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: HIGH EXPRESSION OF SLC26A6 IN THE KIDNEY MAY CONTRIBUTE TO RENAL CALCIFICATION VIA AN SLC26A6-DEPENDENT MECHANISM
Review round: 1
Reviewer: 1

Output:
Basic reporting: 1. In the introduction section, more background information is needed for the biological process and chemistry of kidney stone formation. Also, the authors can move the content about oxalate secretion from the discussion section to the introduction section.

2. References are needed for Lines 37, 38, 42, 44.

3. Bright-field images are needed for Figure 2b.

4. In Table 2, the authors should also provides the sequences of other primers and synthetic fragments.

5. In Line 80, write the full name of the acronym IHC. Also, write the acronym SDS-PAGE in Line 83.

6. In Line 91, in triplicates is more often used than thrice.

7. In Line 119 and other places, it is lentivirus ‘infection’ not ‘transfection’.

8. In all figures, y axis units are missing.

9. In Line 255, is it Figure 3b or 4c that should be referred to here?
Experimental design: 1. In Line 74, the authors need to describe the method here other than citing a previous publication.

2. About lentiviral infection, which lentiviral infection system did the authors use and how was the titer calculated? More experimental details are expected, such as what are the packaging and envelope plasmids, transfection reagent.
Validity of the findings: In Figure 3a and 3b, it is strange that lentivirus infection did not change Slc26a6 gene expression in duodenal tissue. I am wondering if the infection was successful, maybe the duodenal tissue is hard to infect using the specific lentiviral infection system. Did the authors see RFP expression in the tissue? If so, the authors should provide the image and give a hypothesis of the tissue-specific expression.
Additional comments: The authors should check the usage of acronym. For example, in the legend of Figure 3, standard deviation and SD were used alternatively. The same problem happened to IHC, 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: HIGH EXPRESSION OF SLC26A6 IN THE KIDNEY MAY CONTRIBUTE TO RENAL CALCIFICATION VIA AN SLC26A6-DEPENDENT MECHANISM
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript in title of High expression of SLC26A6 in the kidney may contribute to renal calcification via an SLC26A6 dependent mechanism aims to investigate the role of kidney SLC26A6 in urolithiasis. This is a nice manuscript and been well prepared, however, revision should be done before it could be accepted for publication.

More information should be added in the instruction section;
Experimental design: The experimental design is good and been well conducted, however, several issues need be fixed. The method for lentivirus transfection into the kidneys of rats is questionable; regarding the efficiency of lentivirus subcapsular renal transection. Please provide more information about this procedure.
Validity of the findings: 1. Better images should be provided to demonstrate the location of SLC26A6
2. In line 101, please change the word “animal culture”.
3. Images in fig 1D are not comparable, please update them.
4. English editing is needed.
Additional comments: This is a nice manuscript and been well prepared, however, revision should be done.


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: HIGH EXPRESSION OF SLC26A6 IN THE KIDNEY MAY CONTRIBUTE TO RENAL CALCIFICATION VIA AN SLC26A6-DEPENDENT MECHANISM
Review round: 1
Reviewer: 3

Output:
Basic reporting: The paper is well constructed, the hypothesis is sound, te approach is innovative, the topic is relevant.
It is well written, the figures are OK
References OK
Experimental design: The experimental design is appropriate to the hypothesis
Methods are well described
Validity of the findings: Data are robust. The graphics should be changed according to my comments in the section below
Additional comments: All the 10 patients identified as stone formers had hyperoxaluria. Since there are other stone types, please explain if the patients were selected according to previous records of hyperoxaluria or by the analysis of stones previously eliminated.
In those 10 patients was a diet rich in oxalates excluded as a cause for the stone formation?
The transfection of lentivirus with Slc26a6 sequence and lentivirus with siRNA anti-226 Slc26a6 into the kidneys of rats was achieved by injection in the organs. How many injections were done per kidney to obtain a good spread of the vector?

For how long can the vectors remain in the kidney?

The fragments of kidney tissue used for Western blot and immunohistochemical staining were obtained from parts of the organ well apart from the local of the injections? Why two weeks was the time point decided and not 4 or 8?

90%–95% endogenous oxalate is excreted by kidneys. Did the animals in the siRNA-Slc26a6 groups have any evidence of oxalate overload in the organism?
Transfection with lv-Slc26a6 and siRNA-Slc26a6 was carried out in 4 plus 4 animals. This is a relatively low number. Please show individual values in fig 3 and 4 for each one of the transfected rats using a graphic plot as used in fig 1 for patients. This request is for valid for Western Blot,  immunohistochemical staining intensity, urinary oxalate and crystal formation.


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: HIGH EXPRESSION OF SLC26A6 IN THE KIDNEY MAY CONTRIBUTE TO RENAL CALCIFICATION VIA AN SLC26A6-DEPENDENT MECHANISM
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The revised version addresses all my concerns. I recommend the acceptance of this manuscript.


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

Input:
Title: HIGH EXPRESSION OF SLC26A6 IN THE KIDNEY MAY CONTRIBUTE TO RENAL CALCIFICATION VIA AN SLC26A6-DEPENDENT MECHANISM
Review round: 2
Reviewer: 2

Output:
Basic reporting: Authors made nice revision, no more comments.
Experimental design: Authors made nice revision, no more comments.
Validity of the findings: Authors made nice revision, no more comments.
Additional comments: Authors made nice revision, no more 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: HIGH EXPRESSION OF SLC26A6 IN THE KIDNEY MAY CONTRIBUTE TO RENAL CALCIFICATION VIA AN SLC26A6-DEPENDENT MECHANISM
Review round: 2
Reviewer: 3

Output:
Basic reporting: Authors responded to all the queries made and changed the manuscript and figures in a satisfactory way
Experimental design: OK
Validity of the findings: OK
Additional comments: No more 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: ANNOTATIONBUSTR: AN R PACKAGE TO EXTRACT SUBSEQUENCES FROM GENBANK ANNOTATIONS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript is grammatically correct, contains adequate background material to place the current work in the appropriate context, and is liberally referenced. The raw code is readily available on github, and easily installed into an existing R environment. Including a tutorial is a valuable addition. The figures are clearly labeled and described.

There are several instances of awkwardly long sentences that take away from the clarity of the writing; for example, lines 13-16, 61-63, 74-75, 125-129. More importantly, while the manuscript contains a substantial number of references, the literature used to support a central tenet of the study – that a major challenge to obtaining large amounts of sequence data is the highly variable nomenclature of gene names – is 10-15 years old. The field of bioinformatics has progressed dramatically since that time; for example, GenBank now includes synonyms in gene symbol searches, numerous data-specific repositories have arisen to provide gold-standard sequence data for barcoding genes like rDNA, many sites have fairly simple APIs for data access, etc.
Experimental design: The authors have developed an R package to extract data (particularly sequences) from GenBank using annotation text and synonym matching. They argue that this is potentially advantageous over sequence similarity (e.g., BLAST) for downstream analyses, particularly the construction of phylogenies, and allows easier access to underlying sequence data itself.
It is not clear that the current software is a particularly meaningful addition to the field. First, no mention is made of GenBank’s own eUtils, which provides straightforward access to sequence data using url strings. At least a comparison should be presented between eUtils and AnnotationBustR with respect to speed, usability, and accuracy. Second, no data is presented showing how much sequence data in GenBank is present in concatenated sequences versus discrete GenBank entries. Third, annotation errors in sequence data are a significant and ongoing problem, for reasons that are fairly well known but difficult to address (for one example of many, see Ben-Shitrit et al. 2012 Nature Methods 9: 373-8). It is not clear how AnnotationBustR accounts for these errors, which could insert substantial errors into downstream analyses. Fourth, the authors present no data (historical or in the current study) indicating that reliance on sequence similarity is a significant contributor to erroneous phylogenetic analyses, or that text matching such as that employed in AnnotationBustR would show any improvement over existing methods. This may, in fact, be the case; however, sequence similarity has been firmly entrenched in tree building for many years, and it is incumbent upon the authors to provide data to support using annotation strings instead.
Validity of the findings: The benchmark comparisons in Figure 3 are interesting – I expect the large variation around some data points is due primarily to differences in network latency – but I would have enjoyed more discussion around hardware requirements. For example, which is more important to AnnotationBustR performance: processor count, RAM, or network speed? Also, it is not clear how many times each test was performed.

Importantly, there is no analysis of AnnotationBustR’s accuracy or sensitivity. For example, do I retrieve fewer false positives using AnnotationBustR vs. BLAST? What about eUtils? How do results and usability compare between the current software and gold-standard databases like RDP or Greengenes?

