ml of seawater was filtered. This is quite a bit less than most studies do for seawater? Why so little?
l155: was it really 8 minutes?
l159: is there a reference to the ITS primers used?
l190: based on the title, introduction and aim, this section comes as a surprise. It would need some reasoning in the intro why to include this or to leave it out.
Or is it a cut and paste error...?

RESULTS
l267: I understand the categorisation made, but I am not sure if it works out well. Since all samples were collected from a small space, it is very likely that the same 'environmental' fungi will be found in multiple replicates and therefore they may just be transient in many sponge individuals. Here it would be important to compare the fungi found in the sponges to the fungi found in the seawater to help to decide what is transient, resident and 'core'.
And if you use the classification, it would be good to have it consistently recurring throughout the manuscript. For example, now in the conclusion there are hints towards these terms, but they are not actually mentioned anymore.
l306: "OUT_8"= "OTU_8"
l311-313: the way it is stated is not wrong, but it would be more informative to also report that many individuals of these species did not harbour this fungal OTU, because the high averages are solely based on very high relative abundance in one sample for each of the 2 OTUs.
l317: the results should be presented a bit more cautiously here. Here differences in relative abundances are compared, but one sample (T. anhelans 3, 2014) that causes the high relative abundance for OTU63 in this sponge species is based on 8 fungal reads, which makes the relative abundance kind of meaningless. Please also check other number that are presented here in the results, because this one creates a false idea of "difference".

DISCUSSION
l334: "Penicillium can be considered as a 'generalist'...." Would you also imply it makes part of the 'core' fungi isolated? I'm exploring how this matches with the terms you introduced for the paper.
l407: "only other study...". There is now also the study of Chaib De Mares et al (2017) where fungal communities in replicates in Aplysina aerophoba,  Aplysina cauliformis, Dysidea avara and Dysidea etheria.
Front. Microbiol. 8, doi: 10.3389/fmicb.2017.02560
l430: "OUT_8" = "OTU_8"
l431: "OUT_13" = "OTU_13", also OUT41
l466-481: in the discussion data from fungi isolated from sponges are mixed in with speculation on ecological function. This should be done with caution. Also from this study it is shown that there is near to no overlap between the cultivated fungi and fungi observed through cultivation-independent techniques.
Also line 487.
l506: correct the formatting


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: DIVERSITY, HOST-SPECIFICITY AND STABILITY OF SPONGE-ASSOCIATED FUNGAL COMMUNITIES OF CO-OCCURRING SPONGES
Review round: 1
Reviewer: 2

Output:
Basic reporting: The authors describe fungal communities that are associated to three distinct marine sponges. They show that only a small fraction of the fungal community might be species-specific and that various species are accumulated from the surrounding seawater. Moreover, they show that cultivation-dependent and –independent approaches result in the identification of different fungal lineages. The manuscript is well-written and requires minor clarifications/amendments.
Experimental design: The experiments are well described and concise for most parts. Sequencing of fungal ITS regions was conducted in 2014 and 2016. Was the same instrument and chemistry used for both approaches? Different approaches would explain the variation in the results from these experiments. The Material & Methods section includes the description of an amplicon study with 16S gene fragments; however the results from this approach were later not included in the Results and Discussion sections.
Validity of the findings: The findings are sound and well described.
Additional comments: L27: Correct ‘that have so far have not been’ and ‘with in’ in the two sentences.
L130: Brackets are only required for the year of the reference.
L136 & 138 and other parts: Include a separator between values and units.
L73-174: Was the same instrument and chemistry used for both approaches?
L190-207: Why are the results not presented later on?
L229 and other parts: Most journals require italicized writing of all taxonomic ranks with a Latin name.
L289-291: This is the only passage that mentions results from the 16S RNA gene fragment assessment.
L306: ‘OTU’
L308: ‘OTU_14 was’
L314: Rephrase this sentence for better understanding.
L319 and other parts: For taxa with a high variation in abundance it would be better to present the range, e.g. x% - y%.
L359-360: This doesn’t explain why this was not a problem in the approach conducted in 2016.
L386: add brackets for the year of publication
L426: It could also be that these are species without database entries in the utilized reference database or generally without sequenced ITS region although the species might be described already.
L431: ‘OTU’
L461-464: Is it plausible that a lichen-forming fungus occurs at the sampling site? It might be a close relative or a non-lichenized lineage.


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: DIVERSITY, HOST-SPECIFICITY AND STABILITY OF SPONGE-ASSOCIATED FUNGAL COMMUNITIES OF CO-OCCURRING SPONGES
Review round: 1
Reviewer: 3

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: The manuscript generally complies with the editorial criteria: the research is within the scope ofthe Journal, poses meaningful questions and provides a broad discussion with the knowledge in the field.
It is well written, adequately referenced and appropriately structured with data properly considered and nicely presented. This is in dissonance with the fact that the experimental data obtained in two separated sets are not sufficient to validate author’s generalized interpretations.
Additional comments: In this paper, the authors describe the diversity of three co-occurring sponge’s associated fungi, evaluate their incidence stability and host specificity.
Since the knowledge about fungi in these environments is rather scarce the work of Nguyen and Thomas is of great interest for marine ecologist and for biodiversity-interested individuals in general.
The work presented adds valuable information towards the current knowledge in the field of marine ecology and fungal diversity. The comprehensive introduction and competent discussion make the article newsworthy to read. Nonetheless, the results are over-interpreted and conclusions insufficiently supported.
I have the following questions:
-The authors declare to present the first data set with biological replications. Yet, 4 out of 6 Scopalina samples were represented by very low number of reads and practically it is very difficult to treat them as decent biological replicas.
-What is the reason for such a big difference in NGS results standard between data from 2014 and 2016? I am not sure if such data should be concurrently analysed (even after normalization), as can be valued in respective graphics/tables where 6 of them remain NA. The paper would gain a lot if NGS procedure for 2014 samples could be repeated.
-What about the technical repetitions - what is now the standard in such research? Analysing data presented in Tab.1 it seems that ITS PCR amplifications were heavily biased, especially for 2014 samples (e.g., 3 OTUs out of 8 reads vs 2 out of 681 in T. anhelans case). Although in their Discussion authors consider the problem of PCR nonspecific starters for fugal symbionts, lack of amplifications technical details make respectable explication impossible. Were PCR amplifications performed in several independent reactions for each DNA sample? The extremely low fungal reads obtained suggest single reaction.
-Why the cultivation of sponge-associated fungi was not performed in 2016 as well? Since cultivation recovered some organisms not detected by culture-independent approach, these data would strengthen validation of the statements about the in-time stability of fungal population for a given sponge.

Some clarifications in Material and Methods section are needed:
- how many DNA extractions from each sample were done?,
- what was the weight of sponge samples?,
- was the NGS sequencing done in technical repetitions?


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: DIVERSITY, HOST-SPECIFICITY AND STABILITY OF SPONGE-ASSOCIATED FUNGAL COMMUNITIES OF CO-OCCURRING SPONGES
Review round: 2
Reviewer: 1

Output:
Basic reporting: Thanks for the revised version.
Main comments were well addressed.
Some small things listed below.

best wishes
Experimental design: Original Comment:
l190: based on the title, introduction and aim, this section comes as a surprise. It would need some reasoning in the intro why to include this or to leave it out.
Or is it a cut and paste error...?

Response:
We provide now a brief motivation why we also analysed the bacterial community composition:” Given the high variability of fungal communities observed between biological replicates (see below), we wanted to understand if this is a peculiar aspect of the samples we took and how we processed them or if this is due to real biological variation. We therefore also analysed all samples for the bacterial community composition, which has been shown to be very consistent between replicates of the three sponges analysed here (Fan et al. 2012, Esteves et al. 2016).

Comment revision
Alright. That makes sense and I agree. I see that they are also shown now in the results section and included in the discussion.
Please also upload the 16S data then to NCBI or elsewhere so they are of use to the community. As you mentioned before, sponge sample for which both fungi and bacteria were sequenced are rare.
Validity of the findings: Original Comment:
l267: I understand the categorisation made, but I am not sure if it works out well. Since all samples were collected from a small space, it is very likely that the same 'environmental' fungi will be found in multiple replicates and therefore they may just be transient in many sponge individuals. Here it would be important to compare the fungi found in the sponges to the fungi found in the seawater to help to decide what is transient, resident and 'core'.
And if you use the classification, it would be good to have it consistently recurring throughout the manuscript. For example, now in the conclusion there are hints towards these terms, but they are not actually mentioned anymore.

Response:
The definition of “core” is typically applied to OTUs that are found in all (or a very large proportion) of replicate samples for a particular sample type or habitat (e.g. Schmitt et al. ISME J. 2012, 6(3): 564–576.). It is independent of the core organisms being also found in (or derived from) other habitats or sample types. We therefore think the term is appropriate in the current context. We used the term “transient” to specify OTUs that are only once found in any samples type, however we agree that this term has an unintended connotation of a particular kind of interaction. We therefore renamed this category as “occasional”. The same applies for “resident”, which might also suggest some ecological interaction, and our definition of being found more than once, but not in all samples, is better described with the term “variable”. This definition allowed us to focus our analysis on OTUs, for which we have evidence of repeated occurrence in each sample type.

