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: TAXONOMIC REVISION OF DASYDORYLAS SKEVINGTON, 2001 (DIPTERA, PIPUNCULIDAE) IN THE MIDDLE EAST
Review round: 1
Reviewer: 1

Output:
Basic reporting: Illustrations need to be inserted into appropriate part of key, the failure to do this waste so much time usually having to re-edit keys to make them practical.
Experimental design: na
Validity of the findings: ok
Additional comments: Because highlighting lost when pasted here copy uploaded

32 Dorilas and Eudorylas. Dasydorylas was discovered during a comprehensive phylogenetic study
33 of world Eudorylini published by Skevington & Yeates in 2001.
This does not make sense, I think “coined” or first used better.

notopleuron often
39 with dense bush of long setae, femora often with posterdorsal row of long and back setae
Not apparent in any of the three species available to me? Perhaps these are small hairs rather than long setae??

sternite 3–5 with posterior
41 setae
Can’t see any in my specimens

41 setae, syntergosternite 8 with membranous area of normal size
Normal for what? In pipunculidae as a whole this feature ranges from absent to occupying whole width of syntergite. Try median size.

An identification key to Palaearctic and Afrotropical
50 species was provided by Kehlmaier (2005a) and Földvári (2013).
Kehlmaier (2005a) only covers European species, change this to
Either
An identification key to Palaearctic and Afrotropical
50 species was provided by Kehlmaier (2005a&b) and Földvári (2013).
OR
An identification key to European species was provided by Kehlmaier (2005a) and Afrotropical species by Földvári (2013).

70 For several species, only the male genitalia provide characters for accurate species identification
Seems presumptuous  I would prefer - 70 For several species characters for accurate identification have only been found in the male genitalia.

postpronotal lobe with 4‒7 long setae
157 along upper margin;
Not apparent in my specimens, is this the same feature referred to in line 39 above pertaining there to notopleuron?


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: TAXONOMIC REVISION OF DASYDORYLAS SKEVINGTON, 2001 (DIPTERA, PIPUNCULIDAE) IN THE MIDDLE EAST
Review round: 1
Reviewer: 2

Output:
Basic reporting: The language is precise, describes morphological features and species relationships very in a detailed and clear manner.
Literature is used appropriately with references even to the distributional data, which is very helpful.
All figures and the two tables are informative and necessary, they give good support to the statements and species distances.
Experimental design: no comment
Validity of the findings: New species are well described with terms generally used in the field and the relevant reasoning is given with related species mentioned and COI genetic distances given.
Additional comments: A well-written account of the insect group with all the necessary ingredients and supporting information. The photographs provide all the required information and because they are photographs and not drawings they represent the actual structures (not representations of them), and are therefore very useful and advantageous.
Please find attached the few typos found in the text.
-the name Kehlmaier is misspelled 3 times
-table 2. has a value that should be bold


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: SPECIESPRIMER: A BIOINFORMATICS PIPELINE DEDICATED TO THE DESIGN OF QPCR PRIMERS FOR THE QUANTIFICATION OF BACTERIAL SPECIES
Review round: 1
Reviewer: 1

Output:
Basic reporting: "SpeciesPrimer: A bioinformatics pipeline dedicated to the design of qPCR primers for the quantification of bacterial species" by Matthias Dreier et al present a software tool SpeciesPrimer that aims to be a one-stop shop for designing differential qPCR primers.  Overall, the manuscript clear, concise, and well-presented. The introduction sets up the need for better tools to design diagnostic primers in a high-throughput manner. In particular, the specific knowledge gap appears to be the need to primer design to be both species-specific, user-friendly,  but also constrained to the parameters of microfluidic qPCR systems (ie not requiring optimization steps for cycling conditions, etc). All the code is available on GitHub, and the supplementary data is well organized. Overall, it appears that the pipeline has been put together in a logical manner, and the case study of developing primers for 4 cheese-relevant strains seems strong. The documentation on the GitHub repository seems sufficient to get new users comfortable with the interface (well done with adding the notes to help those new to Docker!). The structure mostly conforms to PeerJ standards, though there is some confusion arising from having both a supplementary Data S1 and Dataset S1.

The figures need a bit of work to more clearly convey the information:
Figure 1: Either the leaf names need to be simplified, the figure enlarged, or both. Vector graphics are preferred (https://peerj.com/about/author-instructions/#figures), and would allow viewers to  highlight text, zoom without losing quality, etc.  Perhaps the low quality of the images is an artifact of the reviewing system, but regardless, these trees need to be improved for clarity (see guidelines here http://currents.plos.org/treeoflife/index.html%3Fp=6345.html). Including the newick trees in the supplementary data would be nice as well.
Figure 2: This also should be a vector graphic  (otherwise I can't search for the  strain I am interested in). The color key should be moved next to the color bars on the left-hand side of the figure (or the strain IDs to  the left and  the color bars to  the  right), as they do not line up with the data.  Lastly, the strains should be labeled as they are in the qPCR data (perhaps in brackets?) -- it is tedious to have to  relate the data from Dataset S1 to Data S2 before viewing Figure 5.

The genomic data for the strains in the Agroscope Culture Collection does not appear to be made available. If this is not the case, links must be provided for accessing the data. As it stands, this severely limits the reproducibility of the analysis. In particular, in the case study for P. acidilactici, of the 127 genomes considered, only 9  are  available; for P. pentosaceus, only half are available.  Given the plethora of other possible genomes to consider, I believe the analysis should only contain genomes publicly available, or the genomes for the FAM*  strains be deposited in the public repositories.

That said, I appreciate that the authors included accession number for all strains used, and  the raw qPCR data.
Experimental design: The Code:
It would be a good idea to either (a) add citations in a README file in https://github.com/biologger/speciesprimer/tree/master/pipeline/ext-scripts, or to (b)  use git submodules to use/credit code not written by the authors.  For instance, the DBGeneratory.py script is credited in the  manuscript, but not in the repository.  Similarly,  it would be good to include a README file in the  https://github.com/biologger/speciesprimer/tree/master/pipeline/dictionaries directory, explaining how the files there are generated.

- There are no tests for the code, apart from whether a build passes on Dockerhub. I suppose its not required  for publication, although in the long run the authors might consider employing a test framework to make maintaining it easier.

The Manuscript:
The pipeline presented is well described. Some readers may wish to see an explanation for tool choice -- for instance, why Prokka for annotation, why Roary for pan-genome analysis, why PRANK for alignment, etc. SpeciesPrimer pipeline primarily leverages published tools, perhaps a table containing the features of the different primer design tools would be useful in the introduction as well.

This is primarily a software paper, but the paper lacks the necessary comparison to existing state-of-the-art tools. There is no shortage of various primer design tools (the "primer design" topic on GitHub has 20 repositories https://github.com/topics/primer-design), and no comparisons are given. A failed comparison with RUCS is mentioned in passing on line 83, but I would like to see what data and parameters were used for this test.  Specifically, on line 88 the manuscript says the" RUCS works best for very  similar genome assemblies in the positive and the negative sets.... and was therefore  not suitable for our high-throughput approach", while later in the manuscript on line 313, "The results of the two test cases illustrate that the SpeciesPrimer pipeline performs best on closely related genome assemblies  with a good overall quality"; more information should be provided as to how the two situations differ.

Particular tools that I would like to see a comparison to include:
- https://github.com/quwubin/MFEprimer
- https://github.com/widdowquinn/find_differential_primers
- https://github.com/billzt/PrimerServer
Validity of the findings: As noted above, including non-public genomic data in the analysis prevents reproducibility. Either the data  should be made public, or these strains should be excluded from the analysis.