In general, while this software may prove to be useful, the appropriate comparisons and benchmarks have not been included here. Without these data, the relevance of this contribution is not clear.
Additional comments: Please include hardware and software requirements for AnnotationBustR in the manuscript. I discovered during review that it does not appear to function properly on R version 3.3 (OS X) but does on R version 3.4. Also, running the AnnotationBust step of the tutorial throws several errors of the form:

Warning messages:
1: In if (grepl("unknown accession", full.rec) == TRUE) { :
  the condition has length > 1 and only the first element will be used


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

Input:
Title: ANNOTATIONBUSTR: AN R PACKAGE TO EXTRACT SUBSEQUENCES FROM GENBANK ANNOTATIONS
Review round: 1
Reviewer: 2

Output:
Basic reporting: Because this manuscript describes a software package, I will be reviewing not only the manuscript but also the code and documentation provided.

I find that all data is shared appropriately. In addition to completely open source code, the distribution of a curated set of synonyms for organellar and ribosomal DNA sequences will be very valuable for the community. I was able to execute the example code used to generate the timing figure, with similar results on my Macbook Pro.

ANUNC is used several times in the introduction and abstract without a definition. I had to look it up-- its definition (as a sequence retrieval system) should be explained.

The number of additional synonyms is likely to proliferate as GenBank grows. Do the developers have plans to keep the cpDNA, mtDNA, and rDNA tables updated with the package?

The authors should mention that the mtDNA list they have provided with the package is intended for animal mitochondrial genomes. Many of the loci in the Brassica oleracea mitochondrial reference sequence (JF920286.1) are not shown, including several of the ribosomal proteins (rpl5, rpl16) and the tag arginine translocation gene (tatC). Although mitochondrial coding regions evolve slowly in plants, they are frequently used for deep phylogenetics. See, for instance: Liu, Y., Cox, C. J., Wang, W., & Goffinet, B. (2014). Mitochondrial Phylogenomics of Early Land Plants: Mitigating the Effects of Saturation, Compositional Heterogeneity, and Codon-Usage Bias. Systematic Biology, 63(6), 862–878. doi:10.1093/sysbio/syu049
Experimental design: Here I will report on conceptual issues with the AnnotationBustR package, its utility, and suggestions for minor improvements to the package and text.

Why don't refseq accessions like NC_ work? Is it possible to extract the GenBank accession number given a refseq accession number?

Why are intron sequences not supported? In plants, intron sequences within plastid genes are often quite useful for phylogenetics (for example, within rps2 or matK). Could the second column of the annotations feature column be used to extract the intron from a named gene while also using the synonym utility of AnnotationBustR?

It would be useful if the vignette gave an example of retrieving sequences with different IDs in GenBank. I did not go through all of the mtDNA examples, but the four great ape sequences chosen for cytochrome odixase were all identified by COX1. If there already is such an example, it should be highlighted in the vignette as well as within the manuscript text.

Because the package uses seqinr as a dependency, it would also be useful to see a full example employing a workflow that includes searching for gene sequences (the orange box in Figure 1). For example, one use of AnnotationBustR would be to retrieve all trnG sequences from the plant genus Oenothera. Some GenBank sequences from this genus are deposited as individual genes, while others are full chloroplasts. How would I manipulate a GenBank search using seqinr to fully utilize AnnotationBustR? I realize that GenBank search results will have to be manually curated, but adding this example to the workflow and tutorial would  probably enhance the usability of the package.
Validity of the findings: Here I will report some issues and suggestions I found when running the R vignette or code contained in help documentation.

While the vignette is formatted fine within RStudio, it is not formatted properly in Github, due to a recent change in Git Flavored Markdown-- a space is required between the ## and the header. The images also do not appear on GitHub.

In the vignette, under troubleshooting, fix this sentence:

"if you have internet access yet are still haivng isues, check that these sites and there servers are not down"

The help documentation for AnnotationBust says that the function returns a data.frame containing a list of sequences that “can be turned into an accession table using MakeAccessionTable”. I cannot find any other reference to this function.

As is, the data.frame currently combines multiple accessions from each species into a comma-delimited table, which may not be easily used in other formats. In the tutorial example, the two human sequences are joined into a single row.  Why not have one row for each sequence source?

It should be noted in the documentation that the default behavior is to overwrite existing files when code is re-run. Users should be notified/warned that this is occurring. Users could also benefit from an option to append to existing files.

When I run AnnotationBust for either the R vignette tutorial code or my own example, I always get the following errors:

When the code is first executed:
In socketConnection(host = host, port = port, server = server, blocking = blocking,  :
  pbil.univ-lyon1.fr:5558 cannot be opened

Upon code completion:
Warning messages:
1: In if (grepl("unknown ", full.rec) == TRUE) { :
  the condition has length > 1 and only the first element will be used

The FASTA files all began with a newline character. This will break some FASTA parsers which rely on detecting the > as the first character.

Why are the nucleotides returned in lower case? The sequences are not stored in GenBank this way. Some sequence parsers will treat these sequences as soft masked. Perhaps give the user an option about case?
Additional comments: I approached this review as a potential user of the software package, and as someone who works primarily in phylogenetics of plants. The scripts included with AnnotationBustR will be useful for researchers who wish to extract reasonably well-curated protein coding sequences from GenBank. The suggestions I have made will hopefully improve the usability and documentation of the R package.


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: ANNOTATIONBUSTR: AN R PACKAGE TO EXTRACT SUBSEQUENCES FROM GENBANK ANNOTATIONS
Review round: 1
Reviewer: 3

Output:
Basic reporting: Borstein and O'Meara present a package that provides important functionality: extracting subsequences from GenBank based on the annotations associated with sequences. While there are a few tools that allow for the extraction of features from GenBank files, none of these are particularly easy to, for example, incorporate into scripts and automated workflows.

In general, the manuscript is well written. However, I found the  introduction to be written with a strange audience in mind. For example, shouldn't it be obvious that molecular sequence data is vital for biological research these days. Not sure if that needs to be stated. And is it necessary to say that molecular phylogenies are "prevalent" in biological research? If the reader doesn't know that, then they probably don't know what a molecular phylogeny is. I would recommend rewording some of this for a more consistent audience (e.g., choose whether the reader knows phylogenies / molecular work or not). Then again, perhaps I am off base and this better reflects an audience group that I am simply overlooking.

Otherwise, the basic reporting and writing in the manuscript is well done.
Experimental design: I would mainly question the use of R for this task. R is not traditionally used as an informatics platform for many reasons. R is not particularly fast for text and is somewhat more obtuse for incorporating into informatics pipelines. I wonder if the authors might be able to write a paragraph that describes why this would be beneficial. I am not suggesting that they authors write this in another language, just wondering why R was chosen.

I am also a little surprised that other methods or other packages aren't discussed. For example, do the functions presented here have more functionality or are they better than the SeqRecord class functions in BioPython? Also, while I am less familiar with it, there surely must be similar functions in BioPerl. This may be addressed by responding to the comment mentioned above, but is there a reason that these are not adequate for the authors use? I see no mention of either of these widely used packages in the manuscript. If the functionality isn't there, then it would seem like this fills a gap. If the functionality is there, it doesn't render your R package less useful, but it would be nice to understand the similarities and differences (i.e., perhaps it is the same and it is extending this functionality to R which is growing as an informatics platform?).

