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: MANAGING MIGRAINE WITH OVER-THE-COUNTER PROVISION OF TRIPTANS: THE PERSPECTIVES AND READINESS OF WESTERN AUSTRALIAN COMMUNITY PHARMACISTS
Review round: 2
Reviewer: 2

Output:
Basic reporting: Overall, this was an interesting paper with information presented logically and coherently throughout. The writing is of a very high standard and previously identified typographical errors have been rectified.

The introduction to the paper provides a detailed background to triptan deregulation with appropriate references cited. The authors have addressed my previous comment on providing a summary of products currently licensed OTC for migraine management in Australia.

No issues with any other aspects of ‘basic reporting’
Experimental design: Interesting and topical area of research with appropriate methods adopted to meet the identified primary objectives of the study.
Validity of the findings: Findings have been clearly identified and appropriately discussed. My previous comment regarding restating of results in the discussion has been addressed and the manuscript updated accordingly. Authors have also expanded the discussion to consider the potential impact of triptan deregulation and have discussed other relevant studies in the field.
Authors have clarified in their response why results from ‘additional comments’ have not been presented in the manuscript. Their rationale is sound their methods are appropriate.

This is an interesting study which has been very well presented and I anticipate it will be welcomed in the field, with particular relevance for those working in Australia.
Additional comments: I recommend this paper for publication.


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

Input:
Title: A NOVEL APPROACH TO IMMUNOAPHERESIS OF C3A/C3 AND PROTEOMIC IDENTIFICATION OF ASSOCIATES
Review round: 1
Reviewer: 1

Output:
Basic reporting: I don’t consider myself to be qualified enough to estimate the quality of English in the text of the manuscript.
Experimental design: No comment
Validity of the findings: Except for the points for revision listed below, all experimental data are presented quite clear and properly discussed, raw data are supplied, conclusions are well stated.

Points for revision:

1) For me, the most intriguing result of the work presented in the manuscript is the data on the identity of low molecular weight antigens for 3F7E2-mAb from blood serum revealed after the immunoapheresis procedure. In the manuscript, these components are designated as ‘C3a’ and ‘C3a missing a C-terminal part’ (line 275, 282-283; descriptions for Figure 4 and Table 1); the second one is discussed as presumable ‘C3a degradation product’ (line 283). However, taking that there was no mistake in Table 1 preparation, both of these components could not be referred to as C3a. The C3a molecule has an arginine 748 on its C-terminus (or alanine 747 in the case of C3a des-Arg), while both revealed components have an extension 749-764 according to data presented in Table 1. In my opinion, the smaller component is better to describe as ‘missing a fragment/piece from C-terminal half’ but not as ‘missing a C-terminal part’. Both these components should be referred to as ‘C3 degradation products’. To my knowledge, the presence of such C3-derived species in human serum was not reported previously, and it should be discussed in the ‘Discussion’ section.

2) The data of the proteomic research of C3-interacting proteins presented in the manuscript are undoubtedly of high fundamental importance. That is why they should be described in more detail.
I recommend outlining in Table 2 which of the C3 partners were known previously and which were revealed for the first time.
To distinguish between specific and nonspecific interactions, the authors introduced cut-off criteria, which allowed to describe 39 proteins as specific C3-interacting partners among 278 ones identified totally. It seems worthy to list the remaining 239 proteins (or at least those located in the right half of the plot in Figure 5) in a separate supplementary file.

3) In this work, 3F7E2-mAb was demonstrated to have a complement inhibition activity. It is stated in the manuscript that ‘inhibitory potential of 3F7E2-mAb is shown to be dose-dependent’ (lines 260-261; description for Figure 2). In my opinion, the presented data are not enough to conclude a dose dependency. Only two concentrations of the antibody were tested, and they both revealed identical near-complete inhibitory effects according to Figure 2. Therefore, these results should be discussed more carefully or additional data confirming dose dependency should be presented.