Specific comments:
- 224: Although in the majority of cases your error is pretty small, the use of technical duplicates is concerning.  For instance, for the acuL set "nontarget4", you have one Lactobacillus zeae DSM 20178 (FAM 2136) replicate with a Ct value of 12.30, and the other is empty. It appears that these instances are dropped?  There are several other examples of single replicates having Ct values that don't appear to be reported.  A similar case seems to happen with Lactobacillus buchneri LMG 11439 FAM 23117 for the purD assay.  Perhaps consider https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4779984/  for recommendations on choosing a number of replicates (along with their budget walkthrough document https://jdieramon.github.io/BlueberryProject/optimal_Plan.html).
- Further, at first reading I thought "four qPCR runs" meant that these were run with 4 biological replicates, however the raw data appears to show only a  single biological replicate, and that the 4 runs means it took 4 plates to run all of the non-target samples. This should be clarified.
- In Supplementary data S1, or in the text, no explanation is given for changing the default pipeline parameters for certain runs. How was this decided?
- 292: it is unclear why the authors calculated ANI for the subclusters of E. faecium, P. acidilactici separately from the whole species.  Displaying this as a heatmap might be  more helpful to show the clusters.  Perhaps they were all calculated together --  if so, Table 3 should be changed to reflect that.
- If the stock for FAM20347 is contaminated, it should be removed the the analysis entirely.
- 331: In Supplementary Data S3, it is unclear how you are defining "anomalous alignments".  Why were these sequences excluded?
- 113: I would appreciate a note about database usage. The nt database excludes WGS records (ftp://ftp.ncbi.nlm.nih.gov/blast/documents/blastdb.html), and this may impact the pipeline usage. Noting this caveat and explaining why this does or does not influence the results would be helpful.
- 170: It would be helpful to further explain the target template database, the non-target sequence database, and the target genome databases. Perhaps having a schematic showing the workflow?
- 141: it is unclear why you are citing https://github.com/EnzoAndree/tutorials/blob/patch-1/DBGenerator.py.  This is the fork of the repository  https://github.com/microgenomics/tutorials, and it appears that they should be given proper credit.
- 96: The paper (and the GitHub repo) list the hardware  requirements to be a machine with 16GB RAM  and multiple cores,  suggesting that it can be run on consumer hardware.  The timing benchmarks should reflect that -- the analysis was run on a machine with  double the ram and ~5x the CPUs  available on an average consumer computer (line 202). Especially  as pangenome analysis can be RAM intensive, I  would be interested to see  if the analysis could be run on a 16GB machine.
- Supplementary table S1: it is unclear why cell E2 is  highlighted yellow.
- As SpeciesPrimer includes a tool for updating nt, it is a bit surprising that the last time the db was updated was 20 April 2018.
- Table 1: please note the versions of software used in constructing the Docker image
- Table 2: please note the hardware used for timing
- 219: It might be worth  noting why you use the "nr" database here (as there is a discrepancy between normal blast calling "nr" the protein database and primer-blast calling "nr" the non-redundant nucleotide database).
Additional comments: The paper (and software it describes) is well presented, well thought-out, and I believe is close to where it needs to be for publication.  My final note is about the usability of the tool

It appears that there is way to access the functionality  of the tool from the command line.  The browser-based interface is nice, but is cumbersome working on remote systems.  For instance, without root access, I can't set up xforwarding on the HPC I work on. So, without a command-line interface, I can't run SpeciesPrimer on the HPC;  I don't have space for downloading ~65GB nt database on my personal machine, so trying out the software is currently impossible. One possible solution would be to incorporate a command-line utility program to supplement the GUI for users like myself. It would be a relatively small change that would be both easier to run on shared hardware with  HPCs where users don't have root access and also make the execution of the program more reproducible. Another option would be allowing the user to use a custom BLAST database instead of the full nt. Users on consumer hardware could, for instance, use a smaller database.

SpeciesPrimer promises to be a "batteries-included" solution to the primer design problem, and I hope the authors are able to make the recommended changes to remove some of the final barriers to usage.


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: SPECIESPRIMER: A BIOINFORMATICS PIPELINE DEDICATED TO THE DESIGN OF QPCR PRIMERS FOR THE QUANTIFICATION OF BACTERIAL SPECIES
Review round: 1
Reviewer: 2

Output:
Basic reporting: Overall, well written manuscript with clear English and no grammatical/syntax errors.

According to the author guuidelines, if you are using Mendeley (which I assume you are using), you should change your field codes into plain text by performing the following:
If using Windows:
Ctrl-A (Selects all the text in the manuscript).
Ctrl-Shift-F9 (Changes the fields codes).
If using OSX:
Command-A (Selects all the text in the manuscript).
Command-6 (Changes the fields codes).
Experimental design: The manuscript falls under the Aims and Scope of the journal.
The pipeline is very useful although it would be difficult to apply it to other kinds of samples apart from food (e.g. environmental samples).
Validity of the findings: No comment.
Additional comments: Line 24: Since you are focusing on food microbiology, I would suggest you rephrase the "given system"
and make it more food specific/relevant. It is good that you give the example of cheese, but I would like
to see it a bit more clear before that.

Line 63: You could add information on what these genes encode

Lines 65-66: You can elaborate a bit more.

Lines 67-69: You can also add PrimerMiner (doi: 10.1111/2041-210X.12687)

Line 101: There is no Title (and legend) for your Table. Are you sure you should not include it in the doc file also?

Line 104: How is the specificity for the target species assessed?
If I understand correctly, you donwload genome assemblies for specific species.
A true specificity check would require comparison with genome assemblies for other species also.

Line 166: You should specify where the script is located in your github repository.
Users may not be familiar enough to find it on their own.

Line 142: You should refer better to the script. Don't just put it in the brackets.

Line 147: Rephrase to "using consambig function"

Line 148: Correct to "sequences"

Line 152: Correct to "consensus"

Line 166: Correct "in silico" using italics. Also, in line 214 and line 325.

Line 211: Data S1: The first cell in the "Test case 1 assembly accessions" column is highlighted yellow

Lines 218-220: Why did you choose "BLAST database (nr) limited to sequences from bacteria"
and not another kind of database, e.g. Silva?

Lines 272-273: You can delete it, the reader already knows about it .

Lines 283-284: "were created using Roary and FastTree". You can delete it. This information is already mentioned
in the Materials & Methods section. You should also mention SeaView software in the Materials & Methods section
and not only in the legend of the Figure.

Lines 284-288: Can you re-create the figure and somehow find a way to reduce the node names?
We don't need to see all the node names one by one. They create a lot of noise.

Lines 291-292: This belongs to the Materials & Methods section.

Lines 313-315: This belongs to the Discussion or Conclusions section.

Lines 326: The abbreviation PPC should be explained in the legend Table 4.

Line 331: Maybe you should refer in the specific pages of Data S3 also, to make it easier for the reader.

Lines 393-395: This is very important from my point of view. Did you consider assessing it in your in vitro experiments?

There are no references in the Discussion...
In lines 397-399, for example, you should be able to find a suitable reference.

Lines 401-402: Are you sure this is the appropriate way to cite a website?


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: SPECIESPRIMER: A BIOINFORMATICS PIPELINE DEDICATED TO THE DESIGN OF QPCR PRIMERS FOR THE QUANTIFICATION OF BACTERIAL SPECIES
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors work to clarify areas flagged in review, restructuring (and expansion) of the supplementary data, updating figures, and other changes have greatly improved the readability of an already good work.

I also appreciate the authors efforts to make the Agroscope genomes available to the reviews and to the community through uploading them to NCBI.
Experimental design: Expanding the manuscript to include a primer design comparison with similar software strengthens the work. I particularly appreciate the care the authors took in document the commands used to allow replication of that analysis.

I am not sure what is meant by the statement “The positive genome sets for RUCS and fdp were the same genome assemblies used for the SpeciesPrimer pipeline. In addition, genome assemblies for 224 different species from the species list were downloaded.” Were these different from those used in the SpeciesPrimer analysis?
Validity of the findings: I appreciate that the authors clearly outlined usage cases for the command line usage with Docker. That said, the docker implementation could be improved. As far as I can tell, it requires the user to enter into the image to actually use it. For instance, attempting to run with docker run results in an error because there is no command line option for email:

```
docker run --rm biologger/speciesprimer speciesprimer.py --target Staphylococcus_aureus --path ./tmp_staph/ --qc_gene rRNA --assemblylevel complete

Start searching primer for Staphylococcus_aureus
To make use of NCBI's E-utilities, Please enter your email address.
fatal error while working on Staphylococcus_aureus check logfile
EOF when reading a line
Error report:
for target Staphylococcus_aureus
Error 1:
fatal error while working on Staphylococcus_aureus check logfile
```
Editing “tmp_db_path” requires interactively editing a config within docker – there is no –configfile argument either, where this could be provided. Further, providing a –configfile arg would allow users to tweak the thresholds of the various tools – especially for pangenome-calling, these thresholds are sensitive and never one-size-fits-all.

That said, I have tried to use the software a few times, and I am of the opinion that more effort must be taken to ensure that it is in a usable condition. Especially as it is geared towards casual users, proper error handling will help the tool get adopted. It looks from the GitHub logs like development is reasonably active, so hopefully some of these issues are being resolved already. I have a few concerns that I will outline based on my (limited) attempts at usage.