I appreciate the posting of the code on GitHub and the vignette describing how it may be used.
Validity of the findings: The conclusions presented in the paper are well founded and the limitations are well stated. I would only suggest discussing the intersection of this work with other informatics platforms. Again, not to express that one is better than another or any of that, but instead so that users may better understand what functionality is present and not.
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: ANNOTATIONBUSTR: AN R PACKAGE TO EXTRACT SUBSEQUENCES FROM GENBANK ANNOTATIONS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The awkward sentences have been improved where noted, which improves the readability.
Experimental design: The authors have developed a usable software tool for extracting genomic regions of interest from GenBank records in an automated fashion. This is a fundamental task in bioinformatics that underlies many different areas of modern biology. The software presented here relies on a text matching approach, rather than the more traditional use of sequence similarity. This approach has its merits, which are presented well in the manuscript, but it also includes several serious, well-documented challenges, including spelling differences, errors in annotation, annotation of true orthologs as hypothetical or unknown, generic annotation strings that may not indicate true function, and more. AnnotationBustR uses a synonym lookup table to address spelling and syntax differences, but does not address the subtler challenges. Admittedly, much of this may be beyond the scope of the current work; however, as discussed in more detail below, this manuscript would benefit from some analysis that estimates the impact of those issues on the data being produced by the described software.
Validity of the findings: The software is functional and appears to be fairly straightforward to maintain, assuming continued access to the ACNUC database. It does have the advantage of a relatively simple interface, increasing its audience of potential users, and the choice of platform is appropriate – although some might argue that R has a significant learning curve, the same can be argued of virtually any language. Of more concern is assessing the validity of the data being generated by AnnotationBustR, particularly vis-à-vis phylogeny estimation where high quality data is absolutely required. Since this is a stated motivation for developing AnnotationBustR, the current manuscript would benefit greatly from some effort to benchmark the success of this approach. This is particularly true given the myriad issues with gene annotation, some of which are discussed by the authors, upon which AnnotationBustR relies. For example, one might use a double-blind approach, assembling data sets independently using AnnotationBustR and another method or two and comparing the output. Or it might be valuable to provide a comparison between an AnnotationBustR-informed phylogeny and an existing published tree based on the same loci. While AnnotationBustR may be easy to use, it is incumbent upon the authors to assess the quality of the data that it generates so as not to invalidate their hard work in crafting a usable tool.
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: ANNOTATIONBUSTR: AN R PACKAGE TO EXTRACT SUBSEQUENCES FROM GENBANK ANNOTATIONS
Review round: 3
Reviewer: 1

Output:
Basic reporting: The revised manuscript contains numerous changes to the text, and its clarity has improved. In the interest of reproducibility, please include software version numbers and dates of accession of public data sets, where applicable.
Experimental design: The authors have included a direct comparison between AnnotationBustR and an alternative sequence retrieval approach using PHLAWD. This is a notable addition and serves to strengthen the manuscript significantly. However, please describe how PHLAWD was used; specifically, what settings were employed to generate the alternative data set? This may explain the wide disparity between sequence coverage between the two approaches but it is not possible to evaluate. Also, why was PHLAWD employed instead of a simpler BLAST search using a target sequence and restricted by taxonomy? A reader would benefit from a brief explanation of the rationale behind the experimental design here.
Validity of the findings: While the benchmarking results shown in Figure 2 are interesting, they would be much more useful in comparison to existing methods. Alternatively, assessing the AnnotationBustR code itself (apart from its reliance on external tools) might prove more applicable to the current manuscript; for example, how do speed and memory requirements scale with respect to input size?
Additional comments: AnnotationBustR is a potentially useful tool for sequence retrieval, and a worthwhile addition to the R ecosystem. While the authors seem to bill their software as an alternative to sequence similarity (i.e. BLAST), I suggest that it would be quite useful as a companion tool - particularly given the level of manual inspection required for building a quality phylogeny regardless of the approach.


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: BEETLES IN BAMBOO FORESTS: COMMUNITY STRUCTURE IN A HETEROGENEOUS LANDSCAPE OF SOUTHWESTERN AMAZONIA
Review round: 1
Reviewer: 1

Output:
Basic reporting: Very well done
Experimental design: Super
Validity of the findings: A good look at an important sw Amazon system
Additional comments: Glad to see this finally finished. This manuscript is well written with all the essential elements covered; in addition, it is well illustrated and graphed. There was not even little things to nit-pick upon, except two suggestions: I would call the Guadua bamboo "tall" rather than "large," as at their field site most culms are rarely larger than 2.5" DBH. A second thing is that botanical names require authors and at least the first mention of the species should carry the author(s) name.


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: BEETLES IN BAMBOO FORESTS: COMMUNITY STRUCTURE IN A HETEROGENEOUS LANDSCAPE OF SOUTHWESTERN AMAZONIA
Review round: 1
Reviewer: 2

Output:
Basic reporting: Please note: I have provided detailed comments and suggestions in the manuscript, attached as an annotated pdf file.  The statements below highlight my overall impressions and some of the key issues that should be addressed prior to publication.

This paper evaluates differences in beetle communities between bamboo-dominated and terra firma forest patches in Amazonia, and in doing so provides data on a poorly understood insect fauna in an ecologically important habitat type. The article is mostly clearly/concisely written, although some sections of the discussion could be re-organized or removed for clarity and flow.  The introduction provides adequate background information/literature and clearly states the study objective and hypotheses.

Figures are generally good, but I have some suggestions.  For example, Figure 1 should show the location of each study site/forest patch. Figure 4, in addition of being of poor quality, does not add to the reader’s understanding of the experimental design – it should be removed.  Figure 11 is similarly not useful. Some of the captions should contain additional information.  Some of the tables can be combined.

Importantly, the supplementary information, as presented, does not constitute “raw data”. If more detailed data are not already being provided/made available elsewhere, I have made suggestions in the text for correcting this.  I also suggest a second supplemental table be included to provide detailed information about each of the forest patches examined in the study.  The authors should also make sure that any code used to conduct the analyses is included (if applicable).
Experimental design: This is an original primary research study within the scope of the journal. The research question and the knowledge gap it intends to fill have been defined clearly.  The experimental design and collection methods are sound and rigorous, though some additional information (particularly pertaining to the sites) is required for clarity and replicability.  I also had some minor questions regarding terra firma patch sizes/continuity, and possible edge effects from trap placements.
Validity of the findings: Although the analytical methods the authors used to evaluate differences between forest types and seasonal difference are sound, there is one significant issue with how the raw data were handled prior to the analyses. The authors state that the two forest patches in each pair receive equal numbers of traps, but that patches of different sizes received different numbers of traps – this means the sampling effort is not consistent between patches of the same forest type.  A patch with 1 trap will almost certainly have “fewer” species than a patch with 10 traps, simply because one trap will collect fewer individuals than 10.

Patch-level species richness estimates using raw data, and any measure of community structure that uses raw abundance, are therefore not meaningful. The authors briefly address the sample effort in the context of the species accumulation curves, and I agree that when comparing “total species in bamboo” vs “total species in terra firma”, then the effort is the same (but note that I suggest the accumulation curves be replaced by individual-level rarefaction curves).  Otherwise, I suggest that patch-level richness be expressed as rarefied species richness, and that any abundance data that are not pooled at the level of forest type be standardized by sampling effort (i.e. # individuals/# traps) before conducting any analyses.

These changes may generate very different results from what is currently presented, and could necessitate a major revision of the figures, results and discussion sections.

Assuming for a moment that the results remain largely the same even after the sample effort issue is addressed, the discussion is, for the most part, a reasonable assessment of the findings, although there are some sections that are poorly structured, or too speculative in their current form (they lack supportive information or justification).  The authors’ conclusions relate to the original research question and are placed in a broader conservation context.
Additional comments: Please see the annotations in the attached pdf file for detailed comments and suggestions. My recommendation is “Major Revisions”.  The study design and execution is fundamentally fine and the authors have amassed an important data set, but given the issues with the analysis mentioned above, and the lack of adequate raw data, it require a significant overhaul before it can be considered for publication. Given the potential for major changes in the results, I would need to re-evaluate the revision.


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: BEETLES IN BAMBOO FORESTS: COMMUNITY STRUCTURE IN A HETEROGENEOUS LANDSCAPE OF SOUTHWESTERN AMAZONIA
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have addressed earlier concerns regarding figure presentation/captions, raw data, and requests for additional supplementary data. The paper remains well organized, written, and relevant.
Experimental design: The authors have improved the clarity and repeatability of the experimental methods. I appreciated their detailed response and clarification regarding sampling effort and between-patch comparisons (and the associated revisions to the manuscript) regarding sampling effort.  At this time I am satisfied with their rationale for the analytical approaches taken.
Validity of the findings: No comment (see previous section).
Additional comments: Other minor comments/concerns have been satisfactorily addressed by the authors in the manuscript and/or in their reply to reviewer. I have no concerns with the manuscript as presented.