4) Minor editing
Perhaps, it would be better to spell down some abbreviations, which are not quite commonly used, e.g. TPBS (probably, Tween-PBS; line 133), TEA (probably, triethanolamine; line 174).
It seems that ‘3’ is missed in the sodium azide formula (line 178).
From the description for Figure 2, it seems that lanes 1 and 2 represent identical samples, both loaded on the gel in the same volume. However, these lanes are accompanied by ‘+’ signs of different size. Thus, it should be corrected (or description for the Figure should be clarified).
Additional comments: This is an interesting and definitely significant study devoted to the development of a novel approach to the inhibition of complement activation, which is a serious medical task. The 3F7E2-mAb  antibody seems to be promising for clinical applications aimed at limiting excessive complement activity both as a direct complement inhibitor, and as an immunoaffinity reagent for the apheresis procedure


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

Input:
Title: A NOVEL APPROACH TO IMMUNOAPHERESIS OF C3A/C3 AND PROTEOMIC IDENTIFICATION OF ASSOCIATES
Review round: 1
Reviewer: 2

Output:
Basic reporting: Abstract needs to be structural. Please refer to authors’ instructions on https://peerj.com/about/author-instructions/ for details.

Abbreviations should be written in their complete form when they are mentioned for the first time in the text. e.g. C3aR in line 52, IL-2 in line 62, TPBS in line 133, nL in the 1st line of figure 1’s legend.

Names of providers and manufacturers of consumables and instruments should be stated in parenthesis. e. g. “Roche Molecular Biochemicals, Mannheim, Germany” in different places throughout the text, “BioRad, Germany” in line 149, “Abcam, Cambridge, UK” in line 150.

In line 166, “und” should be changed to “and”.
Experimental design: Tissue culture (line 117) needs to be explained in more details including its culture media, timing, temperatures and etc.
Validity of the findings: No comment.
Additional comments: No additional comments


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

Input:
Title: A NOVEL APPROACH TO IMMUNOAPHERESIS OF C3A/C3 AND PROTEOMIC IDENTIFICATION OF ASSOCIATES
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: In the revised manuscript, all the questions raised and the problems noted were taken into account, all the necessary changes and comments were made. On my opinion, now the manuscript is suitable for publication without further revision.
Additional comments: No additional comments


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

Input:
Title: A NOVEL APPROACH TO IMMUNOAPHERESIS OF C3A/C3 AND PROTEOMIC IDENTIFICATION OF ASSOCIATES
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: No additional comments


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

Input:
Title: SIGNIFICANCE OF TP53 MUTATION IN BLADDER CANCER DISEASE PROGRESSION AND DRUG SELECTION
Review round: 1
Reviewer: 1

Output:
Basic reporting: There are no issues with the basic structure of the manuscript.

However, there are issues with language usage throughout, particularly incorrect word use (e.g. line 132, "scope"; line 76, "astronomical"), lack of definite/indefinite articles, incorrect word forms, & singular/plural disagreement.  Use of informal language, such as "dig out", "nowadays", & "what's more" should be avoided, as well.  There are also spelling (e.g. line 146, "oxigen"; line 229, "cacner") & punctuation (e.g. line 164, ",.") errors in need of correction.

Citation of appropriate background literature early in both the Introduction & Discussion is inadequate, as well.  It would also be helpful to cite the appropriate Figs. when Results are directly referenced in the Discussion (e.g. lines 205-226).

Finally, abbreviations for "GO" & "KEGG" analysis need to be defined.
Experimental design: The study appears to be within the scope of the journal.  Its hypothesis & methodology are also adequately described.

However, the authors missed a huge opportunity to further compare MIBC vs. NMIBC disease patients.  After Fig. 1 introduced a very interesting, significant(?) difference in their TP53 mutation frequencies, the remainder of the paper does not distinguish these different types of bladder cancer at all.  This is a major weakness of their study.
Validity of the findings: The biggest issue with this manuscript lies in the poor presentation & discussion of its findings, which prevents assessment of their true validity.