When re-running analyses, QC checking is ignored. It appears that the program just checks for the output of the QC results, and doesn’t check whether the run should pass (the default file has headers, so re-running detects a non-empty file and assumes it passed):

```
Preparing files for BLAST
Start BLAST
Run blastn -task megablast -num_threads 4 -query rRNA.part-0 -max_target_seqs 5 -max_hsps 1 -out rRNA_0_results.xml -outfmt 5 -db nt
Error: No genomes survived QC
Run: remove_qc_failures(rRNA)
Error report:
for target Chlamydia_pneumoniae
Error 1:
Error: No genomes survived QC
root@b1f14c5644bf:/home/primerdesign# speciesprimer.py
Create new config files or start pipeline with previously generated files?
type (n)ew or (s)tart:
s
Search for config files for (a)ll or (s)elect targets:
a
Please specify a path to use as the working directory or hit return to use the current working directory:

Search in /home/primerdesign
found: /home/primerdesign/Chlamydia_pneumoniae/config/config.json
Start searching primer for Chlamydia_pneumoniae
12 genome assemblies are available for download

Already annotated:
['GCF_001007085v1', 'GCF_001007105v1', 'GCF_000008745v1', 'GCF_000007205v1', 'GCF_001007045v1', 'GCF_001007065v1', 'GCF_001007145v1', 'GCF_000011165v1', 'GCF_001007025v1', 'GCF_000024145v1', 'GCF_001007125v1', 'GCF_000091085v1']
Found rRNA_QC_report.csv, skip QC rRNA
Start pan-genome analysis
...
```

This run (and 2 others I tried) ended with the following fatal error:

```
22 Oct 2019 12:28:05: Run: run_blast - Start BLAST
22 Oct 2019 12:28:05: Run blastn -task dc-megablast -num_threads 4 -query conserved.part-0 -max_target_seqs 2000 -evalue 500 -out conserv
ed_0_results.xml -outfmt 5 -db nt
22 Oct 2019 12:35:48: Run blastn -task dc-megablast -num_threads 4 -query conserved.part-1 -max_target_seqs 2000 -evalue 500 -out conserv
ed_1_results.xml -outfmt 5 -db nt
22 Oct 2019 12:43:20: > Blast duration: 0:15:15
22 Oct 2019 12:43:21: Run: run_blastparser(Chlamydia_pneumoniae), conserved sequences
22 Oct 2019 12:43:21: Run: blast_parser
22 Oct 2019 12:43:21: Run: blastresults_files(Chlamydia_pneumoniae)
22 Oct 2019 12:43:29: fatal error while working on Chlamydia_pneumoniae check logfile
fatal error while working on Chlamydia_pneumoniae
Traceback (most recent call last):
  File "/home/pipeline/bin/speciesprimer.py", line 4261, in main
    config).run_blastparser(conserved_seq_dict)
  File "/home/pipeline/bin/speciesprimer.py", line 2611, in run_blastparser
    align_dict = self.blast_parser(self.blast_dir)
  File "/home/pipeline/bin/speciesprimer.py", line 2496, in blast_parser
    blastrecords = self.parse_BLASTfile(filename)
  File "/home/pipeline/bin/speciesprimer.py", line 2184, in parse_BLASTfile
    record_list = list(blast_records)
  File "/usr/local/lib/python3.5/dist-packages/Bio/Blast/NCBIXML.py", line 626, in parse
    expat_parser.Parse(NULL, True)  # End of XML record
xml.parsers.expat.ExpatError: no element found: line 603955, column 0
22 Oct 2019 12:43:29: > Error report:
22 Oct 2019 12:43:29: > for target Chlamydia_pneumoniae
22 Oct 2019 12:43:29: > Error 1:
22 Oct 2019 12:43:29: > fatal error while working on Chlamydia_pneumoniae check logfile
```

The log file provided no additional information about what caused the element not found error when parsing the blast results. I have attached the log file to the review (peerj only allows pdf files, so I had to convert it, sorry!), and here is the config, for reference:
```
cat ./Chlamydia__pneumoniae/config/config.json
{"target": "Chlamydia_pneumoniae", "offline": false, "path": "/home/primerdesign", "mfethreshold": 90, "remoteblast": false, "nolist": false, "probe": false, "exception": "Serratia_marcescens", "assemblylevel": ["complete"], "blastseqs": 1000, "qc_gene": ["rRNA"], "skip_download": false, "mfold": -3.0, "ignore_qc": false, "maxsize": 200, "skip_tree": false, "mpprimer": -3.5, "intermediate": false, "minsize": 70, "blastdbv5": false}
```

As I cannot get the software to run (whether due to user-error and no error-catching, or due to an actual fault in the code), I cannot recommend the work to be published. There is a good chance that I have something configured incorrectly, but no error messages help me fix that, then I cannot use the tool.

My concerns regarding qPCR with only single biological replicates and duplicate technical replicates remain. I realize that this is considerable amount of work to redo, but I would rather fewer strains being screened with sufficient power than an underpowered cursory screen of many strains. While I largely agree with the 4 points presented by the authors in the response, the very fact that there was variability that did not show conclusive results highlights the benefit of having both biological and technical replication. Yes, biological replication in a perfect scenario would not contribute new information, however this is common practice to control for contamination of the culture or other handling errors – this is valuable information. I would recommend the editor allows the authors sufficient time to run the necessary experiments. That said, I recognize the author’s concern that this is indeed primarily a software paper, so I would defer to the editor if they feel that the data presented sufficiently supports the purpose of the work.
Additional comments: The authors have made strides towards making SpeciesPrimer a useful tool for the community, and I hope that the changes I suggest here aren’t too arduous. SpeciesPrimer will be a good resource for the community. I would suggest asking a few colleagues to try it out, to help identify common user errors.


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: SPECIESPRIMER: A BIOINFORMATICS PIPELINE DEDICATED TO THE DESIGN OF QPCR PRIMERS FOR THE QUANTIFICATION OF BACTERIAL SPECIES
Review round: 2
Reviewer: 2

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: Delete the phrase "The phylogeny was created with Roary and FastTree 2 and displayed with iTOL
(https://itol.embl.de/)" from the legends of Figures 2 and 3. The reader already knows this information
from lines 285-286 of the manuscript.

Figure 4: Recreate the figure using other colors instead of green and red.
You should choose color blind friendly colors.


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: BROAD SIMILARITIES IN SHOULDER MUSCLE ARCHITECTURE AND ORGANIZATION ACROSS TWO AMNIOTES: IMPLICATIONS FOR RECONSTRUCTING NON-MAMMALIAN SYNAPSIDS
Review round: 1
Reviewer: 1

Output:
Basic reporting: No problems in my opinion.
Experimental design: No problems in my opinion
Validity of the findings: No problems in my opinion
Additional comments: I really enjoyed reading this study. It deals with two fundamental and inter-connected issues in comparative anatomy and evolutionary biomechanics: (1) how different/adapted are muscles in animals that function in different ways, and subsequently (2) how should we reconstruct muscles in extinct taxa in order to understand how they functioned? In my view these are the most pivotal questions currently facing the field of evolutionary biomechanics. I think the approaches used are entirely appropriate and the research has been carried out to a high standard. I have very few concerns and I’m excited to see the paper published. Below I have listed some general thoughts about the paper, along with some minor points about specific sentences/issues. None represent major concerns and could be implemented as and where the authors think they will improve the manuscript.

General Comments
1.	The one thing I felt the paper was lacking in slightly was a standalone appreciation of how different the tegu and opossum are in their gross skeletal anatomy and the way they walk. Then subsequently how different they are/might be to the transitional fossils like Massetognathus. Appropriate papers are referenced, but if, for example, I want to gauge how sprawling vs upright the tegu and opossum are then I have to piece that together by comparing several published papers. I don’t think this issue is trivial because the paper builds to a situation where you average the morphology of the tegu and opossum and apply that average muscle morphology to the fossil. So it would be directly helpful to anyone wanting to judge the choice of taxa and validity of the approach generally to be able to easily judge the morphological and functional differences of the tegu and opossum.  Would it be possible to add a multi-part figure that summarises the 3D skeletal anatomy and locomotor dynamics of these two animals? For example, part A could show the 3D pectoral girdles and forelimbs of the tegu and opossum in mid-stance or neutral standing postures (possibly with Massetognathus included too?) and then in subsequent parts provide some graphed 3D kinematics of the two species overlaid on each other, utilising data from published studies?

2.	Following on directly from the point above, the general ‘feeling’ that I think the paper leaves the reader with is a very positive one: (1) the tegu and opossum are excellent bracketing taxa for transitional fossils; (2) their muscles are “strikingly” similar; (3) and as a combined result of (1) and (2) we’re in an excellent position to quantitatively reconstruct muscle properties in transitional fossils. I have made some points below about whether the picture being painted is a little too positive, but regardless of that, my previous point is that more background information would help the reader judge this pyramidal/hierarchical argument. However, a better understanding within the paper of the scale of differences between the chosen extant taxa, and the extant and extinct taxa, would also help in terms of the wider applicability of this approach to other groups and evolutionary transitions. The generally positive picture that comes through here is a bit of contrast to dinosaur and hominid studies where the extant bracketing taxa have quite different muscles and this poses issues when viably reconstructing muscles in extinct taxa. Do we have a fundamentally different situation here with early mammals? Might the approach used herein (e.g. applying a mean of two extant species to extinct taxa) work in some instances but not others? I don’t think it’s essential to add this to the discussion section, but it might make the paper more interesting and widen its appeal.

3.	Discussion, Lines 624-628: I couldn’t decide whether to discuss this here or under minor comments below, but opted for here because addressing this concern could be integrated into the broader discussion I’ve suggested above (if the authors want to). All estimates of body mass in extinct taxa inevitably come with uncertainty. In their original paper, Campione and Evans (2012) included confidence or prediction intervals with their mass equation and encouraged (and still encourage; Campione 2017, Palebiology) people to use them when presenting a mass estimate for an extinct animal. There’s been quite a bit written the issue since (Bates et al. 2015, Biology Letters; Campione 2017, Paleobiology) in the context of dinosaur body mass, but here the uncertainty of the mass prediction is directly relevant because you use the derived body mass to scale your muscle properties. I think some discussion of this issue is warranted. Maybe even some calculations/sensitivity analysis could be done? Also, if you have alternative ideas, then perhaps methods of deriving quantitative muscle properties that don’t require an estimated body mass could be discussed? Also, double check that you are using the correct measurements as inputs into the Campione & Evans (2012) equation. In your supplementary table you have labelled the columns “Mid-diaphyseal Circumference”, but I think that the equation is based on minimum shaft circumference (which might not necessarily be mid-shaft, unless that’s your definition of mid-shaft).