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: LOCAL AND RELAXED CLOCKS: THE BEST OF BOTH WORLDS
Review round: 1
Reviewer: 1

Output:
Basic reporting: 1]	Basic Reporting – The manuscript is well-written:  concise, clear and professional.  It deals with a specialized area, but a wider audience will be able to understand the main points of the paper.
	The subject is clearly introduced.  To be redundant:  this is a narrow topic, but the authors have done an adequate job of describing the general problem and their approach to improving the available analytical tools.
	The figures and table are clear and can be understood.  There is no problem with the raw data or with the availability of the authors’ implementation of their software within BEAST.
Experimental design: 2]	Experimental Design – I find that the subject is within the scope of the journal.  However, and this might be repetitious, the problem addressed is narrow and the authors are improving the analytical software.  The biggest deficit of the manuscript, in my opinion, is that there is NO biology!  Yes, influenza sequences are run through their flexible local clock software.  The authors find that the resulting “fit” is slightly better (higher marginal maximum likelihood values), but it is impossible for the reader to discern if this better “number” means something in terms of influenza virus sequence evolution.  This is a common situation, and the authors should not be held to a different standard than the hundreds of other articles in this area.
	Again, I must stress that the manuscript is clear and that the research merits publication in PeerJ.  The methods and results are clearly presented and there is sufficient detail with specific regard to the specific issue under investigation.
Validity of the findings: 3]	Validity of the Findings – The data/results are sound, although the improvement in marginal maximum likelihood values are not earth-shattering.  The conclusion that flexible local clocks would yield an improved fit to the sequence analysis is warranted.  The manuscript is well-written in terms of focus and ensuring that everything ties back to the specific question being tested.
Additional comments: 4]	Overall Assessment – This is a well-written manuscript that is focused and tightly structured.  Publication is recommended.  At the same time, the authors will in the future – one hopes – integrate their modeling results into the broader question of sequence evolution.


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: LOCAL AND RELAXED CLOCKS: THE BEST OF BOTH WORLDS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The pre-print of this article has been discussed with multiple colleagues, and we agree that the piece of research is mostly well presented and clear. Since this piece includes a package, the readership will largely be broader than specialists and other model-developers. For this reason, I have several comments on the basic reporting.

The description of molecular clock hypothesis should be extended. As it stands, it is only accessible for readers familiar with the field. The concept of auto-correlation is particularly  briefly explained. Similarly, the order of appearance of each of the hypotheses is rather arbitrary. Since local clocks contain less rates than uncorrelated clocks, it seems like they should appear first. It also seems appropriate to acknowledge the pure white-noise model of Lepage et al. (2007), which is the only truly uncorrelated clock model.

The description of the flexible local clock should also be more detailed to reach non-specialists. The fact that the number and location of clocks must be determined before the analysis should be mentioned explicitly from the outset. The parametric distributions of rates across branches that are permitted in the author's implementation should also be outlined. Also, are all the other default model settings (substitution models, tree priors) in BEAST2 also allowed in the add on?

Other than the technical details of the proposed model, there is a clear lack of introduction of the biological interpretation of the model. In which scenarios do we expect abrupt changes in the rate, followed by also possibly abrupt changes within each local clock. Are there any examples of this in nature. Or perhaps more intuitively the authors could say why the existing clock models are unrealistic, and how these gaps are met in the new model.

The paragraph starting on page 79 should be broken down into data sets at least, because it is difficult to follow the point in the present form.

The description of the simulations study requires more detail. For example, a non-expert will be lost after reading sentence 119, which refers to non-specific lognormal distributions.

The description of the package in the GitHub repository is extremely brief. A step-by-step process of installing the package would be helpful, as well as a brief tutorial. Also note that in many machines the Library folder is hidden, making it necessary for users to install the package through the command line. Once it is installed, the template does not automatically appear in BEAST2, so the step-by-step process of installation is fundamental.

It would me more suitable to report the pairwise Bayes Factors between models than the marginal likelihood estimates. It is clear that the difference in marginal likelihoods is small, so the Bayes Factors will provide a clearer representation of the improvement in model fit.

The proposed model is substantially more parameter-rich than any of the other models discussed in the introduction. This can come at a substantial computational cost. Reporting the difference in computational demand across models would help users decide whether it is worth using this model.

Lepage, T., Bryant, D., Philippe, H., & Lartillot, N. (2007). A general comparison of relaxed molecular clock models. Molecular biology and evolution, 24(12), 2669-2680.
Experimental design: In its present form, the manuscript gives little insight into the difference between the existing local clock model and the new flexible local clock.

The new model could be tested much more thoroughly to determine exactly when it is more powerful than other clock models. Presumably it will be more powerful than the local clock when the variance in the rate is high within each local clock. Showing how much variance is needed for the standard local clock to fail would be interesting, as would be testing the impact of different magnitudes of change. Presumably the uncertainty in rate estimates is also greater when using the flexible local clock, making it less precise but more accurate. This follows the bias-variance tradeoff in statistics, and would give readers an insight into how this model fits with regards to other existing models.

The simulations at present are of little use and are only briefly discussed. It would be helpful to be explicit about why they have been included and what they say about the model. A figure with the simulation results would be greatly insightful, for example in the form of a boxplot showing the sizes of the HPDI of the mean rate or the rate estimates across scenarios.
Validity of the findings: The new method presente is a valid and natural extension of existing methods. However, it is possible to be more explicit about the impact that this new model will have when analysing empirical data.

The Limitations and Conclusions section should outline how the model might not be biologically realistic, and any possible extensions that could lead to improvement.
Additional comments: The idea for this clock model is interesting as long as it is well justified biologically. I think this model is a useful and interesting step, but it requires a more thorough justification and demonstration of its power.

The authors have made a reasonable effort in making this new model accessible and showing that it can be useful. However, both of these points should be extended. This would make the method truly accessible and appealing for non-specialists and will give a clear picture of the model's performance for specialists willing to expand or examine clock models in the future.


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: LOCAL AND RELAXED CLOCKS: THE BEST OF BOTH WORLDS
Review round: 1
Reviewer: 3

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: The manuscript entitled “Local and relaxed clocks, the best of both worlds” describes an implementation for the widely used BEAST software that allows the use of relaxed clocks within a context of specifying local locals (that previously used only strict local clocks). In my opinion it is a smart implementation that I am certain will be used in multiple situations and different types of data. The manuscript is clearly written in terms of objectives, methodologies and testing of the model (real data and simulated data) and it is clear about its limitations. Given this I advise for its publication.


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

Input:
Title: PEPTIDE PRESENTATION BY HLA-DQ MOLECULES IS ASSOCIATED WITH THE DEVELOPMENT OF IMMUNE TOLERANCE
Review round: 1
Reviewer: 1

Output:
Basic reporting: As a whole the manuscript entitled “Peptide presentation by HLA-DQ molecules is associated with the development of immune tolerance” is well written in a logical way. Cited references in the Introduction are sufficient to provide the readers with the necessary information and problems about the role of the different HLA molecules. There are 3 figures, 1 table and supplementary materials from 2 tables. The proposed hypotheses are not novel and have only confirmatory character.
Experimental design: This article reports results from the bioinformatic modelling of the binding ability of immune epitopes (taken from database) to the HLA-DR and HLA-DQ molecules. The used methods for the performed analyses are well described. Although the results support previous findings about the role of HLA-DQ for tolerance induction, the lack of any experimental data reduces the merit of the manuscript.
Validity of the findings: Therefore, the conclusions in this case could be treated as speculative, but not as confirmatory evidences. Performed analyses by using 5-mer epitopes and 50% binding ability are far away from the real situation.
The problem is that neither in the title nor in the abstract is written that this is only modelling, which could mislead the readers that the results are experimental. In addition, the results generated by these analyses are not novel. In best case only confirm some of the experimental data published from other authors such as epitope binding and association of HLA-DQ with certain autoimmune diseases. I think that the theoretical models have to precede the experimental results, not opposite (to confirm them).

It is not clear what means the sentence:
“… epitopes presented by DQ molecules are more similar to human proteins…”. which is mentioned few times. Some of these epitopes are part of human proteins. How the epitopes are distinguished whether they are human or not. What about the epitopes that have the same sequence in several organisms?
Additional comments: May be the manuscript will be improved if in the title and in the abstract is stated that this is only modeling (bionformatic analyses) and the proposed conclusion are based on these analyses (speculative), which need to be confirmed experimentally.
Alternatively, these bioinformatic analyses have to be supported at least partially with experimental data. For example with synthetic peptides (epitopes) in vitro/ex vivo or in vivo with animal models.