Fig. 1.  The 3 panels of this Fig. are not explained nearly well enough in either the legend or Results section.  For example, the color coding in the odd, single bars of panels A & C is very difficult to read.  The mutation frequencies should be associated with numerical values on an axis, not just a qualitative bar chart.  Labeling panel A = 50% & panel C = 21% does not provide any true quantitation.  There is also no key to the color coded regions of panel B.  For example, what is "P53_tetr_"?  This is apparently a TP53 gene/protein(?) diagram, although this is not explicitly stated anywhere.  Finally, why wasn't another TP53 gene/protein diagram generated for the NMIBC data in panel C?

Table 1.  "Age" should be "Mean Age"?  Do P-values refer to the Stage/Grade totals?  If so, then those total values should be provided.  If not, how were they calculated?

Fig. 2.  The P-values are not clearly associated with panels B & C.  The color keys labeled generically as "....Query Genes" need to be edited to show "Wild-type" or "Mutant".

Fig. 4.  This data requires much more work on both its explanation & presentation.  The analyses for 16 'functional gene sets' are shown in Fig. 4, while the Results state that 13 were significantly enriched.  What about the other 3 sets in Fig. 4?  Furthermore, the enriched 'mTORC1 signaling' gene set mentioned in the Results does not even appear to be shown in Fig. 4.  In addition, there is no way that readers unfamiliar with this particular analysis could interpret this data without far more explanation of its presentation.  Most importantly, what are the criteria used to establish 'significant enrichment'?  As presented, the 16 traces in Fig. 4 appear to be quite similar.  It may be helpful to show an example of a gene set that was not significantly enriched.  If some of the 16 shown are not significant, then they should be labeled as such & separated from the significant sets.

Fig. 5.  How do the 'functional gene sets' presented in Fig. 4 mesh with the DEG's identified in Fig. 5?  This needs to be addressed explicitly in the Discussion.  As it is, these results don't appear to be linked in any useful context.
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: SIGNIFICANCE OF TP53 MUTATION IN BLADDER CANCER DISEASE PROGRESSION AND DRUG SELECTION
Review round: 1
Reviewer: 2

Output:
Basic reporting: The paper by Wu and colleagues is clearly written and understandable.  Since TP53 is the most frequent mutated gene in human tumors it is very relevant.
Experimental design: The experimental design seems reasonable. However more details would be helpful to learn how the  GDSC database concerning drug sensitivity was searched.
Validity of the findings: No comment.
Additional comments: The discussion should contain a section dealing with relevant clinical studies. It is essential to know if clinical studies with Mitomycin, Doxorubicin or Gemcitabine have been performed and how the outcome was. This should also be put in the context of TP53 mutation.


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: SIGNIFICANCE OF TP53 MUTATION IN BLADDER CANCER DISEASE PROGRESSION AND DRUG SELECTION
Review round: 1
Reviewer: 3

Output:
Basic reporting: English editing is recommended.
The literature references were insufficient. Other groups have reported p53 mutations and p53 related pathways as driver mutations in bladder cancer: for example Cazier et. al. (2014), Nature Communications, and this paper and others were not referenced.
Experimental design: The experimental design and methods were appropriate. The novelty of the study was not obvious, and much of the relevant p53 related literature that has published similar findings was not cited.
Validity of the findings: The p53 literature required more careful consideration and many of the study's findings have been published or would be expected based on the literature. The drug related aspects of the study could be relevant to treatment of bladder cancer patients with mutant p53, but a more careful analysis of other confounding factors is required. The affect mitomycin-c, doxorubicin and gemcitabine has on cells with and without mutant p53 is reasonably well established, but again this literature was poorly cited. Mitocmycin-c has been reported to induce p53 independent cell death. Doxorubicin largely has a p53 dependent cell death response, some bladder cancers have enhanced MDM2 expression and this could be a confounding factor to conclude that doxorubicin would be a suitable drug to target tumors with mutant p53. The expression of RUNX2 could confound the results related to p53 mutations and gemcitabine treatment. Wild-type p53 is stabilized at the protein level and many p53 mutations produce a stable protein, so the analyses related to TP53 expression are limited without an analysis of p53 protein levels, or the expression of downstream targets of p53 such as CDKN1A.
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: ALGORITHMS FOR EFFICIENTLY COLLAPSING READS WITH UNIQUE MOLECULAR IDENTIFIERS
Review round: 1
Reviewer: 1