4.	Five muscles are not directly shared between your two studied species. You could very easily quantify what impact this has on gross functional parameters. For example, how much muscle mass is dedicated to shoulder flexion vs extension, abduction vs adduction etc in the two species? i.e. if a flexor is absent in one species, is this reflected in lower flexor muscle mass overall, or is it simply that one of the other flexors is bigger and compensates and thus overall flexor mass remains similar? I did this kind of comparison in my analysis of birds vs crocs/lizards (Bates & Schachner 2012, Fig. 6A&B). Just a thought.

Minor comments
Abstract: “…we find striking similarities in muscle organisation and architectural parameters.” I feel “striking” is just a little sensational.

Abstract: “…distal muscle attachments are notably similar, while differences in proximal muscle attachments are driven by modifications to skeletal anatomy.” Are you talking about qualitative or quantitative differences in the attachment sites/areas? I would assume quantitative given you’re implying the level similarity is surprisingly high (I wouldn’t think qualitatively the muscle paths/sites would be expected to be particularly different would they?). Also, is “distal” the best word here? I would tend to think of wrist/manus muscles when distal is used, but you’re talking about upper arm muscles throughout.

Abstract: “….are statistically indistinguishable for an unexpected number of muscles.” I think this should be reworded. How many would one "expect", objectively, to be distinguishable statistically? You can’t say objectively as one person’s expectation may differ from another.

Introduction, line 52: I personally don’t like the language (“beyond recognition”) here as it seems a bit over the top, but it’s purely a personal preference.

Introduction, line 89: Charles et al. (2016) seems a slightly strange reference here given the sentence is concerned with modelling fossil taxa and estimating the necessary input parameters and this paper is about modelling extant mice gaits. There are quite a few simulation studies of extinct dinosaurs and hominids that could be referenced in this context.

Methods, general comment: Do you know anything about the specific animals used in the study? e.g. are they all wild, all captive, or a mixture? All adult? All male, female or mixed? Apologies if such things are stated and I’ve missed it.

Methods, Equation 1: When collating data on muscle architecture for my 2018 study on T. rex bite force (Bates & Falkingham 2018) I noted that some studies used this equation strictly regardless of whether pennation angle was 0 or >0. However, other studies used muscle-belly length (rather than fibre length) to calculate PSCA when the muscles were considered parallel-fibred (pennation = 0). Which approach are you using and why? We need to know this given you’re statistically comparing PSCA. Calculating it one way versus the other will impact on the data, and may impact on the results/conclusions.

Results, Lines 210-218: I think the description of limb bone orientation would be aided by a ‘purpose-built’ comparative figure of the 3D whole limbs, either in the main text or supplementary information.

Discussion, Line 509: When you say “overwhelmingly similar” do you mean purely qualitatively similar? Basically, origins/insertions/paths? I’m not personally “overwhelmed” by this myself (terrestrial mammals don’t differ that much do they?), but okay. I wouldn’t, however, be okay, with “overwhelmingly” being used if you’re also including the quantitative data in this statement. If it is just the qualitative morphology you're referring to then could you add “topologically” or “qualitatively” to this sentence.

Discussion, Lines 521-534: The paragraph starts to deal with the big issue underpinning the paper: how adapted, functionally, are the muscles in the extant taxa and what does this mean for reconstructing them in fossil taxa. However, the message that comes through here is not quite as clear or explicit as it could be I feel. The paragraphs starts as if the message is going to be “actually the muscles are quantitatively very similar – the similarities outnumber the differences.” You rightly go on to qualify this by explaining that the situation is more complicated than similar PCSAs equals similar function. But then there’s no explicit conclusion. Nor does one really follow in the next paragraph. What’s the explicit answer to the question you yourself pose – does functional similarity parallel anatomical similarity or not? I think there are quite important functional differences here (which you later point out in a different sub-section, lines 599-601) but the overall feeling one is left with at the end of the paper is that, overall, anatomy and function are “overwhelmingly” similar, and by inference we’re in a relatively straightforward position when it comes to taking the next step: reconstructing the muscles in fossil mammals.

Discussion, Line 619-622: “As non-mammalian synapsids are typically reconstructed as terrestrial generalists, the architectural parameters of the tegu and opossum may provide realistic “bookends” for the phylogenetically- and morphologically-intermediate non-mammalian synapsids.” What is a “terrestrial generalist” in this context? Presumably the ultimate over-arching goal here is to reconstruct how sprawling vs upright the transitional fossils were and constrain their maximal locomotor performance. I’m struggling to reconcile what is meant by “terrestrial generalist” in the context of having modern analogues to deliver these goals.

Discussion, Line  622: I’m being pedantic now but I don’t think you can really justify saying that what you’ve done with Massetognathus is a “test case” because you haven't strictly “tested” if the muscle parameters you’ve derived are capable of yielding viable/stable locomotion, or even that the muscle fascicles are of a length to allow them to contract across what might be considered a realistic length range for skeletal muscle. It’s semantics, but I’d rephrase to remove the implication that you’re actually testing the biomechanical plausibility of the reconstruction.

Discussion, Lines 644-658: This is a great paragraph. However, I think you could go a little further and discuss fibre lengths. And tendon lengths. One obvious issue with applying mass-scaled fibre (and tendon) lengths to an animal with a different size and/or shaped skeleton (=different muscle-tendon unit lengths) is tuning. You may, potentially, end up in a situation where the fibres are implausibly short or long given the force-length properties of skeletal muscle, and a muscle (or muscles) are essentially useless. I’ve had this issue within extant species where I’ve used muscle values from the literature and applied them to a musculoskeletal model built using a skeleton from another individual. While some of this is probably a mixture of allometry, ontogenetic differences and measurement error, I think it’s confounded further by the way we tend to model muscles as a single Hill-type fibres which leads to a bit of an all or nothing situation. I’m not suggesting you get into muscle modelling, but some explicit mention of the tuning issue would be useful for the palaeontological readership, and to reinforce the reality that just scaling bracketing extant taxa is not guaranteed to work even if aspects of their muscle morphology are statistically similar.


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: BROAD SIMILARITIES IN SHOULDER MUSCLE ARCHITECTURE AND ORGANIZATION ACROSS TWO AMNIOTES: IMPLICATIONS FOR RECONSTRUCTING NON-MAMMALIAN SYNAPSIDS
Review round: 1
Reviewer: 2

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: Overall the manuscript is well written and seems  and I have only minor comments for the authors.

Minor comments
Style: I suspect your referencing software is responsible for it, but where you name the author in a sentence before citing them, you probably don’t need the name again in the brackets e.g. Romer (Romer, 1944). Can just do Romer (1944), but please double check with the journal style.

Methods: I know isometry is assumed for the range of body masses that you dissected with all being fairly close in size, however all of the specimens in Lugol’s Iodine are smaller (presumably a constraint of the diffusion/time), and one of the tegus was far smaller (~1/2 mass of the dissected animals). Do you see much variation from the assumed isometry between muscle volumes, and muscle attachment areas in these individuals (allowing for individual variation, and slight shrinkage through the staining procedure)? These stained specimens are interesting individuals as most are close to the 1kg size which you are standardising measures down to for your dissected individuals.

Lines 158-178: For personal/professional interest, with your stains were you able to get muscles stained all the way to the bone? We have had issues mapping attachments through staining particularly for more tendinous insertions. By using the fleshy insertions you presumably avoided this issue?

Ln 186: explain PBS

Ln 383: Capitalise figs.

Ln 454-459: This feels like a figure legend rather than an in text paragraph. Perhaps it should be interwoven with the next paragraph.

Ln 528-531: This sentence is awkwardly phrased, possibly due to the “while pennation”. Consider splitting the sentence, e.g. “Muscle PCSA relates to the force requirements for body support and locomotion (Eng et al., 2008). Pennation is linked to PCSA and thus the force capacity for a given volume of muscle, but may also enable dynamic gearing of mechanical output (Azizi, Brainerd & Roberts, 2008).”

It may also be worth explicitly stating here the implications of your statements about the differences between the species. I.e. to get the same PCSA and similar pennation from different masses, and fascicle lengths you have either:

1)	Parallel muscles that are short vs long between the species or,
2)	Pennate muscles that are similar lengths but different volumes/masses or
3)	Multipennate muscles vs pennate of different volumes/masses

This is what your numbers in Table 1 and descriptions say quite well. Your figures may also support this Didelphis bones being almost twice the size of Salvator so muscles may be longer just to cross the larger distance BUT you should mention in your figures which individual is figured as it might be unfair to be naively comparing the 0.6kg Salvator to a 1.11kg Didelphis.

Ln 540: Italicise Varanus exanthemicus.
Ln 549: Italicise S. merianae and D. virginiana.