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: PEPTIDE PRESENTATION BY HLA-DQ MOLECULES IS ASSOCIATED WITH THE DEVELOPMENT OF IMMUNE TOLERANCE
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript meets all criteria of basic reporting.
Experimental design: no comments
Validity of the findings: no comments
Additional comments: In this paper Manczinger and Kemeny present a large-scale comparative analysis of epitopes presented by DQ and DR variants of HLA class II proteins.  They found that epitopes presented by DQ molecules are more similar to human proteins as compared to those presented by DR molecules and showed around 10% overlap, suggesting lack of functional redundancy. In addition, DQ and DR molecules present peptides derived from different microbial species.  The data presented in this manuscript may contribute to the understanding of MHC II biology, but I have some concerns regarding this study.
 The authors claim that “ the binding of autoimmunity-associated epitopes by DQ confers protection against autoimmune diseases”. I think, this is not entirely correct.
Different alleles of DQ as well as DR have different impact on development of autoimmune diseases. For instance, certain of HLA-DQ heterodimers have central role in celiac disease and type 1 diabetes pathogenesis. Ninety percent of Caucasian patients with celiac disease express HLA-DQ2.5cis encoded by HLADQA1*0501-DQB1*0201 or DQ2.5trans onHLA-DQA1*0505 DQB1*0301/DQA1*0201-DQB1*0202 haplotypes. In contrast, approximately 20–30% of the healthy Caucasian population is HLA-DQ2 positive.
Therefore, it would be more useful to compare epitope-binding of DR and DQ alleles associated with risk to autoimmunity vs protection against autoimmunity (Fig.1).
From the microbe-derived epitope binding study (Fig. 2) the authors excluded peptide sequences of commensals. Since, it has been postulated that HLA-DQ may also have impact on the intestinal microbiome and, consequently, to development of autoimmunity, I think, it is reasonable to include these epitopes as well.


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

Input:
Title: PEPTIDE PRESENTATION BY HLA-DQ MOLECULES IS ASSOCIATED WITH THE DEVELOPMENT OF IMMUNE TOLERANCE
Review round: 2
Reviewer: 1

Output:
Basic reporting: The revised manuscript meets the PeerJ criteria.
Experimental design: Now, in addition to the predictive bioinformatic analyses are integrated also data from in vitro experiments.
Validity of the findings: The manuscript is improved significantly.
Additional comments: No 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: GENETIC POLYMORPHISMS AND FORENSIC EFFICIENCY OF 19 X-CHROMOSOMAL STR LOCI FOR XINJIANG MONGOLIAN POPULATION
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors should mention about several previous studies dealing autosomal SNPs in Mongolians populations (for instance, Xing et al. PLOS Genet. 2013, 9:e1003634, Nakayama et al. Mol Biol Evol 2017, 34:1936-1946, and more). Because autosomal SNPs are now common as targets for studying population diversity.
Experimental design: No comment
Validity of the findings: No comment
Additional comments: Aim of Abstract does not seem to properly show the purpose of this study. The should authors re-write to emphasize that testing the forensic efficiency of X chromosome STRs was their first interest, or include more precise population genetic analyses to depict genetic diversity of X linked loci in Mongolian populations, especially focusing on comparison with populations.

The authors should provide deeper discussion about diversity of X chromosome STRs. Comparison of Mongolians and other Asian populations in haplotypic diversity may be useful to give insights into the demographic history of these populations and it would be of interest to readers with biological anthropology background.


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: GENETIC POLYMORPHISMS AND FORENSIC EFFICIENCY OF 19 X-CHROMOSOMAL STR LOCI FOR XINJIANG MONGOLIAN POPULATION
Review round: 2
Reviewer: 2

Output:
Basic reporting: This paper presents the analyses of the 19 X-STR loci included in the AGCU-X19 STR kit in 267 Mongolian individuals from the Xinjiang province. Allele frequencies, Hardy-Weinberg equilibrium and parameters of forensic interest were estimated and a comparison with data from the literature was conducted. The present work enlarges the Chinese ethnic groups analyzed for this X-STR set in Yang et al. (2016), studying an important minority Chinese group.

Raw data. I thank you for providing the raw data, however I cannot understand the data in Supplementary Table 1. Authors supply the results of the 19 X-STRs in 267 individuals (men and women), but why women have only one allele in each STR? It is absolutely incomprehensible for me!

In general, along the text one can find a lot of mistakes (for instance line 175 DXS10134); expressions like “quite high values or “quite useful” that are very vague; redundant words (Mongolian groups lines 42-45) or sentences (lines 36-38, lines 121-123); not clear information (lines 96-97 “it was worth mentioning that….why it is worth mentioning?); grammatical errors (“these authors are contributed…”, line 60 “humane”, line 137 “wasn’t”)...The language and the structure should be improved.

Figure 2 is not clear or informative, it can be eliminated.
Table 3 should be checked.
Experimental design: The present study has been done following correct ethical and technical standards and lab methods are described with sufficient detail. Although authors should provide more justification about the Quality Control. ISFG guidelines (Carracedo et al. 2014) only indicate the number of samples to be analyzed to publish in ISFG Genetics…it is not a Quality Control described by Parson.

Regarding statistical analyses, these should be improved upod before Acceptance (as I have indicated in the general comments).

Although the study contains a large enough number of samples from an interesting population and therefore is of forensic and population genetic interest, in my opinion the manuscript needs a major revision before being considered suitable for publication in PeerJ.
Validity of the findings: The matter of study is not completely novel. In the introduction authors point out that only a nine X-STRs have been studied in the Mongolian group. But, in fact Zhang et al. (2015) analyzed 34 X-markers (18-STRs and 16-Indels) in this ethnic group and recently Tao et al. (2018) have studied 12 out of the 19 X-STR in Mongolians. Anyway, to study this set of X-STRs is useful provided that other Chinese populations have been previously typed with the same markers.

Although your data are useful in forensic casework and interesting for Population Genetic research, the conclusion that Mongolians are away from other clusters and that they have a unique genetic structure is not supported by the results or by previous studies, where Mongolians showed to be closely related with other Chinese groups (Zhang et al. 2015, Tao et al. 2018 and others). These previous results should be included in the discussion. And, if the authors want to demostrate that with these 19 X-STRs Mongolians have an unique structure, additional analysis should be carried out, for instance STRUCTURE analysis.

In the General comments I have pointed out some points in the statistical methods and their interpretation that could be improved.
Additional comments: I would suggest some observations in order to improve the general content of the manuscript:
1-Tittle. The present tittle is not informative about the subject of the study, it should indicate the number of STRs or even the name of the kit, so readers can easily know which STRs are analyzed.

2-More information of the studied ethnic group should be provided. Origin, language, number of individuals, historical relationship with other Chinese ethnic groups…And, is the present sample representative of all the Mongolian population?

3-Statistical methods
a- Please, check PI values in Table 2. The Paternity Index values are not correct, since the more variable a marker is, the lower PI value is provided. Logically it should be the opposite.

b-My main concern is in the comparison with other populations and the conclusions that authors draw from it.
	-Which has been the criterion of selection of the populations? I agree with the Asian samples, due to the geographical context of the study, but there are other worldwide studies besides Tomas et al. (2012). Probably Greenlanders, Danes and Somalis are not the most suitable populations to be compared with Mongolians.

	-The authors have calculated significant differences (Fst) in each STR…this information can be useful to evaluate the power of the different markers in population structure analyses, but not for evaluate the relationship between populations. Comparisons between populations should be done with the complete set of X-STRs.

	-NJ tree (Figure 3) has not bootstrap values; therefore the significance of the clusters cannot be assessed. The genetic distances between populations would be better represented as a multidimensional scaling plot than a NJ tree of populations as in the present Figure. Such plot is simpler to interpret.

	-The conclusion that Mongolians are away from other clusters and that they have a unique genetic structure is not supported by the results or by previous studies, where Mongolians showed to be closely related with other Chinese groups (Zhang et al. 2015, Tao et al. 2018 and others). These previous results should be included in the discussion. And, if the authors want to demostrate that with these 19 X-STRs Mongolians have an unique structure, additional analysis should be carried out, for instance STRUCTURE analysis.

	-I have received two different abstracts, in the first, authors claim “a close affinity between Mongolian and Chinese Han populations”, that probably is more certain that the sentence in the other abstract “Mongolian groups had a unique genetic structure, which was significatively different from other populations”.

In conclusion, I cannot recommend the manuscript to be published in PeerJ in the present form. I would recommend that the manuscript was carefully checked, improving the analyses, the discussion and the language, and resubmission, providing the points raised were addressed.