Output:
Basic reporting: * Lines 11-12, the sentence "reads on their finding reads" is unclear and should be rephrased.
* Line 15, the author says "We formulate the problem of deduplicating UMIs", I'd advise using the word "reformulate" as the problem of UMI deduplication was already formulated by Smith et. al. [1] on which the author is proposing the improvements.
* Lines 17-18, the author says "which can deduplicate over one million unique UMIs of length 9 at a single alignment position in around 26 seconds.", this sounds great however it's unclear how much computational resource like threads / CPU / memory was used.
* Line 23-24, the author says "By ligating a short, random UMI sequence onto each strand of DNA fragment before PCR amplification, sequenced reads with the same UMI can be easily identified as PCR duplicates.". The author has attempted to summarize a lot of details into one sentence making it dense to read. This can use a little more detail as it motivates the usefulness of the problem of UMI deduplication of which the author is proposing the solution.
* Line 26, Author should cite a paper which uses iClip data for UMI deduplication.
* Line 28-29, "However, due to sequencing and PCR amplification errors, sequenced reads may contain UMI sequences that deviate from their true UMI sequences before PCR amplification", Author should cite relevant paper for this claim.
* Line 33-34 "they must first be aligned to a reference genome, ", this claim is not true, Vasilis et al. [2] and Srivastava et al. [3] aligns to transcriptome for performing the deduplication.
* Line 36, 47, 244 multiple latex citation issues " umis [umis], gencore [2], fgbio [fgbio], Picard [Picard Tools]" and at many other places. The author should recheck the manuscript for all such errors.
* Line 40-42, It's unclear if the author is talking about single-cell UMI deduplication or in-general PCR deduplication of reads. Author's claim " a consensus read must be extracted for each group of reads" is not always true for the single-cell for example in [2, 3] author generates the gene count directly i.e. they don't assign a consensus to read. Assigning "consensus read" and "read counting" are two different yet related computational problem.
* Line 44, "many works focused on discussing algorithms that make the UMI deduplication process more efficient.", It's unclear which efficiency the author is talking about accuracy, resource usage?
* Line 48-49 Starting of the sentence should be as Smith et. al. [18] instead of just the citation number. Also, the sentence is incomplete " in their UMI-tools *method*".
* Line 50 "That allows it to avoid the issue of overestimating the number of true UMIs from directly counting unique UMIs.", why is there an issue of overestimation, needs more elaboration?
* Line 51, "still being error-tolerant" what kind of errors, both SNP and indels?
* Line 54, "For simplicity, we only consider the problem of deduplicating UMIs at a single alignment position." what are the implications of these assumptions ? can it be generalized?
* Line 70, "Most deduplication algorithms attempt to build graphs", cite one such algorithm.
* Line 72-74, "There are three main deduplication algorithms for identifying groups of unique UMIs based on G", Author should be more clear about the mentioned graph-based algorithms here as Srivastava et al. [3] also proposed graph-based deduplication algorithm which is similar to "directional" but not same and is not mentioned in this context.
* Line 79, "and v is not removed", its unclear v is not removed from where? I think this sentence can use some rephrasing to make it more clear.
* Line 150, "Most of them are special data structures that speed up REMOVE NEAR", the data structure is "special" in what sense? It seems unnecessary superlative as the data-structures used were previously used in other problems too having said that I don't mean to question the novelty of the application.
* Line 228-234, More clarity can be added by stating that the n-gram approach follows the pigeonhole principle [4].
* Line 416, It's unclear what does other means by dynamic query problem?
* Line 418, "implemented with this interface no change to their results. " It's unclear to me if the author wants to sell the paper as a proof-of-concept or wants users to adopt the data structure library built in Java. If later, then there should ideally be some form of evidence be provided that compares the output of UMI-tools and UmiCollapse suggesting there is no difference in the generated output, it doesn't have to be formally shown in the paper but I'd recommend having the analysis made available may be as an ipython notebook?
* Table 1. It is fine to use `Combo` and `Subseq.` as the shorter representation of Combination and Subsequence but it should be mentioned in the caption or the text the meaning of these shorthands.
* Multiple problems in the References like line 466, 467.