We do also compare those ‘variable/core’ OTUs found in sponges to those found seawater (see Figure 2), and use the category definition in the discussion to conclude that “Implementing these guidelines, our study revealed that ‘variable/core’ fungal communities in sponges have low diversity (≤28 fungal OTUs were observed in each sponge species), high variability and that the majority of fungal OTUs were not specifically enriched in any of the sponge species investigated here.”

Comment revision
The authors have changed the terminology, which is ok to me, but not really necessary to me as it does not really change it. So for the final version I would like to leave it up to the authors which term they prefer.
In their reply, the authors state that they adopt the definition of Schmitt et al (ISME 2012) and that is fair. I’m not a fan of this definition, but that should not prevent publication. I’m not a fan of it, because I think it has many of the same drawbacks that the authors later use in their discussion on the terminology of ‘sponge generalists’ and ‘sponge specialists’: (l389-392 of track changes version) “This classification is very limited and dependent on a) the number of sponge species studied, b) the methods used (culture-dependent or -independent approach (see above), and c) experimental design (use (or lack) of biological replicates or seawater reference communities).”
But as mentioned before, I think my personal view here should not prevent publication and I know that many don’t share my view here.
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: DIVERSITY, HOST-SPECIFICITY AND STABILITY OF SPONGE-ASSOCIATED FUNGAL COMMUNITIES OF CO-OCCURRING SPONGES
Review round: 2
Reviewer: 2

Output:
Basic reporting: The manuscript fulfills all criteria.
Experimental design: The manuscript fulfills all criteria.
Validity of the findings: The manuscript fulfills all criteria.
Additional comments: The authors have answered all arising questions and they have implemented the recommended changes.


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: DIVERSITY, HOST-SPECIFICITY AND STABILITY OF SPONGE-ASSOCIATED FUNGAL COMMUNITIES OF CO-OCCURRING SPONGES
Review round: 2
Reviewer: 3

Output:
Basic reporting: The manuscript by Nguyen and Thomas adds valuable information towards the current knowledge in the field of marine ecology and fungal diversity.
It is well written, adequately referenced and appropriately structured with data properly considered and nicely presented.
Experimental design: The experiments are now described with sufficient detail.
Validity of the findings: The presnted manuscript broaden the picture of diversity of fungi associated to marine sponges.
In the revised manuscript conclusions are properly linked and limited to the presented results.
Additional comments: The revised manuscript now properly points the area of presented study. I found very sound the addition of data for general 16SrRNA communities profiling.

It is a nice pice of work which deserves publication. Although I still reserve my concern about unique PCRs to produce representative amplicons.


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: D-GENIES: DOT PLOT LARGE GENOMES IN AN INTERACTIVE, EFFICIENT AND SIMPLE WAY
Review round: 1
Reviewer: 1

Output:
Basic reporting: The article is concise and well written except a few sentences where English could be improved (e.g. line 35).
Experimental design: The article describes a set of stand alone and web-based tools to rapidly plot interactive genome-wide dot-plots. All the scripts are provided on GitHub which should enhance reproducibility.
Validity of the findings: Dgenies tools would be beneficial for users interested in pairwise genome comparisons. Authors provide a few pre-computed alignments and a way to change the colormap for better visualization and color blind persons. The website is well-designed and easy to navigate.
Additional comments: A few points that will further improve the tool are listed below:

1. In the discussion, it is not clear how to use an aligner other than minimap2. Is it only possible to use other aligners in the standalone version? It will be useful to provide a way to upload paf files (ideally, other file types too) on the web based application to view alignments constructed by other aligners.

2. SVG output file does not seem to work on my mac computer. I tried using web browser and Adobe illustrator to open it and both give an error. It seems there is something wrong with the format specification.

3. A description of the paf file and what does each column mean in the downloaded paf file must be added in the documentation for new users.

4. Time estimates in Table 1 also include the time taken by minimap2. It is not clear if the dot-plot rendering is also faster using Dgenies compared to other available packages, or is it only due to minimap2? It should be clarified.

5. It will be also be useful to allow users to download the original minimap2 alignments to assess their quality. The original alignments of each region can also be added to the tsv file, or a separate alignment file in downloads.

6. It will be a welcome addition if authors could add documentations on how to read the genome dot-plot e.g. synteny conservations, inversions etc. for students and new users.


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: D-GENIES: DOT PLOT LARGE GENOMES IN AN INTERACTIVE, EFFICIENT AND SIMPLE WAY
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The manuscript by Cabanettes and Klopp describes D-GENIES, an interactive way to generate dot plots for large genomes. D-GENIES employs minimap2 as its backend engine for sequence alignment, and the D3.js JavaScript library for frontend  data visualization. It may serve as a useful new resource for comparing genomic sequences.

* Fig.1 needs to be redone. The header (with a black background) is illegible and the main image is too small. The annotations within the image are repeated in the figure caption. Also, some of the annotations are not necessary since the button labels (e.g., "Hide noice") are already self-evident. It suffices to number a few key items in the main image, and then annotate them in the caption.

* In the introduction, the authors cite several previous work on dot plots without mentioning SynMap from CoGe (https://genomevolution.org/coge/SynMap.pl). The CoGe platform is actively maintained, and SynMap is directly relevant to the present work.

* The acronym PAF (Pairwise mApping Format) is first introduced on lines 137-8 in page 5, but referenced twice on lines 48 and 76.

* D-GENIES may not be easy to install. According to the installation instruction on the web portal, D-GENIES has numerous dependencies. Root privilege is required for installation on Linux and the webserver mode "is only available for Unix systems and will NOT work on MS Windows."

* Inconsistencies: dotplot vs dot plot; javascript vs JavaScript; Fig. 1 (bold) vs fig. 1.


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: D-GENIES: DOT PLOT LARGE GENOMES IN AN INTERACTIVE, EFFICIENT AND SIMPLE WAY
Review round: 1
Reviewer: 3

Output:
Basic reporting: There are numerous minor English errors, but it is mostly comprehensible.  Lines 83-85 are too hard to understand, and should be
re-written.

* Abstract: "to easily generated" -> "to easily generate"
* Line 71: "At the to top" -> "At the top"
* Line 128: sourve -> source?

The paper claims it can handle "lowly similar" sequences.  This must
be made more precise, e.g. human-versus-rodent?
Human-versus-marsupial?  Human-versus-cabbage?
Experimental design: In my opinion, visualization is important, and sometimes underrated.
A nice, interactive dotplot viewer could be extremely useful.

The paper should clarify whether D-GENIES reads alignment endpoints
only, or also reads the locations of gaps within alignments.  PAF
format does not necessarily include gap locations.  If D-GENIES
assumes that an alignment is a straight line between the endpoints,
this could be inaccurate if there are gaps.
Validity of the findings: This paper has a critical flaw: there is no evidence regarding the
accuracy of the dotplots.  As a test, I compared the genomes of
E. coli (NC_000913.3) and Y. pestis (NC_003143.1) using D-GENIES and
also LAST (http://last.cbrc.jp/).  The two dotplots look worryingly
different.  Moreover, D-GENIES claims that these alignments are mostly
< 15% identity, which seems odd because random DNA has 25% identity.

The best solution, I think, is to further decouple the visualization
from the aligner.  D-GENIES should allow formats other than PAF, which
is not a standard genome alignment format.  Its input form should
allow input of pre-computed alignments (e.g. widely-used ones from the
UCSC genome database).

Then, D-GENIES could be used to compare different aligners, which
would be interesting and useful.
Additional comments: * I find it very annoying that an email address is mandatory.  I
  managed to use a fake email address (after wasting some time), and
  it works just fine.

* In the result view (fig 1), what is "strong precision"?

* Care is needed when discussing run time and memory use.  Many
  aligners have tunable options to allow arbitrarily low run time and
  memory use, at a cost in accuracy.


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: D-GENIES: DOT PLOT LARGE GENOMES IN AN INTERACTIVE, EFFICIENT AND SIMPLE WAY
Review round: 2
Reviewer: 1

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


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

Input:
Title: D-GENIES: DOT PLOT LARGE GENOMES IN AN INTERACTIVE, EFFICIENT AND SIMPLE WAY
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: I am satisfied with the revisions of the manuscript, except for the following minor typos/inconsistencies:

* Fig. 1 (line 69) vs. Fig 1. (lines 53, 87) and Fig 2. (line 101)
* Lines 106-7: "The Gallery menu item *give* access to several examples ..."
* Line 135: "command *and and* run with the"


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: D-GENIES: DOT PLOT LARGE GENOMES IN AN INTERACTIVE, EFFICIENT AND SIMPLE WAY
Review round: 2
Reviewer: 3

Output:
Basic reporting: Lines 90-93 are still too vague.  How are the bin boundaries defined?
Why need they be "scanned in increasing size order" to "find the most
represented one"?  This is too vague: "the one corresponding to one
percent of its count".
Experimental design: D-GENIES has a bug for reverse strands in MAF files.  One way to see
this: align the human and fruit fly mitochondria at
http://lastweb.cbrc.jp/, download the MAF, and upload it to D-GENIES.
Validity of the findings: I must apologize for saying that the dotplots "look worryingly
different".  I cannot now replicate this.  I suspect I did not notice
that LAST's dotplot is upside-down relative to D-GENIES!