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

Input:
Title: GENETIC POLYMORPHISMS AND FORENSIC EFFICIENCY OF 19 X-CHROMOSOMAL STR LOCI FOR XINJIANG MONGOLIAN POPULATION
Review round: 2
Reviewer: 3

Output:
Basic reporting: This study analyses the 19 X-STR markers included in the AGCU-X19 STR kit organised in 7 linkage groups. In this work, 267 individuals from the minority Mongolian group in China are genotyped. Allele frequencies, Hardy-Weinberg equilibrium and forensic parameters are calculated for intrapopulation analysis. Interpopulation comparison is also performed.
Along the text, the English language should be improved. There are grammatical and typo mistakes for examples in lines: 15 (are), 36 (and), 60 (humane), 127 (but), etc. There are also problems with the conjugation of verbs (line 85, were drew drawn). In general, there is a mix of present and past tenses that would be good to equal. Sometimes the phrasing is redundant and make is difficult to follow (lines 121-122, 128,131,etc.).
In the introduction, I have missed more information about the population, historical events and how this minority group has been in contact with the other groups in China.
The reference citing has some mistakes in the text (lines 87, 120, 125, 165, 193…) and in the references part (line 225, this reference has three authors, please revise it).
Tables and Figures:
Figure 1: There is a small square in the bottom right part of the figure that I cannot see what it is. Do the different green colours in the map have any meaning? If they have should be specified in the Figure legend, if not, I would recommend putting all the regions in white.
Figure 2: This figure is not relevant. Can be deleted.
Figure 3: This figure gives not clear information. Since it is based on genetic distances, I would recommend changing it for the genetic distances matrix, then the interpretation of the results will be much clearer to see.
Figure 4: There is a problem with the nomenclature of the populations. It would be good if the authors always use of the name of the country or the gentilic (name of the inhabitants) but not a mix of them (this also happen in lines 141-147 in the text). Besides, the inhabitants of Denmark are called Danish, not Dane.
Table 1: In this table would be useful to label those alleles which are described for the first time. There is also some “O” instead of zeros (Allele 29 of DXS101, allele 31 of DXS10135). The frequency of allele 31 of DXS6809 has a “/” instead of a dot.
Table 2: The expected heterozygosity is described as HE at the bottom and called He in the Table. The PI value has no sense. It should be higher as more polymorphic is a marker, revise.
Table 3: There is different number of decimals in the table, I would recommend having the same number for all the data in the table. The format (the columns are not wide enough for all the data) makes it difficult to read the table.
Supplementary Table 1: The table presents the raw data of supposedly 267 individuals (111 women and 156 men). The main problem is that there is just one row for sample. Considering that X-chromosome markers are studied, should be two alleles per woman for each marker, and just one for males. So, I can’t understand how these markers have been genotyped and what is worse, how all the analyses in this paper have been done.
Supplementary Table 2: In this table should be specified, which markers belong to each Linkage group (at least in the legend), since the naming of the linkage groups is different that the proposed in Argus X kit (Qiagen) and even different from the used in Yang et al. (2017).
Supplementary Table 3: The format of this table is different from the other (the decimals in the frequencies have commas instead of points).
As I have mentioned before, Raw data is supplied (In Supplementary table 1) but the data has no sense. For that reason, is difficult to be sure if the analyses done in this study are done with this Raw data, or there is a mistake just in the table. Please check it, because it could do none of the analyses trustable.
Experimental design: Even though is not the first study on X-STR in this population, this paper is the first using this specific kit. I have missed an explanation on which marker contain this kit, I only could find it in the Tables. The number of samples is adequate.
Apart of the issue of the Raw data, and so of the number of chromosome studied, It seem strange for me, why arlequin software is used for allele calculations, but other parameters as haplotype frequencies are made by hand.
Regarding allele frequencies the best way to pool male and female frequencies is using the formula ( (2*femalefreq + male freq) /3), since woman has 2/3 of the chromosomes of the population. I am not sure how authors have pooled male and female data in this study.
I also wonder if POPTREE calculations are made in the correct form. This software need the number of chromosomes studied and allele frequencies in every population to calculate the genetic distances. These genetic distances are much better to compare populations differentiation that the Fst distances marker by marker (Table 3).
Validity of the findings: Since the result are interesting mainly in forensic genetics, and in population genetics (however only eleven markers are used for that part) the discussion of the paper is poor. The discussion is mostly a repetition of the data presented in results (lines 106-122 and 184-188, for example). I would expect the comparison with other studies using this X-STR kit in other populations, and also results obtained with other set of markers in this Mongolian population.
Then the authors would have more argument to state whether the Mongolians are similar to other groups in China or if they are statistically different from all of them. Since the conclusions they state are not supported and well argumented with the data they present.
Additional comments: Apart from all the commented before there are some points that should be addressed:
Line 175: DXS10134 is present in the Argus kit, the one that the author have studied and is not in the Argus is DXS10162.
Line 39: I miss a reference here, it is not clear which is the previous study you are referring to.
Lines 49 and 56: The name of the region is Borxtala or Bortala?
Line 103: There is an extra “0.” In the Ho value.
Line 126-128: Do this population have any historical relation with the Somalis? If not, this sentence is not relevant.


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: HONEY REDUCES THE METASTATIC CHARACTERISTICS OF PROSTATE CANCER CELL LINES BY PROMOTING A LOSS OF ADHESION
Review round: 1
Reviewer: 1

Output:
Basic reporting: Abel et al in this manuscript have attempted to demonstrate the anti-metastatic activity of various honey from N. Zealand (Thyme, Manuka and Honey Due) using prostate cancer cells PC-3 and DU-145 cells.  The authors do not highlight any mechanisms to suggest their hypothesis. Reported literature already suggests honey as an anti-metastatic agent having ability to alter matrix metalloproteinases. Therefore the work performed by authors is not new to this area of investigation though their experiments have a direct approach suggesting the anti-metastatic action of honey.
Experimental design: The authors list the phenolic contents of honey. The authors have not accounted for the difference in the contents in all three honey: Thyme, Manuka and Honey Due . This might suggest the difference in the activity which may differ from the other.

Figure 1-5 have not used a vehicle control in their experiment to make a comparison.

The authors did not show the effect of honey on the proteins involved in metastasis (invasion or migration or adhesion) to effectively draw conclusions from a mechanistic point.
Validity of the findings: The authors have used two aggressive prostate cancer cell lines to prove their hypothesis. It would be ideal if they use some more metastatic cell lines along with a normal cell counterpart.
Additional comments: Additional experiments are needed to substantiate these results.


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: HONEY REDUCES THE METASTATIC CHARACTERISTICS OF PROSTATE CANCER CELL LINES BY PROMOTING A LOSS OF ADHESION
Review round: 1
Reviewer: 2

Output:
Basic reporting: Background is clear
Experimental design: Research question well defined and methods as well
Validity of the findings: Data is robust and the conclusion are well stated
Additional comments: In this paper, Baird present that Honey can reduce the metastatic characteristics of prostate cancer cell lines by promoting a loss of adhesion .

The manuscript is well written. Although the manuscript contains some valuable information obtained in vitro experiments. So, the article possess a practical value. In the background of the study the purpose of the article is clearly stated, data is robust and statistically sound, the conclusion is well stated and limited to supporting results. However some specific comments are cited below;

1.	Catenin is a protein, originally described as an integral part of intercellular adhesion system. Today it is considered an oncoprotein likely to activate proliferation and inhibit apoptosis. why it was not studied

2.	Since honey is mixture of phenolic compounds how it’s possible to exprime the concentration in nM ? Table 1 shows the concentrations of free and total phenols found in the thyme, manuka and honeydew honeys, as both µg/100g of honey and in nM (page 11line 255 and 256)

3.	Gallic acid concentration at 1082.88 ± 16.25 µg/100g is the equivalent of 10,8 mg/g it seems very low to exert any activity (page 11 line 259)
4.	How Sugars can act as antioxidants in the body, in a similar way to that reported for polyphenols as reported in page 19 line 450 (It was necessary to carry out the DPPH test to evaluate the antioxidant activity of honeys).

5.	The polyphenols found in honeys are the same found in propolis from the same regions. why the authors did not compare the honey and propolis activities ?

6.	Indeed, The honey inhibitory activity of MMP3 and 9 is low compared to that observed with a phenolic compound of Algerian propolis (Segueni et al, 20011 Planta Medica). Chicoric acid inhibits MMP-3, MMP-7 and MMP-9 with IC 50 of 0.63, 0.10 and 2.38 ug / ml

7.	why the authors did not realize western blotting to quantify the variations of the tumoral proteins like integrins, intracellular signalling proteins, fibronectin…?