[1] Smith, Tom, Andreas Heger, and Ian Sudbery. "UMI-tools: modeling sequencing errors in Unique Molecular Identifiers to improve quantification accuracy." Genome Research 27.3 (2017): 491-499.
[2] Ntranos, Vasilis, et al. "Fast and accurate single-cell RNA-seq analysis by clustering of transcript-compatibility counts." Genome Biology 17.1 (2016): 112.
[3] Srivastava, Avi, et al. "Alevin efficiently estimates accurate gene abundances from dscRNA-seq data." Genome Biology 20.1 (2019): 65.
[4] https://en.wikipedia.org/wiki/Pigeonhole_principle
Experimental design: * Although not a requirement but the manuscript could benefit from an independent validation of the simulated data generated using the tool from Sarkar et. al. [5].

[5] Sarkar, Hirak, Avi Srivastava, and Rob Patro. "Minnow: a principled framework for rapid simulation of dscRNA-seq data at the read level." Bioinformatics 35.14 (2019): i136-i144.
Validity of the findings: no comment
Additional comments: Author discusses the reformulation of the UMI-deduplication problem using efficient data structure which improves the overall running time of the current algorithm by a significant margin. Apart from minor edits in the background section, I find the paper to be very well written however author should consider revising the manuscript with some more experienced latex user as there are multiple syntactical problems throughout the manuscript. I find the methodological contribution in the manuscript to be very interesting and useful for the community but I am little curious about the validations as they are performed on a custom simulations. The manuscript could benefit from an independent validation of the simulated data generated through already published tools.


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: ALGORITHMS FOR EFFICIENTLY COLLAPSING READS WITH UNIQUE MOLECULAR IDENTIFIERS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript is excellently written, with good, professional English through-out. The text is to the point, avoiding unnecessary verbosity without excluding the relevant explanation. There is a sufficient review of the literature to allow the reader to appreciate the problem at hand and the work proceeding this one. One small quibble, the author cites Schirmer et al (reference 16) as an example of previous work on the UMI deduplication task, but although the paper does use UMIs in one of its library preparation methods, it doesn’t really examine the task of deduplicating reads based on these. The references are in an odd style, with numbers for peer-review literature references, and names for citations to grey-literature such as github repositories. However, I understand the journal will reformat these?  Figures and tables are well presented, with a sufficient font size, and acceptable legends. There is little “raw data” to speak of for this work and so raw data sharing is not really relevant.
Experimental design: The work is primary research, investigating the performance of a range of different algorithms/data structures, some novel, and thus fits within the Aims and Scope of the journal. The research question (which data structures allow the fastest performance in the UMI deduplication problem) is well defined and the study clearly addresses the question.

The methods are well described in terms of the algorithms that the author implements, but parts of the process are less well described. I don’t see any reference to code used in the study beyond the code of the tool itself. For example, the author might want to include the code that generated the simulated datasets used in the study. Benchmarking takes place against UMI-tools, but the author does not describe the version of UMI-tools used, nor the command-line. This is important, because, as the author notes, early version of UMI-tools used the “naïve” approach, while later versions implemented a version of the n-grams structure described in the manuscript. Also various command line setting can have a large effect on the performance of a tool.
Validity of the findings: The author compare their different algorithms to each other and to UMI-tools on simulated data. UMICollapse and UMI-tools are also compared on two real datasets.  One thing which I did not understand about the simulated datasets is the number of unique UMIs. For example, the authors state that they generate a set of simulated UMIs by taking 10^5 different 10-bp UMI sequences and generating 20 random sequences that are within 1 edit of each of these 10^5 “centre” UMIs. The author says this generates 1,550,871 unique UMIs. But unless I’m mistaken, there are only 1,048,576 possible 10nt UMI sequences. Is each “centre” UMI and its child sequence fed into the algorithm separately. If so this should be explained. This seems unlikely however as the data in Table 4 is described as “many unique UMIs at the exact sam alignment position”, but has one position with 1,114,686 unique 9nt UMIs, when there are only 262,144 unique 9nt sequences. More likely the author has counted some sequences twice when a single change from two centre UMI results in the same new sequence.