Line 613: Is it possible that sprawling amniotes have a greater mobility of the humeral head relative to the glenoid fossa so require less stabilisation?

Line 640: How did you measure Massetognathus origin areas?

Figures: All are very hard to read even zoomed in on a computer screen when they embedded with text (e.g. a pdf) due to the size of the text. Also check your colours with some of the yellow text being potentially difficult to read. I would suggest checking on print-outs.

Tables: Whilst I appreciate you measured the pennation angle using ImageJ, I would caution against the level of precision you are claiming with tables showing 2-3 decimal places, particularly with any variation in camera angle, muscle “squishing” etc. going to have an effect on pennation angles in muscles that are no longer in situ. Consider reducing to 1 d.p.

Sup. Info: No legends? Also check formatting for table S1. Is it possible to make the table landscape?

Sup text: Check spacing between “which  were” in m. scapulohumeralis line 3.

Raw data: Check what Excel has done to your specimen labels as Sep 71, 74, 85, 87, 88, 92 have all become dates. Either change formatting from general, or put an apostrophe before the text e.g. ‘Sep71 which will freeze it and the apostrophe disappears from standard view.

Also why are skin measurements included in the excel sheet? Please check what is in the spreadsheet, and whilst some of it is great data to have, it should probably have a caveat in the sup text or at the top of the excel file to explain its presence (and that of the muscles that may not have been discussed, and random photo notes too)


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: BROAD SIMILARITIES IN SHOULDER MUSCLE ARCHITECTURE AND ORGANIZATION ACROSS TWO AMNIOTES: IMPLICATIONS FOR RECONSTRUCTING NON-MAMMALIAN SYNAPSIDS
Review round: 1
Reviewer: 3

Output:
Basic reporting: The present research presents a functional myologic analysis of the musculature of two similar-sized species which constitute a phylogenetic and morphofunctional bracket for inferring four non-conserved myologic characters of a fossil species, Massetognathus pascuali. The manuscript is self-contained and presents relevant results as it is the first estimation of PCSA in a fossil species. This character is fundamental for a confident functional comparison among species and the authors provide a strong base for myologic parameter´s calculation by dissecting two similar-sized species as “brackets”. Although the general interest is not new (evolution of pectoral girdles in vertebrates, associated to habitat and locomotor changes has been largely studied), the approach is original and worthy of publication.

The paper has a good structure that allows easy reading and interpretation, also, structure is ok according the Journal requested format. The English writing is good and unambiguous. Although I am not a native English speaker, I found three or four things that did not sound correct, so I left some suggestions in the attached file. Please, make sure a native speaker checks on them.

Cited literature was pertinent and sufficient. Regarding background, I believe the authors could add some small details to help scientist who are not specialist in vertebrate anatomical evolution, better understand the anatomical differences between species (particularly pectoral girdle). Also, no functional description is made of the fossil about its posture and locomotor mode, more information about this is needed.

Article´s structure is ok and most raw data is available in well-structured tables, but I was not able to find availability of scanned 3D images throughout the manuscript.

My only suggestion about Table 1 is to mark significant differences (may be the usually used *), even when they are in Fig. 6, as this information helps readers.

Figures are all necessary, in a general point of view they are appropriately described and labeled, but I would suggest the following changes:

All figures: The journal format ask for labeling each part of a multi part figure with an uppercase letter, this requisite is not present in the figures. Also, as requested by the journal, Figures must be cited in the order they appear in the text, please check that the last figures appear cited first in your manuscript (see also comments in attached file).

Please see other specific changes in the attached file

Additionally, I would suggest if possible, another figure (should be the first or in supplementary material) with osteological structure compared between the two studied species and the fossil, again to make it easier to read and understand for a non-specialized vertebrate anatomist, but also to make a more complete functional reading of your findings. Alternatively, authors could consider adding in Fig. 1, the names of the bones mentioned in the text (may be with coding) and address the reader to Fig. 1 in Lai et al., 2018 (doi: 10.1111/joa.12766) to see the osteological structure of the fossil species.

All results are the ones relevant to the hypothesis.
Experimental design: Primary research falls within Aims and Scope of the Journal. The manuscript presents a clear question, i.e. calculating non-conserved myologic parameters like PCSA in fossil species. Methods were described with sufficient detail & information to replicate.
Validity of the findings: The data on which the conclusions are based are statistically sound and controlled. Conclusions are well stated, linked to original research question and extremely limited to supporting results. Actually, I would like the authors to use a bit more of the Journal´s allowance for speculation to see more interpretations with previous analysis. For example, how do you think your knew approach influences previous morphofunctional interpretations of the fossil species? (I mean besides the posture, are there other ecological factors that can be associated to these changes?) And in a broader way? How would the possibility of calculating these parameters will affect future fossil´s research?

Finally, results are strong and with biological sense (not only statistical), and as a first approach to solving an extremely difficult problem, it is well sustained and practical.
Additional comments: This is a well-supported study that provides a new approach to solving an important issue in inferring fossil species eco-morphology. It provides a practical and useful example on how to calculate important myologic parameters fundamental to complement typical fossil´s reconstructions.
A have mostly small suggestions to make about the paper and they are disclosed in the attached file, but here I summarize the most notable ones.

1)	The paper is very well written, but in a certain way I feel the authors are writing for a very specialized public, I would suggest some small changes that can make reading easier for non-specialists, e.g., adding the names of bones in figures (or a new figure; see attached file) or adding more background of Massetognathus pascuali inferred ecology and ecomorphology.
2)	Discussion section is extremely limited to supporting results. I would like the authors to use a bit more of the Journal´s allowance for speculation to see more interpretations including information of previous analysis.
3)	I don´t know if I missed it, but I haven’t seen availability of the 3D scanned images (as requested by the Journal)
4)	Improving size letters in figures and change format according to the Journal


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: BROAD SIMILARITIES IN SHOULDER MUSCLE ARCHITECTURE AND ORGANIZATION ACROSS TWO AMNIOTES: IMPLICATIONS FOR RECONSTRUCTING NON-MAMMALIAN SYNAPSIDS
Review round: 2
Reviewer: 1

Output:
Basic reporting: Absolutely fine.
Experimental design: Absolute fine.
Validity of the findings: Fine.
Additional comments: Many thanks to the authors for responding so comprehensively and addressing the queries I raised about the first submission. As I noted in my first review, a lot of my points were suggestions rather than issues or obligatory ‘corrections’ and so I’m fine with the authors arguments not to implement some of them. The new Fig 1 is an excellent addition!

I think the only thing left to resolve with me is the issue around muscle length vs fibre length to calculate PCSA in parallel fibred muscles (Equation 1). I fully appreciate that this is going to feel like a very annoying niggle to the authors, but this issue also needs to be confronted in a very similar paper by the authors that I am reviewing for a different journal. So we might as well deal with it here and hopefully kill two birds with one stone.

Firstly, please could the authors remove the citation of Bates and Falkingham 2018 from the following sentence:
“We therefore opted to follow the common practice of applying Eq.1 to all muscles, regardless of pennation (Bates & Falkingham, 2018).”

I feel citing us here implies that we are advocating that fibre length should be used to calculate PCSA when pennation = 0. That may not be what the author were trying to do by citing us, but I feel that it carries that implication and I’d rather avoid it (because we don’t advocate it. We sit on the fence in that paper). Secondly, I’m not completely satisfied with the justification added to support the authors choice of using fibre length. This is partly because I think that, in principle, the justification should be based around consideration of the links between muscle architecture and force generating capacity, rather than simply what was or wasn’t convenient to measure. I accept that in science we sometimes have to pick less accurate but more practical options, but in those circumstances, we should explain why that sub-optimal method is the best we can do and what effect it might have on the data/conclusions. So, are the authors saying that based on the links between muscle architecture and force generating capacity we should be using muscle belly length, but they can’t do that here because measuring muscle length is too subjective? That might be okay (i.e. I would just ask the authors to provide an expanded and more explicit explanation of the situation), except in the other paper I’m reviewing by the authors (same methods, same muscle groups, but additional species) they have included an analysis of muscle fibre length:muscle length in forelimb muscles. The fact they are measuring and analysing muscle length values for the same muscles in another paper makes it difficult for me to accept the justification they’ve added here (“we can’t meaningfully measure muscle length”) for using fibre length instead of muscle length to calculate PCSA. As the authors will see from my comments on the other paper, I lean towards using muscle length rather than fibre length because the forces do not sum in parallel fibred muscles in the same way that they do in pennate muscles. But I’m not a muscle physiologist. If the authors can counter this with a stronger theoretical argument about how muscle architecture = force then that’s fine. Please do so and include that in the paper. I’m not trying to force the authors to use muscle length, but I do think in light of the above they need better justification for using fibre length than they’ve offered in their resubmission.