Nevertheless, my main concern remains.  D-GENIES uses minimap2, a very
new aligner: it is not primarily designed for aligning
distantly-related genomes, and to my knowledge there is no public
evidence regarding its accuracy for this task.  More to the point, the
D-GENIES paper mentions no such evidence.

I performed more tests, by giving somewhat distantly-related sequences
to D-GENIES.  I gave it the human mitochondrial genome (NC_012920.1)
and the mitochondrial genome of

fruit fly (NC_024511.2): "Sorry, we did not find any match"
sea squirt (NC_017929.1): "Sorry, we did not find any match"
lancelet (NC_000834.1): "Sorry, we did not find any match"
sea urchin (NC_001453.1): it aligns just a tiny fragment.

In all the above cases, I get extensive alignments and dotplots at
http://lastweb.cbrc.jp/ (with default values for the "Advanced
parameters").

This suggests that minimap2 (as used by D-GENIES) cannot handle
somewhat-diverged sequences.  This is not fatal for the D-GENIES
paper, but the paper must be clear and honest about this.

In a similar vein, it is unacceptable that the RESULTS section has run
time and memory usage only, with no comment on the actual results.  I
tested the first example in Table 1 by giving NC_000913.3 and
NZ_CP008918.1 to D-GENIES.  The resulting dotplot is very sparse,
almost empty.  It is trivial to achieve high speed if you find almost
no alignments!  I suggest the RESULTS should include one more figure,
showing all the dotplots (from D-GENIES, Gepard, and r2cat)
side-by-side.
Additional comments: It is not critical to address the following points.

D-GENIES is already quite user-friendly, but it would become more so
by minimizing the mandatory inputs.  The authors make a good argument
for an optional email address, not a mandatory one.

When plotting a pre-made alignment, only the (PAF or MAF) alignment
file should be mandatory.  The "target file" and "query	file" provide
the sequence lengths, but these are present in MAF and PAF.  The only
case where the target and query	files provide extra information	is
when some sequences participate in no alignment.  In such cases, if
only an alignment file is provided, presumably D-GENIES would only
show sequences that have alignments, which seems OK.


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: ADAPTIVE GENETIC VARIATION AT THREE LOCI IN SOUTH AFRICAN VERVET MONKEYS (CHLOROCEBUS PYGERYTHRUS) AND THE ROLE OF SELECTION WITHIN PRIMATES
Review round: 1
Reviewer: 1

Output:
Basic reporting: Basic reporting is acceptable.
Experimental design: Experimental design is appropriate, although analyses would benefit from a more targeted approach - see my general comments.
Validity of the findings: The findings and interpretation are largely in line with the results. See also my general comments.
Additional comments: In this study, the authors examine diversity of three genes (TLR4, TLR7 and ACR) in vervet monkeys across South Africa. Population genetic analysis was undertaken to examine the distribution of diversity across habitats, although overall diversity was low. The researchers also examined sequence variation among species by comparing their new data to sequences from 11 – 21 primates and conducting various selection analyses. Overall this paper is well written, however the analyses are hampered by very low levels of diversity overall (although I note that low diversity should not necessarily preclude publication). The addition of cross-species comparisons provides an additional angle to the work that supports the main findings. I have a few comments that relate to the analysis:

At present, the paper comes across as though the authors attempted to conduct as many analyses as possible, to try and make the most of the data they had. While it is commendable that the authors should explore all aspects of their data, unfortunately when diversity is low this also poses a risk of over interpreting data. I believe the paper would benefit from a more targeted approach to the analysis. For example, I am not sure that population-level substitution analyses (such as Tajima’s D) would be informative for a dataset that contains only a handful of SNPs, although welcome the authors’ perspective on this. Similarly, seven codon-based sequence analyses are conducted (5 datamonkey methods, plus 2 Paml methods) – this is a lot.  Many of these methods overlap in their objectives, and are therefore not necessarily independent lines of evidence for selection. It would be helpful to point out which methods are similar to one another, and justify why using each is important. What different information do they provide? What are the pros and cons of each method?

The authors allude to a “selective sweep” as a possible explanation for the low diversity they observe (mentioned in the Abstract, and otherwise just once, in the Discussion at L355). This is an interesting idea relating to the low diversity that was observed, and which could therefore be expanded. What evolutionary mechanisms would cause a sweep for these genes? Is it likely in this widespread species? Are there precedents in the literature? What additional data would be required in order to confirm the sweep?

Do the statistical methods used throughout the paper account for the fact that TLR7 is on the X-chromosome (and that males therefore are hemizygous, not homozygous)? This is important, because much of the ecological analysis relies on haplotype diversity, which is calculated from haplotype frequencies and therefore depends on an accurate count of gene copies in the population. It would be appropriate to reiterate the suitability of methods for sex-linked loci throughout the Methods.

Minor comments:

L26: “Low levels” – be explicit, give the numbers of synonymous and nonsynonymous SNPs
L35-36: Briefly explain how this data could be used.
L109: Suggest changing “the original paper” to “a previous study using mtDNA analysis”
L222: In the current version, the source of the samples is explained 3 times – suggest deleting from here to avoid repetition
L129: Explain how these “partial segments” were chosen for targeting
L172: Explain what kind of GLM was used and the rationale – was it a logistic regression?
L290: “It is well known…” please provide references and context
L312: Why is the non-significant result surprising? The paragraph L311-332 draws a lot of inference from a non-significant finding. I believe it would be more appropriate to shorten this substantially.
L334: I do not understand what information is being conveyed in the parentheses.
L343-347: Note that purifying selection is identified at the codon level, not the SNP position. Thus, I’m unclear how these results support evidence that synonymous substitutions could have adaptive significance. It is the codon that is under purifying selection.
L380: It would be useful to explain how the different evolutionary histories of these taxa could drive the different patterns – what evolutionary process would you hypothesise?
L390: Typo “selection”
L394: Missing word “different immune-linked genes”
L400-401: As “conservation” is also a rationale for this study (Intro L106), it would be helpful to have some information on the conservation status of the species and current threats. Please provide more detail of how the results of the current study could be used to inform conservation of this species.
Figure 1: Please explain the importance of the difference symbols used for the sampling sites
Figure 1: The position of the sampling points in C is shifted relative to their position in B – is this intentional?
Figure 3: According to the hypotheses posed, I think it would be more appropriate if Hd were the dependant variable (response, y-axis) and precipitation were the independent variable (predictor, x-axis) – is this how the statistics were conducted?


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: ADAPTIVE GENETIC VARIATION AT THREE LOCI IN SOUTH AFRICAN VERVET MONKEYS (CHLOROCEBUS PYGERYTHRUS) AND THE ROLE OF SELECTION WITHIN PRIMATES
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript is well-written in clear, professional language. Although the figures and tables clearly present the materials and the outcomes of the analyses, the presentation of additional data would be useful, especially the following.
•	A table should be added that summarizes all vervet polymorphic sites that were identified in the study.
•	Raw sequence data is unnecessary, but an additional supplementary table containing genotype data from all sequenced animals would be useful.
The Introduction well summarizes the background information and the rationale behind this study; however, it could be supplemented with what is known about the genetic diversity and adaptive genetic variation in the genus Chlorocebus.
Experimental design: The study is original research, within the scope of the journal, and approved by the appropriate ethics committee. The overall experimental design is clear and implements standard methodology that properly addresses the research question about the effects of selection on genetic variation in South African vervet populations. The study takes an interesting approach, combining genetic data from vervet monkey populations and local environmental data supplemented with natural selection analyses in vervet populations and 23 other primate taxa. The study targets three preselected candidate genes that are potentially linked to fitness and climatic conditions. A limitation of this study is that it analyzes only partial gene sequences, including only 1‒2 exons with adjacent introns.
•	Line 224: Haplotype sequences mentioned here as being deposited in the GenBank are not available in this database. Please add them.
•	How were the target sequences within the selected genes chosen? If it is available, provide information on the functionality of the gene region covered by this analysis. Do they code any specific protein domains?
•	Supplementary Table 1 suggests that multiple animals were drawn from each of several troops. Comment on the possible effects that the relatedness among the animals in this study has on its results.
Validity of the findings: The data are robust, and the findings are valid. The authors properly comment on the possible limitations and biases that are due to analyzing partial gene sequences and to the relatively low sample size for each haplotype (the overall sample size is impressive for studies in wild primate populations). This study generates important findings beyond vervet monkey evolution. The data obtained can inform primate management in South Africa.