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: SPATIAL CHANGES IN ZOOPLANKTON COMMUNITIES IN A STRONG HUMAN-MEDIATED RIVER ECOSYSTEM
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript has a clear structure which meets the requirements of an international journal. It is written in professional English, and the literature mentioned in the introduction is relevant and reflects the state of the art. This shows that the authors are experts in their field.
The figures and tables are relevant and are high quality, but some of them could be improved to make it easier to compare the sampling points with the flow direction of the river. Figure 3, Figure 5 and Figure 8 would be easier to understand if the order of the sampling stations were similar to the flow direction of the river Barycz, with RI, T1… starting on the right side of the graph.
Another comment concerns Table 2, where taxa, groups and, in the last line, the number of taxa are listed. It would be helpful to provide information regarding how the groups are related to the number of taxa. Maybe you could add a sentence in the table heading.
The raw data offered corresponded to the PeerJ policy, but please add the units.
Experimental design: The focus of the paper is to provide information about the development of metazooplankton in a river, based on river dams and the outlets of carp ponds. The findings are new, because previously published studies have only considered the influence of dams on the plankton community in large rivers, whereas the influence of ponds used for fish culture on the plankton community in rivers has not been considered until now. This means that this paper includes original primary research which is within the scope of the journal.
The standard of the investigation is appropriate, however,  the manuscript would be improved by adding some more information about the sampling sites: 1) information about why you used these sampling points (and not others), 2) information about whether the water samples from the free-flowing section were collected through successive sampling using a Lagrangian sampling strategy and 3) information about the interaction between the carp ponds and the regulated channels. Perhaps an example outline would be helpful.
Do you have information about the water from the outlets (e.g. plankton, nutrients and seston)? I had the impression from your description that the water samples are “only” from the channels.
Validity of the findings: The findings are new (see the paragraph regarding the experimental design) and meaningful for understanding the riverine community.
There are two additional points which could be discussed:
1) What influence the observed development will have on the Oder River is one point for discussion. Do you think that the River Barycz is a promotor of the plankton densities in the Oder zooplankton community?
2) Your findings could also be interesting for the European Water Framework Directive.

The data were analysed with appropriate mathematical methods, and the latest literature is included.

I recommend the publication of this interesting and important paper. The expected changes are minor ones which could help to improve the clarity and the scope of the paper.
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: SPATIAL CHANGES IN ZOOPLANKTON COMMUNITIES IN A STRONG HUMAN-MEDIATED RIVER ECOSYSTEM
Review round: 1
Reviewer: 2

Output:
Basic reporting: Abstract:
Should be rewriten. There is no one word on methods used, and results are not sufficiently described. If an aim of the paper was to examine the effects of carp ponds and dams, then what was a hypothesis? It should be given in Abstract and Introduction.
Table 2. Should be Brachionus calyciflorus and Scaridium longicaudum
Figures are not generally bad, but extremely difficult to understand if you have not enough time to spend on looking for explanations.
The manuscript is full of unnecessary introductory phrases as:
„regarding”, „it is worth”, „moreover”, „it must bepointed” etc.

The are also my remarks in the manuscript (pdf) attached
Experimental design: Well performed investigations sufficiently described in the manuscript
Validity of the findings: No comment
Additional comments: No 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: SPATIAL CHANGES IN ZOOPLANKTON COMMUNITIES IN A STRONG HUMAN-MEDIATED RIVER ECOSYSTEM
Review round: 1
Reviewer: 3

Output:
Basic reporting: There should be a more carefull use of English language. They way it is, the authors are trying to explain their point and repear the same thing in consecutive sentenses with different words. Proper language use will allow for this repeatition to be avoided, the text to become more concise and to the point.
Literature reference mostly OK
Structure of the article OK, raw data available
results relevant to hypotheses
Experimental design: The manuscript deals with a very interesting subject, within the Scope of the Journal, the effect of the dams on species distribution, especially zooplankton which lately understudied in freshwater ecosystems due to the fact that it is excluded by the WFD 2000/60. Also a very strong asset of this study is the large data set.
They define the research questions and apply standard methods sufficiently described
Validity of the findings: As I already stated,  a very strong asset of this study is the large data set.

But in the wordy text the authors fail to link their results with the conclusions
Additional comments: My specific comments can be found directly on the PDF


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: RECOMBINATION BETWEEN T-DNA INSERTIONS TO CAUSE CHROMOSOMAL DELETIONS IN ARABIDOPSIS IS A RARE PHENOMENON
Review round: 1
Reviewer: 1

Output:
Basic reporting: This paper is written clearly and easy to follow.
Experimental design: No comments
Validity of the findings: Technologically, genome deletion is significant not only for producing multiple mutants but also for functional analysis of non-coding sequence. Inspired by the authors’ early discovery about MEKK1, they designed a strategy based on recombination between neighboring T-DNA to delete genome sequence.  Deleting genome with high efficiency, even at the age of CRISP/Cas9, is still challenging. Although the result is negative, the efforts are helpful for us to understand T-DNA recombination.
Additional comments: The major points are:
1.	The term of “T-DNA directed chromosomal deletion” may be misleading. Recently a report showed heterogeneous T-DNA integration can result in major chromosomal rearrangements including large deletion (Hu et al., 2017). Accordingly, the so called “T-DNA directed deletion” in this work may occur at the stage of T-DNA integration or later stage (through recombination between T-DNA). I would suggest the authors cite that study and emphasize recombination process in the term.
2.	Generally, crossover between two homologous chromosomes at a specific site is rare. The finding about recombination between MEKK1 and MEKK3 may result from their unique T-DNA structures which may increase the recombination rate. In the original report about MEKK1 (Su et al., 2013), the authors suggested multiple copies of T-DNA were inserted head-to-head with left border at both sides. Whether features like multiple-copy insertion and orientation would influence the rate? This work did not tell us any structural features of T-DNA insertion sites.


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: RECOMBINATION BETWEEN T-DNA INSERTIONS TO CAUSE CHROMOSOMAL DELETIONS IN ARABIDOPSIS IS A RARE PHENOMENON
Review round: 1
Reviewer: 2

Output:
Basic reporting: The article is concisely written. The Introduction and Materials & Methods sections are well-presented and contain necessary information. The references appear appropriate and up-to-date. The structure conforms to the PeerJ standards. The figures, which all appear to be vector graphics, are of sufficient quality, well-labeled and also sufficiently described, as they are basically self-explaining. The figures are relevant for the understanding of the manuscript and are thus of importance, but the main findings are summarized in Table 1, rather. The inclusion of raw data is not really applicable to the current article, as the raw data is presented in table 1 as numbers, and there is no original data beyond that.
Experimental design: The current submission has a very simple question at its basis: Can T-DNA insertions, present in two different lines, be exploited for the creation of deletions via recombination between the homologous sequences (the T-DNAs) upon crossing of the respective lines? This had been observed in a previous report (Su et al 2013), where a deletion of ~ 25 kb had been detected upon crossing two T-DNA insertion lines.
In the current study, T-DNA insertion lines with insertions flanking FUS genes were crossed to provide a visual output of such recombination events, by elimination of the respective FUS gene(s), visible by photomorphogenesis in dark-grown seedlings (vs etiolated seedlings). The phenotype is well-chosen, as fus mutants can be easily identified among wild type, etiolated seedlings, and also the repertoire of FUS genes allows testing of different genes/T-DNA insertions in a unified experimental design.
The underlying questions is of interest, as the proposed technique might represent a valid approach to eliminate clustered genes and/or gene families, and fits the aims and scopes of the journal. The research question is very simple, as stated above (can the proposed approach be used to create deletions?), and well-defined. The analysis appears solid and the methods are well-described.
Validity of the findings: The findings are presented in a clear manner, although the outcome surely is somewhat disappointing for the authors: The tested approach does not work; the previously observed chromosomal deletion was a rare event picked up by chance, apparently. The presented data appears robust, as several different crosses / T-DNA insertion lines were tested. Statistics are not really relevant, as no recombination events were detected. The conclusion that such recombination events are rare is clearly stated and answers the research question: This approach is not very promising. Alternative strategies for obtaining such deletions via sequence-specific nucleases (CRISPR/Cas) are indicated in the conclusions section, which provides guidance for those intending to generate deletions.
Additional comments: Overall, the article is well-written, and provides a clear answer to the previously stated hypothesis that adjacent T-DNA insertions might give rise to chromosomal deletions upon crossing of respective lines. Considering that the previous statement that such phenomena could be exploited for genetic engineering yet remained unanswered, it is important to have this information available in the public domain. However, a few aspects should be clarified and/or corrected:

- gene names should be consistently written in italics, also e.g. Arabidopsis thaliana or Agrobacterium tumefaciens as species names should be in italics

- there are a few typos, as for example CONTITUTIVE PHOTOMORPHOGENIC in line 164 (constitutive),

- as far as I am aware (although I cannot provide a suitable citation here), inverted repeats (T-DNA insertions in opposing senses) rather than direct repeats (T-DNA insertions in identical orientations) may give rise to the "desired" recombination events. I think that it would therefore be important to include this info in the text: What was the orientation of T-DNAs in the previous report (Su et al 2013), and which orientations were tested in the genetic screen presented here? For Su et el., this can be mentioned in the text, and indicated for combinations tested here in figure 2.