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: BROAD SIMILARITIES IN SHOULDER MUSCLE ARCHITECTURE AND ORGANIZATION ACROSS TWO AMNIOTES: IMPLICATIONS FOR RECONSTRUCTING NON-MAMMALIAN SYNAPSIDS
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors have made most of the changes I requested and I think the manuscript is pretty much ready for publication. I have a couple of very minor formatting things that should be corrected.

ln 660: spelling - gsupraspinatus
Figure 7: Numbers from the tests for significance are still very small and difficult to read.
Raw Data: Some of your specimen numbers are still in date format. See previous review comments for how to fix.
Experimental design: No comment
Validity of the findings: No comment
Additional comments: No additional comments


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

Input:
Title: BROAD SIMILARITIES IN SHOULDER MUSCLE ARCHITECTURE AND ORGANIZATION ACROSS TWO AMNIOTES: IMPLICATIONS FOR RECONSTRUCTING NON-MAMMALIAN SYNAPSIDS
Review round: 3
Reviewer: 1

Output:
Basic reporting: Fine
Experimental design: Fine
Validity of the findings: Fine
Additional comments: I’d like to thank the reviewers for indulging me in the issue of muscle length vs fibre length when calculating PCSA. I asked for a justification and they’ve provided one so I’m happy to see the paper published in its current form.

To explain my mild obsession with this issue: Two years ago I was asked by two reviewers to justify using fibre length rather than muscle length to calculate PCSA when pennation = 0. I responded in near identical fashion to the way you have here. My paper was rejected because both reviewers subsequently responded with a mechanistic argument to support the idea that it should be muscle length rather than fibre length. I’ve since spoken to several biomechanicists and muscle physiologists that agree. As I’ve said, I’m leaning this way now too, but its something I want to look further into myself with data. The rationale is as follows. Equation 1 (calculation of PCSA) is usually followed by the equation

Muscle force = PCSA x Force Per Unit Area (Equation 2).

Equation 2 is why we bother to calculate PCSA, even if (as the current study) we don’t actually carry out the calculation directly. If pennation = 0 then either:

(1)	fibres run in parallel from origin to insertion (=muscle length; e.g. muscle length = 10cm, fibre length = 10cm),
(2) they are arranged ‘perfectly’ in parallel end-to-end and when added up they equal muscle length (e.g. muscle length = 10cm, 2 sets of fibres 5cm long arranged end-to-end to make up the 10cm), or
(3) the fibres are partly arranged in series and partly overlap.

If muscle fibres work in series (fully or partly), the muscle model presented in equations 1 and 2 becomes an abstraction as it assumes muscle fibres do not to work in series.  For fibres arranged in series it is therefore probably more realistic, given equations 1 & 2, to treat them as spanning muscle length. Equation 1 (and then 2) allow us to take into account the greater number of shorter muscle fascicles that can be packed into a pennate muscle. These fascicles act in parallel so their forces sum. An assumption of the above PCSA and force calculation is that the individual fascicles run the full length of non-pennate muscle and from aponeurosis to aponeurosis in a pennate muscle. If there are fibers or even fascicles arranged in series (as they must be in some your muscles), they do not increase PSCA or muscle force in the linear way that results from using fibre length (instead of muscle length) in equation 1. I found these two papers quite useful when thinking about the issue after my paper was rejected.

Anapol F, Shahnoor N, Ross CF. 2008. Scaling of reduced physiologic cross-sectional area in primate muscles of mastication. In: Vinyard C, Wall CE, Ravosa MJ, editors. Primate Cranio-facial Function and Biology. New York: Springer. pp 201–216.

Infantolino, B.W., Neuberger, T. and Challis, J.H., 2012. The arrangement of fascicles in whole muscle. The Anatomical Record, 295(7), pp.1174-1180.

Anyway, congratulations on a great study.


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

Input:
Title: NOMOGRAMS PREDICT SURVIVAL OUTCOME OF KLATSKIN TUMORS PATIENTS
Review round: 1
Reviewer: 1

Output:
Basic reporting: 1.	A flowchart of inclusion and exclusion criteria is required.
Experimental design: 2.	For analysis, “age” was converted from a continuous variant to a categorical variant as “< 50” and “≥ 50”. The authors need to explain why age is classified this way. Use software such as X-tile and R or refer to the NCCN guidelines to find the best cutoff value for the continuous variable.
3.	In the statistical analysis, the author should note the format for the presented results, such as the mean ± S.E or median (range).
4.	Continuous variables in normal distribution and homogeneity of variance were compared by using the t test, otherwise, the Mann-Whitney U test was performed.
Validity of the findings: 5.	A nomogram without external validation is only a visualization of Cox model. Therefore, the nomogram in this study should be placed after the results of Cox model.
6.	In terms of model diagnosis, C-index alone is far from enough. First, the calibration curve for the nomogram is necessary. Besides, have you considered using some other penalization methods such as Elastic Net and Lasso regression to ensure the model not overfitting? One can look at the Cox-Snell residual plot, or Martingale residual plots as ways to assess model fit. ROC curve can evaluate the accuracy of the model.
7.	The authors remove all patients with any kind of missing data. Maybe the authors want to consider using multiple imputation so they can include more patients. This way they could create two independent large datasets from the SEER data and use the small local dataset as a second independent validation set.
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: NOMOGRAMS PREDICT SURVIVAL OUTCOME OF KLATSKIN TUMORS PATIENTS
Review round: 1
Reviewer: 2

Output:
Basic reporting: As described in general comments.
Experimental design: As described in general comments.
Validity of the findings: As described in general comments.
Additional comments: This is an interesting study by Qi et al on Klatskin tumors. Albeit its scientific significance, this study includes several major issues that should be addressed.
-	The English language should be improved to ensure that an international audience can clearly understand the text.
-	Why were patients only after 2005 included? Moreover, since the follow up period in the KM curves in 5y why patients until 2015 and not 2014 were included?
-	Moreover, what was the follow up length and rate?
-	Could the authors please specify their inclusion/exclusion criteria?
-	Would the authors consider assessing the era effect?
-	Could they provide more info regarding the treatment plan of the patients?
-	Authors state that t-test was used, but there were 3 groups. How did they control for a error?
-	In all Figures, authors are encouraged to provide patients at risk
-	Could the authors provide a flowchart of the study showing total No of pts, reason for exclusion and No?
-	There were significant differences among the 3 groups regarding patients’ demographic data. Have the authors considered performing propensity score matching analysis? Or a Nelson Aaalen cumulative hazard function including a multivariate analysis so that an adjusted risk can be calculated instead of a simple KM?
-	Could the authors provide p-values between each 2 group comparison?


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: NOMOGRAMS PREDICT SURVIVAL OUTCOME OF KLATSKIN TUMORS PATIENTS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The manuscript can be accepted in this form
Experimental design: The manuscript can be accepted in this form
Validity of the findings: The manuscript can be accepted in this form
Additional comments: The manuscript can be accepted in this form


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

Input:
Title: NOMOGRAMS PREDICT SURVIVAL OUTCOME OF KLATSKIN TUMORS PATIENTS
Review round: 2
Reviewer: 2

Output:
Basic reporting: OK
Experimental design: OK
Validity of the findings: OK
Additional comments: OK to publish


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: NO ISLAND-EFFECT ON GLUCOCORTICOID LEVELS FOR A RODENT FROM A NEAR-SHORE ARCHIPELAGO
Review round: 1
Reviewer: 1

Output:
Basic reporting: Very well writing and concise. In some areas the references and context could be expanded and I give specific comments below in the General Comments section. The figures and tables look good but could use some additional details about statistical differences to aid readers. Raw data are shared. More comments in the General Comments section.
Experimental design: Fits Aims and Scopes of the journal and is original research. The research question is defined but predictions need some elaboration (I detail this in the General Comments below). The authors nicely describe the knowledge gap about "island syndromes". The Methods need more details and I make specific suggestions in the General Comments below.
Validity of the findings: There are some issues with the statistical approaches and more details are needed to evaluate these. I discuss this and provide suggestions below.
Additional comments: This manuscript examines aspects of the “island syndrome” in wild deer mice in islands and mainland areas in Canada. The island syndrome is characterized as the constellation of phenotypic traits that differ between individuals living on islands versus those conspecifics on the mainland. For example, the authors describe how rodents typically show increased body size/mass on islands vs. mainland areas and, as the authors indicate, this may be because of differences in the abundance of food, interspecific competitors, or predators

The authors quantified body mass of mice on islands and mainland areas as well as two measures of their stress physiology (hair corticosterone and fecal corticosterone levels). The authors indicate that differences in stress physiology between island and mainland populations have not been investigated in detail. Mice were captured on different islands (n=11) and mainland areas (n=5) over a few months in the spring and/or summer in two consecutive years. Hair corticosterone and fecal corticosterone levels were measured from samples collected during trapping and body mass was recorded. The density of mice was also estimated.

The authors show that mice captured on islands and those on mainland areas did not differ in body mass, fecal corticosterone, or hair corticosterone. They also did not find a difference in mouse density on islands vs. mainland areas. However, they do show seasonal shifts in fecal and hair corticosterone levels and that heavier mice had higher hair corticosterone levels. They also report a weak but significant positive association between fecal corticosterone and hair corticosterone.