Other Points
•	Supplementary Table 1: The “Shamwari NR, Dump, EC” site lacks geographic coordinates.
•	Figure 3: It would be useful to include p-val and slope in the figure legend.
•	Supplementary Figure 1: Clarify in the figure legend whether it is a haplotype analysis.
•	Line 28: The sentence “Population genetic…” seems too vague for the Abstract.
•	Line 267‒270: It would be easier to read the text if both the text and Table 3 used the same codon numbering system. For clarity, perhaps you could just add human codon numbers to the text.
•	Line 276‒279: Based on Table 3, the highest haplotype diversity levels seem to be for TLR4 (0.996), not TLR7 (0.988), as stated in the text.
•	Line 305: To which study does “from this study” refer?
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: ADAPTIVE GENETIC VARIATION AT THREE LOCI IN SOUTH AFRICAN VERVET MONKEYS (CHLOROCEBUS PYGERYTHRUS) AND THE ROLE OF SELECTION WITHIN PRIMATES
Review round: 1
Reviewer: 3

Output:
Basic reporting: Overall the language is clear and professional, but there are some areas that need clarification or correction, cited here for convenience

1) Lines 35-36 regarding impact of the study on conservation agency decision making are not very clear. Please reword to better connect to the rest of the abstract. The impact described is not obvious.

2) Line 76 the organisms listed here are not PAMPs. PAMPs are pathogen-associated *molecular patterns *. Fix this error by replacing "gram-positive bacteria,..." etc. with the relevant PAMPs (i.e. peptidoglycan etc). It would helpful to put the pathogen type in brackets alongside those PAMPs.

3) Since the paper largely focuses on two TLRs and proposes connections between gene diversity, environmental conditions and the pathogens-detected by these TLRs, the authors should be citing original citations when describing the PAMPs in lines 75-77 rather than a single review. Invivogen has actually done most of this work for you. They state the original papers identifying these molecules as ligands for TLRs in their product inserts for TLRs. That's how you can find those papers, review them and cite them (as you have later in the paper, e.g. Kurt-Jones et al. 2000; Diebold et al. 2004).

4) Related to #2 and #4 lines 77- 79 describing what the two major groups of TLRs recognize is not accurate. Extracellular TLRs recognize a broad range of extracellular threats, including bacteria, parasites, fungi, not "mainly bacteria". TLR4 also recognizes respiratory syncytial virus. Similarly, intracellular TLRs recognize mainly nucleic acids, including self-nucleic acids. So while they certainly recognize viruses, they also recognize unmethylated DNA from intracellular bacteria, fungi, parasites and have been found in experimental to recognize host DNA. Revise this description as "extracellular threats" with examples and "intracellular threats" with examples, and the misleading writing here will be resolved

 5) lines 83-86 The broad statement that Gram-negative bacteria can cause a range of infections is poorly backed up by examples and citation. Given the focus on TLRs as receptors, these examples should be name pathogens with the manifestion following. As worded it sounds like E. coli is major cause of meningitis.

6) Throughout the article when genes are clearly being discussed, they are not italicized in text as is required. Please fix this

7) Line 253 possible mistyping. "D- loop". TLR7 has a Z-loop. Is that what you mean here? It otherwise sounds like you are discussing mtDNA, which has not yet been brought up in the paper.

8) descriptions of sources of DNA, here described only as "tissues" and unattributed "samples" are not specific enough to help a reader understand the amplification methods you then pursued. Please describe these samples further in text, or provide a supplementary table. What are the tissues? Are any of these samples not tissues but
fecal?

9) Line 302-310 This section requires develop. The connections being made between environment and "pathogen-specific" molecules are not obvious. Moreover, the paper focuses on TLRs, which detect "pathogen-associated" not "pathogen-specific" molecules, so the meaning of this section is a little confusing given prior pages. This distinction matters because TLRs are innate immune broadly sensing sentries of threats, and the term "pathogen-specific" is usually reserved for descriptions of adaptive cellular responses to an identified epitope.

10) Line 317. TLR4 also detects a range of heat shock proteins in humans (e.g. hs70), which is likely a more relevant example. In both mice and humans TLR4-mediated responses to heat shock proteins are very well established and very common.

11) and probably most importantly, there is a complete lack of clarity regarding the regions of these genes that being amplified. Given that the paper is focused on diversification and evolution of a very small number genes based on only partial amplification of those genes, the bp positions within the gene and the genome sequence should be identified here. Simply stating partial sequence from exon 4 and not detailing in a table or anywhere in the paper where that sequence is in the gene makes it very difficult for a reader to assess the results. Along with that, the domains of the TLRs in this paper are well defined. The domains of these genes have been found to be evolving at different rates. The authors need to clarify in the primer table what domains are being addressed here. Otherwise the results can't be fairly interpreted by the reader.

Article structure is professional, figures, tables are shared. Raw data has been submitted to Genbank

Paper is self-contained with results that inform the hypothesis.
Experimental design: The primary research is within the aims and the scope of the journal. The research question is well defined and relevant an meaningful. The authors have stated that the research fills a knowledge gap for this species.

From an investigative standpoint, though, the paper has problems. Along with the lack of clarity regarding methods, mentioned above, there are some other issues that need to be addressed. The authors analyzed the sequences of only 3 genes from 81 samples. One gene is reproductively linked and the other two are well studied genes associated with the immune system. This is very little data to draw some of the conclusions that the authors have drawn here. If they did test other genes and did not get a result, it would be preferable to list those genes as well. Otherwise, the paper appears insubstantial. Given the relatively low cost, it is difficult to understand, for example, why they did not address other TLRs that are more strongly connected to the background examples they give as a justification for this research. Those papers make connections between climate and parasite infections, for example. Additions like TLR2/1, TLR5, TLR9 would have been logical genes to include.

More importantly no explanation is given for why only partial sequences of genes are examined here. For example, TLR4 is a ~6100 bp gene encoding around 820 amino acids. It's a small readily amplifiable gene, the domains differ significantly in their variability within and between species. The TIR domain and small cytoplasmic tail of TLR4 appear to be able to handle a great deal of variation, with humans, rhesus monkeys and mouse, for example, differing in the length and construct of the this domain considerably. Between rhesus monkeys and humans, for example, a quick alignment of publicly available sequences shows a 13 amino acid deletion in this area. Within species differences can be substantial as well (e.g. this old review. Please note that LRRs are structural repeats, not domains. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2215765/)

 The authors acknowledge that their results may have been shaped by the regions of the gene that they amplified. They most certainly will be, and this has been established in the past, so there needs to be a justification for why only partial gene regions were assessed, what protein domains they represent and why those regions were chosen. This problem holds for TLR7 and ACR as well. Once described, it would be preferable to put the results in the context of what is already known about the evolution of these sequences in mammals or primates, regarding the diversity of the sequences from these domains.
Validity of the findings: See above. The findings are not valid if the authors cannot address why they used partial sequences and put it in the context of what is known about the variability of the domains they amplified either across or within primates, mammals. There could be perfectly reasonable rationale. It needs to be up front.

Data is not particularly robust at the gene level. Only three genes are assessed, when the question would be better addressed by the consideration of more genes. At the within species level, the authors have a very robust data set of 81 samples. They should make clear whether or not those 81 samples represent 81 individuals.

The analysis and are discussion are very simple. Though the results are minimal, there are means of demonstrating their importance to the reader that the authors have not used here. It is strongly suggested that the authors map a reference Chlorocebus sequence to the human TLR4 (3FX1; 4G8A) and TLR7/8 (4QC0) sequences in PDB (there is a boar ACR sequence) and highlight where the variation is occurring in the protein (where applicable). This can be done by homology modeling which is very straightforward. Since the model is not to be assessed or argued, it wouldn't need to be verified. It would serve as a mannequin on which to illustrate a) the data that was actually gathered and b) where the variable hotspots are. Given that the available structures are bound to ligands, this would allow the authors to discuss the implications for pathogen detection.  If the amino acid sequences are very similar to humans, then the authors should use a human construct label it as such. Regardless, there is a need to connect the experimental findings to the data that was recovered in a stronger way. This is the method suggested here.
Additional comments: This is an interesting pursuit. To be accepted, the methods of the paper need to be more clear. The rationale for the limited sequence length needs to be stated. The results needs to be interpreted in the context of the protein domains in which the sequence occur because it is known that these domains vary in their diversity between and within species and by domain.


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: ADAPTIVE GENETIC VARIATION AT THREE LOCI IN SOUTH AFRICAN VERVET MONKEYS (CHLOROCEBUS PYGERYTHRUS) AND THE ROLE OF SELECTION WITHIN PRIMATES
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: In this paper, which I reviewed previously, the authors examine TLR and ACR diversity in populations of vervet monkeys across southern Africa. Overall the paper is much improved over the previous version. The paper is much improved, and the authors have taken my suggestions on board. I have only two minor comments:

The authors indicate in the Abstract that ACR had no exonic variation [L31], but selection was nevertheless detected [L34].  Selection tests should be undertaken on the coding sequence only (refer http://www.datamonkey.org/help), so I am unclear how selection was detected at the intron.