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: NOVEL INFLAMMATORY CELL INFILTRATION SCORING SYSTEM TO INVESTIGATE HEALTHY AND FOOTROT AFFECTED OVINE INTERDIGITAL SKIN
Review round: 1
Reviewer: 1

Output:
Basic reporting: This manuscript reports a histopathological comparison of interdigital skin lesions of healthy and footrot affected sheep.
The manuscript correctly matches with the scope of the journal.  It is correctly described and English language is properly used. The general structure is according to the standards of the journal and is clear for the reader.
Figures are of great quality and represent the main findings that the authors stated in the manuscript.
Minor editing points of criticism have been detected in lines:
236 to 238: necrophorum (Egerton et al. 1969). Parakeratosis may trigger the release of pro-inflammatory cytokines such as IL-1β (Chang et al. 1992) which in-turn MEDIATES the recruitment of inflammatory cells.
278. 278 polymicrobial disease of bovine feet, digital DERMATITIS, no association was found between
358: Total DNA was extracted from sequential 40 µm horizontal sections of 6mm punch biopsies of from…
360: enumerate total bacterial DNA AND detect…
Experimental design: The authors should reconsider the title of the manuscript because it doesn’t reflect the findings of the study. What is novel in the research is the “inflammatory cell infiltration scoring system”. The rest of the study shows that the histopathology findings are quite similar both in healthy and affected sheep. Nevertheless, the title can also include all the descriptive work that has been done.
About this second affirmation, is missing in the discussion part why the histopathological findings are similar in healthy and footrot affected animals. Are the clinical findings correctly diagnosed?. Are we underdiagnosing footrot animals?. The animals are suffering an subclinical disease?. Please, reconsider all this topics and discus in the manuscript.
The investigation presented is in agreement with the scope of the journal. The methodology is rigorously described and the experiments are reproducible.
Validity of the findings: Although results are presented whose findings are inconclusive, it is considered that it can help the scientific community to continue with this study in a rigorous manner.
The data presented is strong and the statistical analysis is well applied.
Conclusions are well stated.
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: NOVEL INFLAMMATORY CELL INFILTRATION SCORING SYSTEM TO INVESTIGATE HEALTHY AND FOOTROT AFFECTED OVINE INTERDIGITAL SKIN
Review round: 1
Reviewer: 2

Output:
Basic reporting: The paper by Agbaje et al. studies by histopathological methods natural cases of ovine footrot together with etiological and some immunological approaches in relation with the lesions. Not too much work has been done on this particular disease, especially in the pathogenesis of the lesions. In this sense, the paper would contribute
This is a well-written and clearly presented paper that includes representative and high-quality figures that clarify the histological findings reported in the paper. The literature review is accurate and up-to-date.
Experimental design: There are some concerns concerning this paper:
-	The aim of the study is not clear at all, more than performing a grading of histological lesions.
- It is not clear enough the aim prosecuted by the study of IL1beta mRNA expression in association with the lesions. Considering the samples and material, they should have made an attempt to go further into pathogenesis of the lesion, as they try to, when measure IL1betta expression. It could be examined the relationship of its expression with the presence of different cell populations, location of lesions, etc.
-	Why the authors have chosen this particular cytokine and no other, including inflammatory mediators, that would have given more information on the development of the lesions in relation with the host immune response?
-	The methods for the histological studies are classical and well-presented. In relation to HE staining, why the authors have decided to treat the samples with acetic spirits and ammoniated water? Which are the advantages respect to conventional HE staining?
-	Methods for bacteriological and statistical analysis seem to be appropriate.
-	Which criteria have been taken into account for the gross classification of the samples into healthy and footrot affected? Please, state in the material and methods section.
Validity of the findings: The main lack of this work is the scarce novelty of the scoring system the authors have proposed. They classify the inflammatory infiltrate according to the intensity, following a very classical pathological scoring system. Moreover, there are other facts that they also consider, but separately. Putting all together, they do not find any difference between healthy and diseased samples. All the histological findings considered are classical and have been previously well described and reported, as is mentioned in the paper.
 The authors should have made a more ambitious attempt of classification putting considering all the changes together and giving a particular score to each animal, and try to find a cut-off value to discriminate between healthy and footrot affected animals. This would help to the diagnosis and future studies on pathogenesis.
Regarding the role of IL1beta, the authors have just measured the mRNA expression of this cytokine. It would be of interest to assess the protein expression by immunohistochemistry, in relation with the lesions.
The study made on the presence of different bacteria in association do not provide new or relevant findings. The role of the different bacteria have been elucidated in others papers of the group. The presence in the various depths of the lesion is the only new finding, but its relevancy is not well-stablished.
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: RESPONSES OF AN OYSTER HOST (CRASSOSTREA VIRGINICA) AND ITS PROTOZOAN PARASITE (PERKINSUS MARINUS) TO INCREASING AIR TEMPERATURE
Review round: 1
Reviewer: 1

Output:
Basic reporting: This manuscript explores the effect of increasing air temperatures on the interaction of the eastern oyster (Crassostrea virginica) with the protistan parasite Perkinsus marinus.  The number of infected oysters, the parasite load and oyster immune responses were determined.  In addition, oyster mortality was monitored.  Results show that at temperatures above 35°C, host survival decreases and oyster hemocytes are not affected until lethal temperatures are reached.  In addition, P. marinus infection intensity increased at 35°C compared to the other temperatures.

The language is clear and unambiguous, the introduction and background are appropriate and the literature is cited appropriately.  All figures and tables are relevant and well labelled.  Raw data is supplied with the exception of temperature variation in the exposure chambers.
Experimental design: The research question is well defined and relevant.  Methods are described in sufficient detail but see following comments.  The experimental chamber description is hard to follow.  Is it possible to provide a photo or diagram of the setup?  Did the temperature vary more than 0.5°C?  The use of a temperature data logger is indicated but there is no mention of the temperature variation in actual experiments.
Validity of the findings: The conclusions are valid, and data is robust.  Conclusions are well stated and support the results.
Additional comments: The authors state that the average surface air temperature will increase from 0.3 to 4.5°C by 2090 and that the average in the area under study is 33°C.  Why then were temperatures up to 53°C used for the experiments as this is well above the 37.5 projected temperature.  The reasons for the specific temperatures chosen should be discussed.

The authors speculate that increased temperatures may in fact cause an increase in immune responses of the host.  Isn’t it also the case that this increase in immune response is harmful in and of itself?  Isn’t it possible that an increase in immune response damages the host more than it protects the host?

Line 321 – remove are considered


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: RESPONSES OF AN OYSTER HOST (CRASSOSTREA VIRGINICA) AND ITS PROTOZOAN PARASITE (PERKINSUS MARINUS) TO INCREASING AIR TEMPERATURE
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript by Malek and Byers describes interesting results on the potential effects of rising atmospheric temperatures on host/parasite interactions and survival. The study will be of broad interest because of the not only because of the topic, but also because of the host is an economically and ecologically important species and the pathogen is a member of a genus that infects bivalves worldwide. The design and range of experiments, and the variety of methods including pcr, rftm and tests of phagocyte activity, is a strong point of the study. The ms is clear and well written. Figures and tables are appropriate. I recommend acceptance after very minor revision to address the issues raised below.
Experimental design: The design and analysis of experiments is very good. The variety of approaches to the problem is a strong point of the study. I have only 2 minor comments that should be addressed:

line 192. Add the explanation that using gill and mantel tissue in addition to rectal tissue increases the probability of detecting low-intensity infections.

line 199. Could Perkinsus be dormant – not dead? Omitting the highest temperature treatments would still be the appropriate step, but the implication could be different if infections could become active again as temperatures cooled in autumn.
Validity of the findings: The data are robust and the interpretation of the findings is sound.  The conclusions are logical extensions of the results and are not overly speculative.

Some minor suggestions for the authors to consider:

For Perkinsus infections, increased phagocyte activity does not always correlate with decreasef infection prevalence (Keppel et al., 2015). Please include a comment on this.

Volety has worked on temperature effects on this host/parasite system. This is relevant even though it focused on subtidal oysters.

line 314. For the sentence:  ‘As temperatures rise in the intertidal, oysters may suffer not only from temperature-related mortality in the short- term, but also from higher intensity P. marinus infections over the long-term.’ – please explain what is meant by short- and long-term a bit more clearly. Do you mean days (survival) to months (disease) or over decades as temperatures rise.
Additional comments: No additional comments