I enjoyed reading this manuscript and it addresses an interesting question about physiological differences between island and mainland populations that has been rarely addressed and uses a comprehensive approach to do so. The fieldwork undertaken is non-trivial and it was impressive to see that the authors had multiple replicates of the island and mainland populations and did the study across two different years. Although the predictions were not supported, the manuscript is still a valuable contribution in my opinion.

I have a few major and minor comments for the authors below.

Major comments:

I have a few requests for clarifications regarding the statistical methods. First, in the island-mainland comparisons, why was island included as a random effect nested within habitat type? Shouldn’t habitat type be a fixed effect in these comparisons? Second, the rationale for the a priori interactions being included needs to be described. Currently (line 193), it is not adequately described and it seems unusual that preliminary analysis was used to assess which interactions were included. Adding references for why interactions were included for the statement “based upon a priori knowledge” (line 193) also seems needed. Third, the procedures to select the ‘top’ model is not very clear or transparent and needs to be described in greater detail (lines 196-200). Why not use model selection under this approach? Finally, whether or not the fixed effects in the models were or were not standardized should be reported here.

I find the basis for the predictions presented in the Introduction to be slightly unclear (lines 88-95). Specifically, why exactly would you predict that if mice on islands were more abundant a and larger than mainland mice that they would have lower hair and fecal corticosterone levels? That seems quite divergent from what is presented above (and the literature as a whole) where animals in poor condition (such as lower body mass) or those experiencing higher densities would have higher stress measures.

As a whole, I find that the Methods could use more details in certain areas and provide a number of specific comments for where to add detail in the minor comments shown below. More space for Methods is available as it is noticeable that the Methods (lines 97-211) are substantially shorter than the Discussion (lines 302-446).

It isn’t clear to me why trapping occurred at very different time periods between the two years. Capture of mice in 2015 (July-August) and 2016 (May-June) was very different and it would be helpful for the authors to indicate why this occurred because seasonal changes in reproductive activity or within-season changes in density could affect the results. How were the effects of reproductive condition on hair or fecal corticosterone levels controlled for here? Additionally, how synchronous were the sampling of the mainland and island areas? I’m just wondering if the negative results might be due to variation in fecal or hair corticosterone levels due to differences in the seasonal time of sampling.

Whether or not the assays to measure hair and fecal corticosterone levels were validated should be reported in the Methods. As indicated in my minor comments below, it would also be useful to provide more details on the hormone analysis methods.

Other minor comments:

As a general comment, the introduction is nice and concise but in some areas more details would be helpful. For example, when you write “morphological and behavioural traits associated with island syndrome… (line 44), could you indicate what specific traits you are referring to here? What have these studies found?

Additionally, the Introduction has a sharp transition to the stress physiology component and how island syndrome and stress physiology are linked should be made clear before this paragraph.

Line 52 – it seems odd that the references for the effects of density on stress levels are from a primate species and birds when this is a topic that has been studied at length in rodents including mice (reviewed in Creel et al. 2013 Functional Ecology).

Line 56 – it seems like there should be a caveat here for elevated GCs “can result” in negative health and fitness implications? This McEwen and Wingfield paper is arguably from the perspective of lab animals and humans. Throughout the rest of the manuscript, the authors have done a great job at referencing primary research articles (not reviews) but here they should continue that trend and give some examples where long-term elevations in GCs resulted in a reduction in health and fitness in a wild animal.

Line 64- whether its 2 or 72 hours, this is a long time difference and is highly dependent upon the species.

Line 86 – what is the basis for the statement that corticosterone is the major GC in mice and rats? Is there a reference for this?

Line 113-114 – because of my other comment above about the timing of captures and how it might vary between island mainland sites, it would be useful to provide details on when trapping occurred and at each site. How much synchrony occurred between trapping at the island and mainland sites?

Line 121 – I would think that estimates of density would only be accurate with at least 3 nights of trapping. Could you show some more details about how much trapping effort existed between island and mainland sites and if it differed? Did trapping effort impact density estimates?

Line 135 – this level of detail is nice but could the rationale for why this variation in coat colour might impact the hair stress levels be reported here?

Line 138 – is that 6 hour period from collection to freezing likely to impact the fecal stress levels? How do you know if the samples were collected at ~1800 h (when traps were set) or 645 h the following day (when traps were checked)?

Line 141- does time in the freezer impact fecal stress levels?

Line 146 – could you report here the trap nights at the different sites and if it differed among the island and mainland sites?

Line 154 – the basic details of the extraction procedure should be provided here instead of just the two references.

Line 155 – does time spent in the freezer impact the hair corticosterone levels?

Line 167 – are these CVs for hair or fecal corticosterone assays? Can you report how many assays in total were run.

Line 183 – it would be very useful to describe how reproductive condition was assessed and it how might impact the measures of fecal or hair corticosterone. How was pregnancy status in females assessed?

Line 211 – the version number of all the R packages used here should be indicated.

Line 255 – this “condition-dependence” where hair stress levels and body mass are positively correlated would negate one of the statements in the Introduction that high stress levels negatively influences health and fitness.

Tables – I suggest reporting exact P-values unless they are <0.0001

Figures – thank you for reporting the raw data in these figures but why not show the statistical differences (e.g., seasonal effects) or correlations among some of the variables? It makes it challenging for readers that only look at figures to discern if these are statistically significant or not.


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: NO ISLAND-EFFECT ON GLUCOCORTICOID LEVELS FOR A RODENT FROM A NEAR-SHORE ARCHIPELAGO
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript ”No island-effect on stress for a rodent from a near-shore archipelago” assesses whether near-shore island populations of white-footed mice show population-level (density) or morphological and physiological indications of “island syndrome”. Island syndrome is a group of systematic differences in characteristics that tend to be associated with insular living, including higher population densities, larger body size, longer longevity, lower reproductive output, and lower aggression towards counterparts. Three main factors have been highlighted as essential for the evolution of island syndrome: reduced local interspecific competition thus allowing for increases in population densities, isolation from other suitable habitats thus reducing or eliminating dispersal opportunities so that high densities are maintained, and low density-depressing forces such as lower predation pressure, which along with high population densities, tends to select for larger body size, lower sexual maturation rate, and lower reproductive rates, as well greater tolerance of conspecifics and lower aggression levels.
While many differences in morphological and life-history traits between mainland and island populations are well described in the literature, physiological differences have been less explored. Here, the authors investigate whether insular living is also associated with consistent differences in HPA regulation. They do this by assessing long-term corticosterone deposition levels in fur samples, and short-term corticosterone secretion via faecal metabolite concentrations, in captured white-footed mice from 11 islands and 5 mainland sites. Lower predation pressure and lower reproductive rates may be associated with lower GC levels for instance, while higher population densities might be expected to associate with higher GC levels. How these population- and individual-level differences, that together lead to the island syndrome, balance each other out to affect individual GC levels is still not fully understood and is very much worth investigating.

The authors tackle three main subjects in this manuscript:
1) assess whether island populations are found at consistently higher densities and are morphologically and physiologically different from mainland populations;
2) investigate whether island syndrome characteristics are influenced by island size and distance from mainland, and
3) explore associations between hair and faecal CORT with individual characteristics (sex and size), habitat and seasonality.

MAIN COMMENTS:
This study is generally well written and referenced, although it lacks important information at times (see below).

The research questions are well defined, and they are certainly interesting. However, I fear that this study has a major flaw that is hard to get around. The authors acknowledge that the islands are neither isolated (mice may swim between the islands in the summer and walk over the ice between them in the winter) nor entirely free from terrestrial predators, who are capable of moving between mainland and the islands, especially in the winter, and some small populations of predators may be established in some of the chosen islands. This study seems to be a good study, the research question is interesting, and the study seems to have been carried out correctly with a good sampling regime and decent samples sizes. Why did the authors choose this set of islands to test these questions? The choice of location appears to me to make it impossible for the authors to test the questions they set out from the start. Without isolation and reduced mortality pressure (in this case from predation), it stands to reason that white-footed mice would not show any population-level or morphological adaptations to island life as expected according to the island syndrome. Without these, there must be very little reason to expect the same populations to nevertheless show differences in glucocorticoid levels.
The authors point out that island syndrome has been detected in other rodent species at the same national park, although the studies were performed on different islands that were less clustered and thus more (truly) isolated. This point highlights how isolation may not mean the same thing when referring to dispersal opportunities, which directly affects population densities and have knock on effects on body size, behaviour and reproductive rates, and when referring to isolation from predators, which tends to select for smaller body sizes. Dispersal opportunities are likely affected by nearby islands and so potentially distance to nearest land mass rather than distance to mainland should be estimated. Adler & Levins 1994 provide very good explanations and details regarding the importance of sinks in determining morphological responses to island-living. As for predation pressure, I agree that distance to mainland may be the best estimate, at least with regards to terrestrial, larger predators.
Another important point to clarify is that the explanation provided for the species in which island syndrome characteristics have been detected (meadow voles and short-tailed shrews, Lomolino 1984) was that larger individuals were more likely to walk longer distances on ice and found populations at islands that would have average larger body mass.  This reasoning is very different from the usual assumption that larger body sizes are an adaptation to the local conditions, such as low predation rates and higher population densities at said islands. As the potential mechanism responsible for the larger sizes of individuals in these species on some of the islands is different from that assumed to bring about larger body sizes and other island syndrome characteristics, I’m not sure one should consider those results as evidence of island syndrome in rodents at the Thousand Islands National Park. However, regardless of the means by which these species attain larger body sizes at the studied islands, I would like the authors to provide a bit more information on whether there was any overlap in the chosen islands in both studies, why they did not choose the same islands and why they did not work on those species for which body size differences had already been documented, as a start point to explore physiological differences between mainland and insular individuals.
Similarly, the authors did not find significant effects of island size on island syndrome characteristics. As islands need to be considerably large for their size to have noticeable effects on population densities, it is likely that the range of sizes of the islands chosen for this study was not broad enough to detect such differences.