Paragraph beginning L344: full regression statistics should be provided, perhaps in supplementary material.


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: ADAPTIVE GENETIC VARIATION AT THREE LOCI IN SOUTH AFRICAN VERVET MONKEYS (CHLOROCEBUS PYGERYTHRUS) AND THE ROLE OF SELECTION WITHIN PRIMATES
Review round: 2
Reviewer: 2

Output:
Basic reporting: .
Experimental design: .
Validity of the findings: .
Additional comments: My comments were properly addressed, I recommend the revised  manuscript for publishing.


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: TELEOST AND ELASMOBRANCH EYE LENSES AS A TARGET FOR LIFE-HISTORY STABLE ISOTOPE ANALYSES
Review round: 1
Reviewer: 1

Output:
Basic reporting: The paper is well written in clear and unambiguous English, although some terms may be unfamiliar to the non-specialist and could benefit from brief definition.

All references in the reference list are cited in the text although I found 8 references in  the text that are not in the reference list: Schloesser, Hanson, Estrada, Kroger, Matthiessen, Bron, Lynnerup, Kjeldsen. In some parts of the text, Wallace et al (2014) is referred to as Wallace (2014) - lines 68 and 519.

The Introduction provides a very good review into the topic, particularly for this who are unfamiliar with the field of vertebrate eye structure, as the authors summarise very clearly the relevant detail on eye structure, how the lens is formed and why it is a suitable repository of isotopic data. What is less clear is how the aims of the study (lines 212 - 218) relate to, and lead on from the Introduction. In the present draft they stand alone from the preceding Introduction. I was left asking myself 'why' after reading these points. Please provide some background detail/explanation

In my opinion there are some statements in the text that require supporting references; e.g. lines 155-156, 157, 183-184 (in fact the whole paragraph on elasmobranchs line 183 and following) could do with better referencing), 224, 284 and line 565. In lines 52-56, rather than pick a couple of select references it might be better to cite some recent reviews covering larger range of studies?

The paper is well structured with excellent figures that present the data very clearly with appropriate legends of sufficient detail.

I think that the authors should structure the methods/Results sections so it is clearer which samples are being used for which analyses. This is especially so with the stable isotope analysis as the text reads as if measurements were obtained for all species (by my reading of line 275 and following) when this is not the case.

Line 315 - provide statistical support for the statement that standard runs between labs were similar - presumably data were compared?
Experimental design: It is clear as one reads the paper that the sampling design for this study was opportunistic based on various species caught on various cruises and archived and the authors should acknowledge that. I am sure if they were planning this work again they would think about sample sizes and size ranges and redesign their sampling. I an not criticising this as a flaw to the study but I think that this should be acknowledged. Also I think that some 'signpost' statements would be useful. For example, explicitly stating although sample sizes are extremely small in the lens morphology section of the paper, the aim was to compare lens structure across a range of fish taxa and so the data collected provides a good indication of variability in lens structure and permeability (especially as I am guessing that  nobody else has ever done this).

As I mention in the previous section, the research questions defined (lines 212-218) need better defining as they stand alone from the Introduction. It is clear that this study fills an identified knowledge gap and is the first step in developing a refined method to use the fish eye lens as a tool to monitor trophic geography and movement patterns.

The Methods provide sufficient detail.
Validity of the findings: This study provides a very useful advance in the development of the  fish eye lens as a tool to monitor trophic geography and movement patterns - it is novel and contains valuable data whose publication would help develop this recent field of research.

Due to samples sizes and the questions being asked, some of the results are descriptive and/or lack statistical analysis, however I do not see this as an issue given the novelty of the subject, and the absence of any other data for this topic.
Additional comments: This is a nice first step in trying to develop methods to use the fish eye lends as a recorder. The limitations of the data and  approach need to be fully recognised


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: TELEOST AND ELASMOBRANCH EYE LENSES AS A TARGET FOR LIFE-HISTORY STABLE ISOTOPE ANALYSES
Review round: 1
Reviewer: 2

Output:
Basic reporting: The authors present a well written and informative manuscript on a topic of high importance.  While the authors need to address several minor edits as listed below, a revised version of this manuscript will add a timely and necessary contribution to the literature considering the relatively recent interest in the use of eye lenses to study life history characteristics in fishes.  There are several editorial concerns throughout, which are listed here for the attention of the authors:
1)	The term “fish” should be used when referencing a single species, whereas the term “fishes” should be used when referencing multiple species.  This needs to be addressed throughout the manuscript and changed accordingly.
2)	The term “between” should be used for comparisons between two subjects.  The term “among” should be used when addressing three or more subjects.  This needs to be addressed throughout the manuscript and changed accordingly.
3)	Several assumptions and conclusions throughout the manuscript need to be qualified with references, data, or an explanation by the authors.  In several examples, the authors need to allow for alternative explanations as well as their own.  Individual instances are listed in the following sections.

Further, several specific issues are listed for consideration as well:
1)	Line 22:  This language is misleading.  The archives that are being referenced are actually of elements/minerals/isotopes/etc… I recommend rewriting this sentence to clarify that the archives of the analytes represent behavior and physiology.
2)	Line 24:  Please consider removing the comma after the word “unfortunately.”
3)	Line 45:  This sentence makes it sound like the insights are only derivable in marine animals.  This is obviously not the case, so I would suggest either clarifying, or re-writing in a non-ambiguous manner.
4)	Lines 46 – 48: Please consider adding additional information regarding other positive components of this method.
5)	Lines 51 – 56: As written, this sounds like these are the only examples that exist.  I suggest either a thorough review of all examples, or clarifying language to make it clear that these are only several examples.
6)	Line 57:  I recommend clarifying that the isotopic analysis of the organic component in the otolith in this study was not derived as a chronological recorder.  As written, this is not clear.
7)	Lines 49 – 64:  This entire paragraph reads like a fact sheet instead of a story that has a narrative throughout.  Please consider re-writing the paragraph to highlight important components in a logical and clear manner.
8)	Line 68:  I recommend citing Tzadik et al. 2017 that offers a review on this topic.
9)	Line 81:  Please consider qualifying this sentence with a logical transition.  As written, the discussion of the cornea does not flow logically from the previous statement.
10)	Line 100:  Please consider explaining further how growth rate varies, and whether the reference is to the course of an individual’s lifetime, or whether this is a comment that refers across individuals.
11)	Lines 110 – 111:  This is very confusing.  Several sentences earlier, the authors declared that growth rates are variable.  I recommend clarifying this point.
12)	Lines 155 – 156:  Is this the opinion of the author?  Can this statement be qualified?  Please consider adding a reference, or other reasoning to qualify this sentence.
13)	Line 187:  I recommend adding a reference or additional qualifier to this sentence.
14)	Lines 195 – 196:  This sentence along with the previous one make it sound like the authors are only referring to pelagic fishes.  I would recommend clarifying whether this is in fact the case, or not.
15)	Line 230: Please consider changing “of the west Scotland…” to “off the west Scotland…”
16)	Line 711:  Please consider clarifying the page numbers for this citation.
Experimental design: The authors adhered to a strict protocol during sample preparation and present their results in a clear and scientifically-responsible manner.  Minor edits are suggested below:

1)	Lines 246 – 254:  I recommend qualifying the assumption that water temperature will not affect metabolic processes, including mineral/protein deposition within the eye lens.  In addition, the assumption that small sample sizes are representative should be qualified.  These assumptions should be qualified in the discussion section as opposed to here.
Validity of the findings: The findings in this study are valid and well-supported.  However, there are several instances where the authors fail to recognize alternative explanations for their findings.  Minor edits are presented below:
1)	Line 366:  Is lens porosity the only explanation?  If so, please clarify why, or cite previous studies.
2)	Line 381:  Can the authors please explain how the A and B terms were derived?  Where they derived pre-analysis?  Were they derived by weight ratios?  I recommend clarifying this point.
3)	Lines 382 – 384:  Please consider validating this assumption.  Could this assumption present a problem in the analysis of fishes that live in highly contaminated waters?  I agree that the assumption is valid for this study, but I would recommend validating the statement to clarify that the values were obvious for this study, but that future studies should at least consider the possibility that the division will not be so clear.
4)	Lines 433 – 434:  Please consider quantifying this variability.  The figure does not clearly qualify the statement.
5)	Line 441:  Please consider quantifying this variability for consistency purposes.
6)	Lines 464 – 465:  I recommend adding that this has implications for age as well if a length-age relationship has been previously determined.
7)	Lines 521 – 522:  Please consider qualifying this statement with a citation or further examples.
8)	Line 546:  Please consider qualifying why this is considered an “isotopically constant nutrient source.”  I can imagine very erratic behavior from a pregnant mother that may lead to altered feeding behavior.
9)	Line 571 – 573:  Have the authors considered siblicide as another explanation or isotopic variability?
10)	Lines 588 – 589:  Could siblicide be a consideration here as well?
Additional comments: Overall, the material is presented in a concise yet comprehensive manner.  The figures, tables, and supplementary materials are all straight forward and easily understood.  The authors do a good job of reporting their results, stating their analysis, yet not over-reaching in their interpretations of their findings.


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: BULLYING VICTIMIZATION AND CHILD SEXUAL ABUSE AMONG LEFT-BEHIND AND NON-LEFT-BEHIND CHILDREN IN CHINA
Review round: 1
Reviewer: 1

Output:
Basic reporting: 1. Minor grammatical errors and misuse of punctuation and prepositions (e.g. line 91, 125); need to proofread.

2. Unclear writing/irrelevant content here and there:
- line 31, “bullying victimization was higher risk factor for CSA”. Higher compared with what?
- line 34, "CSA was higher among school girls," but the supporting numbers are for "left-behind CHILDREN vs. non-left-behind children", should be "left-behind GIRLS vs. non-left-behind girls"
- line 82, "the link.. remains to be explored". Do you mean "unexplored"?
- line 90, target population aged 11-18 from middle schools", should be "middle school and high school"?
- line 125, assessed by 10 items, but only listed 5 items?
- line 130, does "home place" mean the child's current place of residence? or place of household registration?
- line 186: "direct and indirect effects of CSA": what are they? Which ones are direct? Which are indirect?
- line 216, unclear meaning of "a rebellion stage" and how it relates to your discussion.
- line 219, "children having good relationship with family are less depressed", seem irrelevant as depression was not mentioned anywhere in your lit review/study.
- line 247, the measurement for "social economic level" was discussed as a limitation but was not part of your measure/study.

3. Literature review needs more context, details of existing studies, and re-organization.
- line 60, what year was this national prevalence for? Are the numbers up to date?
- line 69, "left-behind children, a special group in rural China..." How are they special? Special in terms of what? Need a little more context here.
- line 79, you cited Meinck's study, what was their target population? What was the strength of prediction? Need more details.
- some parts in discussion should be moved to lit review: line 192-203.
- need more literature to support why you choose the covariates.

4. Citations should use last names only.

5. The table 1 formatting makes it unclear between which groups the difference was significant.

6. Titles for table 2 & 3 are misplaced.
Experimental design: 1. research questions and hypotheses are unclear.

2. rationale for the study: your identified research gap is “the link between CSA and bullying victimization remains to be unexplored” (line 81), but you cited studies that already explored this link (line 53 & 67)? Perhaps highlight your contribution is no existing studies looked at Chinese left-behind children.

3. method: specify how did you recruit participants, how to select sampled schools, what are the school characteristics, and which grades are the students from.

4. ambiguous explanation for dropped cases: is it because missing data (line 96) or age over 18 (line 155)?

5. ethical concern: need to provide child oral assent form/template

6. did any parents/guardians refuse to participate, since their child may be sexually abused by them?

7. was the survey translated into Chinese? Were the questionnaires culturally applicable?

8. how was the survey administered? By whom? You mentioned it’s done in computer labs, so was it a group setting? Would that bias your results if students were uncomfortable to report when their peers were in the same room? Were anyone on site to answer questions during the survey? Were severely-abused/bullied cases referred to social worker/counselor?

9. unclear measurement: “parental educational level”, of father or mother?

10. data analysis: need to report relative change between crude and adjusted estimates in order to assess effects of confounding variables. Right now you only discussed table 3 results, table 2 was not discussed at all.

11. data analysis: since your main IV is bullying victimization while controlling for all other covariates, why not report multivariate regression tables?
Validity of the findings: 1. If you did not measure the time point of bullying/sexual abuse, then you cannot establish causal inference (as briefly noted in your limitation section). Need to avoid using causal language.

2. Some findings cannot be supported by your study:
- line 204, “the worse the degree of being bullied, the higher risk of sexual abuse” –you did not test the degree of bullying in analysis, only being bullied or not.
- line 238: you did not measure marital status in your analysis.
- line 247: socioeconomic status, you did not mention in your tables/measures.

3. Discussion can be more focused on your main IV (bullying victimization), rather than the covariates (gender, age…)

4. potential selection bias: your sample was from middle school and high school, would younger left-behind children and those dropped out of school be more vulnerable? Peers and being in school are protective factors. In other words, your sample may be in better status than other left-behind children. Maybe add as a limitation.

5. what does your findings mean? What policy/practice implications do they bring?
Additional comments: This study discussed an important issue for a large high-risk population in China: left-behind children. It potentially could make contribution to literature, but does require more introduction of the context and existing literature, clearer research question, more rigorous reporting in methodology and results, and elaboration on findings and limitations.


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: BULLYING VICTIMIZATION AND CHILD SEXUAL ABUSE AMONG LEFT-BEHIND AND NON-LEFT-BEHIND CHILDREN IN CHINA
Review round: 1
Reviewer: 2

Output:
Basic reporting: 1.	It seems there are few studies focused on CSA among LBC and this paper is likely to fill this gap of knowledge in some way. However, it is better if the authors could provide more review on studies on factors that are related to CSA and bullying victimization, justifying the inclusion of covariates in the current study.
2.	There is a lack of theoretical review in this paper and theoretical framework is missing. The authors should also justify the theoretical logic under this study.
3.	Please clarify the statistics used at line 45, are they description for China or for the whole world?
4.	Languages should be improved or re-edited in some places. For example, at line 56-57, “victims of bullies” and “bully-victims” are reiterated for the same meaning. The description of findings in a previous study at line 67-68 is better to be further clarified or restated. The meaning is not clear.
5.	The formats of in-text citations and references are not correct in some parts of this paper. For example, at line 199, should be “Duncan (1999) reported that…..”, while should remove (Duncan, 1999) at line 2000. Same thing for Mansbach at line 200. The authors should check these kinds of in-text citations over the paper. In addition, many papers were not referred correctly in style in the section of references, including but not limited to line 275, 283,295,305,317,319,327, 330 etc.
Experimental design: 1.	Research questions may better to be clearly defined although the knowledge gap has been identified in the introduction section.
2.	The research design has been illustrated in detail. However, regarding measures of variables, it is quite confused for measurements of child sexual abuse. The authors may need to further clarify it. At line 125-126, the authors mentioned 10 items but only listed five here, or it should be five aspects and each was measured by 2 forms, thus in total 10 items? In terms of covariates, it seems in the later part of this paper (see line 150,163, 223 and 241) the authors mentioned parents’ marital status but no relevant information was shown in the section of Covariates (line 129). Nor in the tables. The authors should explain for that.
3.	It seems the statistical analysis described does not match what was shown in Table 3 and results at lines 175-183. The results at lines 175-183 seem to present results for binary logistic regressions for the total sample, LBC and non-LBC, respectively, controlling for covariates. However, what has been shown in Table 3 and described in statistical analysis (see line 150-151, e.g. subgroup analysis) seem to be conducting analysis for each group of gender, age, home place, parent marriage etc. and investigate the associations between bullying victimization and CSA. The authors need to be clear of that.
Validity of the findings: 1.	Despite the confusing presentation of Table 2 and 3, the results written since line 152-183 seem to show the associations between bullying victimization and CSA, as well as its difference for LBC and non-LBC. In addition, other associations for covariates were also illustrated. As mentioned above, the results of parental marriage cannot be obtained from the current table and where are they from? Also, pay attention to it in the discussion part.
2.	The authors explained most of the findings connecting with existing literature. However, what is the theoretical contribution of this study?
3.	Limitations were not identified enough. Particularly, the authors simplify the variable of CSA as a dummy variable, for which it is coded as one if any of those items were answered with yes. CSA with the five aspects at line 126 may hold different levels of severity from each other. Simply condensing information in one dummy variable may lead to biased results of the current study. The authors should identify and explain the potential biases of results in the current study. I would also suggest trying multinomial logistic regression.
Additional comments: This paper focuses on an interesting topic that is related to bullying victimization and child sexual abuse, especially contributing to relevant knowledge for left-behind children in China. For most parts of this paper, it is well written in English. However, the current study seems not sound enough and there are some points suggested being improved and clarified.