MINOR COMMENTS:
Figure 3: Since year does not significantly affect body mass (as main effect or in interaction with habitat) and in itself (2015 v 2016) is not a main focus on the study, I would merge the data and combine the two years, presenting only sex and habitat. The same goes for hair CORT results, and although fecal CORT levels are higher in 2016 than in 2015, unless you have a particular hypothesis or explanation as to why this might be, I do not think this is your most interesting result and that it needs to be visualised. Sample sizes for mainland females seem quite low.
Experimental design: EXPERIMENTAL DESIGN:
Be very transparent with your sample sizes for each response variable. Hair CORT analyses have smaller sample sizes relative to body mass analyses. Is this discrepancy due to the removal of all samples that had > 10% CV? How many samples were omitted in total? How many used in the analyses?
I would like to see more information on how the islands were selected as well as their distance to their nearest land mass, not just mainland. It seems to me that Mermaid, Beaurivage and Camelot Islands add very little to the overall dataset. In addition, Mermaid Island uniquely had its traps placed along transects and Camelot Island artificially appears to be the most isolated island (from distance to mainland) when it is actually very close to multiple other islands (Figure 1).

Methods:
The paragraph starting on line 144 (relative abundance) requires more explaining. For instance, please provide the correction factor equation you used to control for tripped traps. Also, what is your interpretation of the tripped traps occurrences? Would they have been tripped by white-footed mice or by other species?  None of this is explained and it is not obvious to the regular reader.
Validity of the findings: Results:
I am not sure I fully understand how you analysed your data, or at least how you have chosen to present it. For instance, does Table 1 present the results of 2 models or multiple models within each dataset. It looks like you present the results from 1 final model with all resulting significant effects, however, you state that the effect of year was tested using summer data from 2015 and 2016, while you test the effect of season using 2016 spring and summer data. Each of these analyses requires a different subset of your data, and therefore could not have been done in just one model. So either the statistics are not presented correctly, or they are not described correctly (paragraph starting on line 225).

Paragraph starting on line 279: As the season by sex interaction is significant, you should not report on the main effect of sex. The two factors are not independent.

Discussion:
Paragraph starting on line 317: the authors should clarify what they mean by “a high degree of isolation”, explain whether isolation was measured in similar ways and how it compares between the studies cited and the current one. How low a degree of isolation is required for population differences to be detected?

Line 333: The authors found that males were heavier than females independently of where they were sampled (mainland or islands). As is, it is unclear whether “in the Thousand Islands” relates to the whole national park including mainland areas or refers to the sampled islands only. If the latter, then the statement is not fully correct: yes, males on the islands are heavier than females, but so are males at the mainland sites.

 Hair corticosterone increased with body mass: The first paragraph in this section needs some clarifications.
Do you mean?:
1 – older individuals are heavier
2 – heavier individuals are more likely to breed
3 – breeding is stressful resulting in higher corticosterone levels
4 – corticosterone inhibits moulting
5 – resulting in breeding individuals having had their current coat for longer and put on weight since their last moult (though not clear why this is relevant in this context – line 389)
6 –incomplete moults do not replace all fur, thus older hairs store higher CORT
7 – concluding that heavier individuals are more stressed due to breeding which also inhibits moulting resulting in incomplete moults which means that individuals have had their hair for a longer period of time allowing for more CORT to be deposited in the hairs.
8 – and in parallel: younger individuals, which would be smaller, “have more recently grown their adult pelage” = lighter individuals may have moulted more recently and thus have lower CORT levels in their fur?
If I did understand all these arguments correctly, then they need to be explained better in that first paragraph.

Line 403: The correlation between CORT hair and CORT faeces, as reported by the authors, cannot be “similar” as the one reported for eastern chipmunks, because the latter is not significant and thus the two CORT measurements are not correlated.

I find the interaction between sex and season in CORT hair levels very interesting. The authors have, understandably, focused on the females, as this is the sex that differs between seasons. However, it takes both sexes to make the interaction significant. I would like to know the authors’ interpretation for males maintaining high CORT levels in both Spring and Summer. What is it about the mating or social system of this species, that the authors argue that females can have reduced CORT levels in the Spring, despite being pregnant, yet males’ levels remain high? A little more information regarding the species’ reproduction (number of litters, number of young per litter, etc) in the methods section would be quite useful.
Additional comments: This study clearly has value but, as is written, I think it highlights what I see as a fundamental flaw in it – that you could not possibly test your main questions in the set up (Thousand Islands National Park) that you used because the location does not have the basic requirements/conditions for the island syndrome to occur. Your results relating to island syndrome thus cannot provide any evidence for or against your main hypotheses. I wonder whether you could instead, focus on the link you found between CORT hair and CORT faeces with seasonality and sex, and having established that there are no differences between the populations, use that dataset to explore further questions. For instance, you discuss that both matrices reflect basal metabolic rates, but faecal CORT is additionally more strongly influenced by short-term stressors. Would you expect the measurements for females in the Spring, when faecal CORT levels are lower to correlate more strongly with hair CORT?


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: NO ISLAND-EFFECT ON GLUCOCORTICOID LEVELS FOR A RODENT FROM A NEAR-SHORE ARCHIPELAGO
Review round: 1
Reviewer: 3

Output:
Basic reporting: Overall the paper was strong in this respect.  I have some minor comments to improve the figures and tables (see attached comments).
Experimental design: The field methods were appropriate.  I do however have substantive questions and potential concerns with the statistical analyses. These concerns (and suggested improvements and points of clarification) are detailed in the attached comments.
Validity of the findings: Assuming that questions regarding statistical approach (detailed in attachment) are addressed, the findings are interpreted appropriately.  I have some minor feedback (detailed in comments) where additional information about the system will facilitate reader understanding of data interpretation.
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: NO ISLAND-EFFECT ON GLUCOCORTICOID LEVELS FOR A RODENT FROM A NEAR-SHORE ARCHIPELAGO
Review round: 2
Reviewer: 1

Output:
Basic reporting: The manuscript is very well written and clear. All of the revisions help to guide the reader - thank you for these.

The figures and tables are all appropriate but the one issue that I wanted to mention is that Figure 4 has a regression line that doesn't match up with with the data (it's origin goes beyond the lowest value of body mass observed). Similarly, Figure 6, the regression line goes beyond the maximum value of fecal CORT observed.
Experimental design: In the original review, I asked the reviewers to provide more details about their validations of hair and fecal corticosterone levels and asked to provide more details on the hormone analysis methods. I don't really think that their response (adding two citations) is adequate and we still need more details. For example, what does it mean that the corticosterone antibody used in this study was "similar" to one used previously? I think that this is a very important point and needs more detail for the authors to justify their use of the antibodies used for faeces and hair.

The authors indicate in their response letter that "During the 6-hour period from collection to freezing, the samples were kept on ice packs, which slows down any degradation of the hormones in the feces until the samples can be frozen" but provide no references for this statement. I would like the authors to justify this statement and add some sentence to the main text of the manuscript. Similarly, the authors indicate in the response letter that "Long-term storage in a –20C freezer can cause some degradation of glucocorticoids in the feces. However, tests in our lab on the re-extraction of fecal samples over time did not show any significant changes in fecal glucocorticoid metabolites within one year at –20C." but where are these analyses? Shouldn't they be provided for us to judge ourselves?
Validity of the findings: These all seem appropriate and thank you for the clarifications to the statistical analyses.
Additional comments: Thank you for making all of these detailed revisions to this manuscript. In light of the recent MacDougall-Shackleton et al. (2019 Integrative and Comparative Biology) paper about how "stress" is not the same as "glucocorticoids" and here you are only measuring glucocorticoids, might it be worth to revisit the title and use "glucocorticoids"?

In the original submission, I thanked the authors for providing citations to the original research (rather than reviews) and I appreciate that they have continued this approach in the revised manuscript. However, in my original review, I asked the authors to provide some references that show how elevations of GCs in wildlife result in a reduction in health and fitness. They now provide two citations (Wright et al., 2007; Hing et al., 2016) but after looking at these, I'm not really sure these meet that aim and they are reviews. E.g., the Wright et al. just reviews how anthropogenic noise may induce physiological stress. Hing et al. is another review that focuses on One Health and doesn't necessarily address this point. Are there better references here to justify this point. Note, DOI for the Hing et al. citation that was added doesn't work.