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: BULLYING VICTIMIZATION AND CHILD SEXUAL ABUSE AMONG LEFT-BEHIND AND NON-LEFT-BEHIND CHILDREN IN CHINA
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment
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: N-ACETYLCYSTEINE PROTECTS AGAINST MOTOR, OPTOMOTOR AND MORPHOLOGICAL DEFICITS INDUCED BY 6-OHDA IN ZEBRAFISH LARVAE
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript by Benvenutti et al reports findings that N-acetylcysteine, a drug with a polypharmacological profile, blocks the motor effects of 6-OHDA in zebrafish larvae, a putative model for Parkinson's disease. The text is clear and unambiguous, with good and professional English, and professional structure of the article; the raw data was shared (although in a format that precludes automated analyses; CSV should be used, and columns organized as such). The article appears to be self-contained, but the hypothesis is generic and not directly tested.
The manuscript partially follows ARRIVE guidelines; the abstract and introduction are OK, but there is a need for more minute descriptions of the hypothesis and objectives. As it stands, the hypothesis (“we hypothesized that NAC may be a strong candidate for the prevention of PD”, lines 95-96) is generic, and not directly tested. The authors state that NAC could be a good candidate for repurposing due to the role of oxidative stress and neuroinflammation in Parkinson’s disease, but these endpoints have not been analyzed in their model.
A secondary issue is the lack of clear definition of primary and secondary outcomes assessed (also a recommendation from the ARRIVE guidelines). From the literature (e.g., 10.1016/j.neuro.2016.11.006; 10.1002/pmic.201500291; 10.1016/j.nbd.2010.06.001), we should expect motor alterations to be more relevant to modeling PD in zebrafish than, say, the morphological variables. What behavioral endpoint (from either the first or second behavioral assays) the authors consider the primary outcome? (This is also relevant for sample size calculations [see below], which should be made considering the primary outcome).
Third, the authors do not report why unequal samples sizes were used for each group. Incidentally, the group with the smaller sample size (and below we will discuss that this is not and independent replication) is the one treated with 6-OHDA, suggesting adverse events and/or censored data. If that  was the case, what was the rationale for removing the data point? Did treatment results in death of one animal from this group? Were any gross anatomical alterations found in one animal? Were the data produced an outlier? If so, how was “outlier” defined?
A minor problem is that the authors did not report whether the statistical analysis used weighted or unweighted means analysis (they used unweighted-means analysis, the only way to reach the values found in Table 1). This is crucial for reproducibility.
Experimental design: The experimental design proposed by the authors is interesting in that animals were tested in both behavioral assays, therefore reducing the overall number of individuals used. There are some important problems with the design that need to be addressed.
-Your most important issue is that of randomization and blinding. Masca et al. (2015; doi:10.7554/eLife.05519), examining the problems of reproducibility in biomedical research, observed that one of the main reasons for irreproducible research is the lack of blinding/masking and convenience samples. From the text, it is not possible to know whether or not these crucial steps were taken. Were interventions (NAC) and conditions (6-OHDA) allocated at random to animals? If so, what was the method of random allocation? If not, why? Are there any other possible confounders (e.g., dishes) to which the units may need to be randomly allocated? Were care providers (lab technicians or anyone who cares for the animals) blind to treatment? Were experimenters blind to treatment? Were data analysts blind to treatment? If not, why? How was allocation concealed, and how was blinding maintained? Under what circumstances will the data be
unblinded? These questions need to be addressed, and clearly stated in the methods section.
-There is an important issue of pseudo-replication. From lines 123-127, it appears that animals were exposed as a group to each treatment (that is, animals from the 6-OHDA/NAC group were exposed to these treatments in a group of 8-11, animals from the H2O/NAC group were exposed as a group, and so forth). From the definitions and recommendations made by Lazic (2010; doi: 10.1186/1471-2202-11-5), that is a hierarchical/nested design, with a high risk for pseudoreplication in that the experimental unit is not the individual larva, but the treatment well. In zebrafish research, it is common for larvae to be treated individually in microplate wells to circumvent this problem. Please explain why the authors chose a different design, and clearly state it in the methods section.
-How was sample size calculated? Was it based on a pilot experiment to determine effect sizes? How were calculations performed? It seems that the sample sizes used for each group are small, leading to an underpowered study that inflates both Type I and Type II statistical errors; describing how the authors reached these n would be helpful in avoiding this conclusion. A good introduction to sample size calculation is Prajapati et al. (2010; http://tiny.cc/i0s6qy).
-Why did the authors choose the specific morphological endpoints they used? Using total length is, although very coarse-grained, acceptable from a developmental toxicology point of view; however, what information can be gained from analyzing head length? For that matter, what information can be gained from analyzing forebrain and midbrain width?
-A minor point is the use of the same animal in both behavioral assays. I was not able to find anything in the manuscript that affirms that, but it stands to reason, given reported sample sizes. If that was the case, it should be clearly reported, and Figure 1 should be changed to reflect that. Moreover, the authors should state whether all animals followed the same test order, or whether they were randomly distributed between locomotor first and optomotor second (and vice versa), with balancing.
-Finally, while the raw data was shared, it appears to simply be an Excel file in which values from GraphPad were pasted. International standards were not used, as commas separate decimals instead of points. Data are organized in a way that precludes quick and automated analyses (i.e., interested parties would need to copy and paste results manually, instead of automatically importing results, to programs other than GraphPad). While I strongly commend the authors for their attempt to share raw data, therefore increasing reproducibility and rigor, the file needs to be changed to a two files - one with behavioral data, and other with morphological data -, in CSV format, organized with one column per endpoint, and one line per subject.
Validity of the findings: Even though there are problems with the experimental design or its reporting, the validity of the findings is good. For example, it seems clear that 6-OHDA produced a general locomotor impairment (decreased total distance, mean speed, and maximal acceleration, and increased absolute turn angle and immobility time; decreased responsivity to an optomotor stimuli) and produced gross anatomical alterations (slightly decreased head length, decreased total length [which I infer from Figure 4F was caused by the appearance of a spinal curvature]) with no gross neuroanatomical changes; and that NAC blocked these effects, while at the same time producing gross neuroanatomical alterations (increasing forebrain width, eye distance, and total length; these later results are reported in Table 2, but the authors affirm the opposite in the text [line 194]). The small sample sizes suggest low statistical power, which could result in inflation of effect sizes and in the false discovery rate. Therefore, while valid, the results are very preliminary, and should be labeled as so, awaiting confirmatory research. The results from the neuroanatomical analyses are very coarse-grained, and appear to be not very informative (as a matter of fact, the authors sparsely discuss them) on either the neuropathology of PD, the specific effects of 6-OHDA, or the proposed neuroprotective effect of NAC; while they should be reported, the limitation of the analysis should be clearly discussed in the text.
Additional comments: As a final comment, the authors need to deeply review their Discussion section. Main issues follow:
-The language from the conclusion is boisterous, and does not reflect the actual findings. We cannot judge whether NAC exerted a “powerful neuroprotective effect” because effect sizes were not reported (and are likely to be inflated due to low statistical power). While NAC does appear to have polypharmacological profile, this was not actually tested in the experiments; and the authors did not study PD, but an animal model, which provides only weak evidence for clinical efficacy; finally, NAC itself produced gross neuroanatomical alterations (which the authors did not address); therefore, sentences such as “NAC is a strong candidate for the prevention and treatment of PD, acting in different aspects of the disease” are inaccurate and represent improper spin.
-There are many findings in the literature reporting neuroprotective effects of drugs (e.g., McKinley et al., 2005, doi: 10.1016/j.molbrainres.2005.08.014), antioxidants (e.g., Chong et al., 2013, doi: 10.1016/j.neulet.2013.02.069; Zhang et al., 2010, doi: 10.3892/ijmm.2010.571), or plant extracts (e.g., Zhang et al., 2011, doi: 10.1007/s10571-011-9731-0) on 6-OHDA-treated zebrafish. NAC was also shown to protect DAergic neurons from 6-OHDA neurotoxicity (Muñoz et al., 2004, doi: 10.1002/jnr.20107). The findings from the authors should be critically discussed in relation to this literature. Moreover, findings from the mammalian literature should also be appreciated (e.g., Zbarsky et al., 2009, doi: 10.1080/10715760500233113), as well as clinical trials on antioxidants (e.g., Snow et al., 2010, doi: 10.1002/mds.23148) and systematic reviews on anti-inflammatory drugs for PD patients (Rees et al., 2011, doi: 10.1002/14651858.CD008454.pub2); these will help put the results in context.

Ethical approval statement – OK

Necessity and ethics of experiments – OK

Animal research policies – Some aspects of the ARRIVE guidelines were not followed; critically, randomization/blinding was not reported, nor was the rationale for the small sample sizes.


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: N-ACETYLCYSTEINE PROTECTS AGAINST MOTOR, OPTOMOTOR AND MORPHOLOGICAL DEFICITS INDUCED BY 6-OHDA IN ZEBRAFISH LARVAE
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: The present version of the manuscript greatly improved its reporting in terms of transparency and rigor.


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: CRANIAL ANATOMY OF BELLUSAURUS SUI (DINOSAURIA: EUSAUROPODA) FROM THE MIDDLE-LATE JURASSIC SHISHUGOU FORMATION OF NORTHWEST CHINA AND A REVIEW OF SAUROPOD CRANIAL ONTOGENY
Review round: 1
Reviewer: 1

Output:
Basic reporting: I really like to see that paper coming out, as describe a very important, almost unknown, sauropod that has a great relevance for sauropod evolution and possible the origin of some lineages (e.g. Neosauropoda). I found the paper really informative, well written and very well figured.

I found the description extremely interesting and detailed, and I do not have comments on this section, as is well figured as well. Maybe the authors can call figures in some parts of the text (specially to clearly refer to some morphological character), and not solely at the title of the described bone.

In the discussion the authors include a very complete list of ontogenetic changes, based on previous studies and own observations. This section is very informative but probably could be re-accommodate in order to better identify each transformation.
For example, using sub-headings (e.g., in Line 1236 they start describing ontogenetic transformations in the frontal, whereas lines 1248 to 1257 looks more like an introduction to basal knowledgment, and in line 1258 the changes in preantotbital fenestra are described, in line 1272 the tooth number…
Additionally, in line 1511 the authors describe the problems in the identification of the preantorbital foramen. I suggest to move this part of the text to here maybe, as in this part the authors state that this fenestra is present in Bellusaurus, but discussing at the end how to identify it. Also, maybe you can include here the orientation criterion used by Wilson and Serene (posterior maxillary foramen opens posteroventrally whereas the preantorbital fenestra opens anteroventrally).
Taking into account c the similarities observed in the frontal articulation (for nasal and prefrontal) between Bellusaurus and Europasaurus.. Do you think this characterwill be much different in adult Bellusaurus specimen (and therefore still diagnostic of adult Europasaurus) or you beileve that Bellusaurus will not have so strong transformation and that probably this character will be similar to Europasaurus?, questiongin therefore its usage as autapomorphy (depending in the final position of Bellusaurus).

Dividing this in sub-heading will allow clarifying, at the end of each sub-section, its relevance for the interpretations in Bellusaurus (which are now in a different sub-section all the bones together).

In line 1431,  as well as in other sections of the manuscript, the authors mention the absence of ontogenetic trends in Camarasaurus, as the case of tooth row migration. My major concern is about the ontogenetic stage of the Camarasaurus skulls, and the possibility of being them too advanced for having great changes, even if they are from “juvenile specimens”. In the axial skeleton of Europasaurus foe example it was observed that most of the final adult characters were present just prior to the “adult” stage, therefore this implies a morphological maturity previous to the neurocentral closure. Could happened something similar in the skull…?

In the phylogenetic section the authors discuses the possible problems in scoring juvenile taxa. In that section, specially lines 1499 to 1510 the authors mention some derived characters observed in the skull of Bellusaurus, and some of them were previously discussed to do not be affected by ontogeny (or affected but, as in the case of the tooth row length, not plausible to change to the plesiomorphic state). I think that this paragraph could be a bit more detailed, mentioning some more detailed the distribution of these derived characters observed in Bellusaurus (e.g., preantorbital fenetrsa present in Jobaria + Neosauropoda).. As I mentioned above the last part of the discussion seems to be more properly placed in the discussion of the preantorbital fenestra, and here solely include its presence in Bellusaurus and its significance in terms of phylogentic position. Here, the authors can also mantion, shortly, the age of Bellusaurus, as one of the most important inferences made with this taxon as a neosauropods, was the Middle Jurassic origin of this clade, whereas the new age here reported does not change so drastically the origin of Neosauropoda (if Bellusaurus finally is a neosauropod).

I really like the tables included. In table 2 maybe you can include some abbreviation making reference to the phylogenetic position and ontogenetic stage of mentioned taxa (for phylogenetic position e.g., BSM, Basal sauropodomorph; BS, Basal Sauropod; ND, Neosauropod diplodocoid; BM, basal Macronarian, TF, Titanosauriform..), and some information on the ontogenetic stage of the specimens, Marpmanet al used MOS, maybe a similar criterion or any other, at least late juvenile, early juvenile, adult…..
In this way will be much more easy for the reader to identify the possible phylogenetic-related changes and ontogenetic-related changes
Experimental design: My single comments are related to the order of the discussion, as mentioned above. The design of the reseacrh, taxa included in the discussion
Validity of the findings: The findings are relevant, and the manuscript will be certainly highly cited.
Additional comments: I hope to see the work published soon.

Good Luck

Jose


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: CRANIAL ANATOMY OF BELLUSAURUS SUI (DINOSAURIA: EUSAUROPODA) FROM THE MIDDLE-LATE JURASSIC SHISHUGOU FORMATION OF NORTHWEST CHINA AND A REVIEW OF SAUROPOD CRANIAL ONTOGENY
Review round: 1
Reviewer: 2

Output:
Basic reporting: This paper is an important contribution to our knowledge of sauropod anatomy, and brings one of the least understood taxa in a crucial point of sauropod evolution (the radiation of the crown group Neosauropoda) onto the stage by describing in great detail its cranial anatomy. Thanks to the detail, this study will be of long-lasting impact in the community and follows or even sets new standards concerning basic reporting. There are a few typos and peculiar formatting decisions (e.g. many specimen numbers are in bold), but nothing serious.
Experimental design: The paper provides original research, clearly states its significance, and puts everything into the context of sauropod anatomy, ontogeny, and phylogeny. It reviews ontogenetically changing characteristics in sauropod skulls in great detail, and adds significant new data.
Validity of the findings: Discussion and stated implications are well-supported by the data. I especially like the part on the influence of ontogenetic characters on phylogeny, and how to best cope with juvenile specimens in a phylogenetic dataset.
Additional comments: Line 127: Bellusaurus, not Bellusaursu
Line 186: you mention that at least three individuals are represented. It is unclear if you intend that these three individuals are within a particular specimen number – further above you mention that there are 17 individuals in total. Please clarify
Line 190 (and many other notions): The study of Carballido & Sander on Europasaurus axial morphology was officially published in print in 2014.
Line 210: I didn’t understand if Bellusaurus has subdivided pleurocoels in the cervicals or Klamelisaurus. If it’s Bellusaurus you might want to state that this is opposite of what would be expected in a juvenile individual (see Carballido et al. 2012; Carballido & Sander 2014 and Tschopp & Mateus 2017 for more detailed information on this).
Line 256: an elongate scapular blade might also be ontogenetic – in the juvenile sauropod SMA 0009, the scapula is very long (Schwarz et al. 2007; Carballido et al. 2012)
Line 273: Bellusaurus really has LAGs in longbones? That’s unusual for what I know
Line 282: Interesting thought that you can distinguish individual from ontogenetic variation. You might want to add a table of the morphological features that change individuals vs. ontogenetically. But maybe this could also be a paper on its own.
Line 284 (and later): catalog numbers of specimens you use for comparisons are often in bold, you should probably “un-bold” them.
Line 302: I can’t really see that lateral expansion of the nasal process of the maxilla you mention here in the figures, it just seems to project posterodorsally from the maxillary body to me
Line 317: the foramen in the nasal process of the maxilla would probably also qualify as local autapomorphy for Bellusaurus if Shunosaurus had the same opening – in the end they’re very far from each other in the phylogeny.
Line 344 (and elsewhere): the specimen number for the cf. Brachisoaurus skull should probably be USNM 5370. Please check and correct throughout the MS. If this is a typo, it might be a good idea to check if there are others too I didn’t spot because I’m not familiar with Asian taxa/specimens.
Line 513: add “a” between “and the medial,” and “less acute facet”
Line 536: the presence of a cartilaginous palpebral element is highly unlikely, because the palpebral and other supraorbital bones are of dermal origin in crocodiles and lizards, so that there is no cartilaginous precursor (see Maidment et al. 2010; Nesbitt et al. 2012). It is true that we proposed this, so feel free to correct it or just delete it (we will address this in a study that is currently in preparation also).
Line 697: delete one of the opening brackets
Line 732: add “the” between “total height of” and “quadrate. In lateral”
Line 891: the exit of the hypoglossal nerve seems to be variable within Camarasaurus: AMNH 5761 and SMA 0002 have a single one, CM 11338 seems to have a double exit (I’m not sure if this is published somewhere, maybe in some study with Larry Witmer that uses CM 11338? Otherwise feel free to cite as pers. comm.)
Line 917: “paroccipital”, not “paroccipitial”
Line 957: “diplodocoids”, not “diplodocids”, because Amargasaurus is a dicraeosaurid. You could also use flagellicaudatan instead.
Line 1012: “Läng”, not “Lang” (in the bibliography it’s correct)
Line 1117 (and elsewhere): the study of Wiersma & Sander was published in print in 2016
Line 1131: delete first closing bracket
Lines 1150-1153: consider using the terminology used by Holwerda et al. (2015), who described for the first time (to my knowledge) in detail tooth wrinkling patterns
Lines 1219-1221: you might want to add also Chure et al. 2010; Whitlock et al. 2010; and Poropat & Kear 2016
Line 1235: “Camarasaurus”, not “Camarsaurus”
Line 1493: Tschopp et al. (2015) mention that the use of implied weighting should downweight ontogenetically variable characters. However, if juveniles are scored for these characters, the potentially misleading information from them still influences the algorithm, so I’m not so convinced anymore that these scores should be included.
Line 1536: “interpreted”, not “intepreted"
Table 2: is the ratio basically a comparison between anterior and posterior width of the frontal?
Table 3: why are many SI values missing even if you have both apicobasal length and transverse width stated?