The author might also like to comment on the method for generating the simulated datasets, which results in a particular structure, where each “child” UMI is only one hop away from the parent UMI, and there are no chain of child UMIs, such as AAAA -> AAAT -> AATT, or other, more complex network structures. Might this be why UMI-tools is only 3x slower than UMICollapse in the data presented in Table 3, where constructing the network might account for less of the time spent?

The author convincingly shows that on the simulated dataset, at a single position, the n-grams and BK-tree structures outperform other structures, and that their combination, n-gram BK-tree performs best of all, being 2x as fast as n-grams and 20x as faster as BK-tree on the largest number of UMIs tested on all three. UMICollapse is also orders of magnitude faster than UMI-tools on simulated data from a single, highly dense, alignment location. If this is due to the algorithm/data structure used, then it is surprising because UMI-tools uses a version n-grams, which Table 1 shows to only take 2x as long as the n-grams BK structure used for UMICollapse in Table 4. Thus I don’t think the claim that the massive improvement over UMI-tools is due to the data structure used can be entirely supported. What role might other factors, such as the edit_distance calculation routine, the efficiency of code implementing the same algorithms or the choice of programming language make in this difference?

The authors do not report whether their algorithm returns the same results as UMI-tools. Although not returning the exact same reads is understandable, the author should report if the same or similar number of reads is returned for both the simulated and real datasets.

The discussion mentions the memory efficiency of the different algorithms, but no data is presented in the manuscript. The author should make clear if the discussion of memory usage is based on a theoretical assessment of how much memory the different algorithms should use, or a measurement of the actual memory usage.
Additional comments: In several use cases it is useful to not just select and output a single read for each group of reads with a similar UMI, but the output all reads, tagged with the “parent UMI”, or UMI group that have been associated with. Because the methods described in this paper imply the UMI network, rather than explicitly constructing it, it is not clear to me if this is possible, particularly if a child UMI is several steps removed from its parent. Could the author clarify which data structures/algorithms would be compatible with this?

In their description of the directional adjacency algorithm, the author refer to the inequality 2f(v)+1≤f(u). In fact the inequality in the UMI-tools paper is 2f(v)-1≤f(u). It is not clear if this is a typo in the paper or if this is in the code.

I have successfully downloaded, installed and run the UMICollapse software using the example data. The authors are to be congratulated on the easy installation.

Summary

The size of datasets currently being used is getting to the point where it is becoming impossible to use error correcting UMI deduplication algorithms due to their time and memory requirements. This is thus an important and timely contribution. The author is to be congratulated on a through and insightful study. I recommend publication with minor alterations. In order of importance:
	1. The author should clarify the status of the frequency inequality.
	2. The author should explore the similarity/compatibility of the results from UMICollapse and UMI-tools as well as just the timings.
	3. The author should clarify the number of unique UMIs in the simulated datasets
	4. The author should re-consider their claim that the bottleneck in UMI-tools is definitely in the edit distance calculations, and at least discuss the fact that their own computations show that the n-grams bk-tree structure is only 2x fast than the n-gram structure, but UMICollapse appears to be orders of magnitude faster than UMI-tools.
	5. The methods, outside the description of the data structures/algorithms should be more detailed. Most importantly the version of UMI-tools must be specified.

The following would improve the manuscript but might be considered optional:
	1. The author could discuss the scheme for simulating data and how that might affect their comparisons.
	2. The author could discuss if these algorithms are suitable for assigning a parent UMI to every read and outputting that as a tag on the read.
	3. The authors should clarify the status of the memory usage estimates, and include real usage estimates for the difference algorithms if possible


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: ALGORITHMS FOR EFFICIENTLY COLLAPSING READS WITH UNIQUE MOLECULAR IDENTIFIERS
Review round: 2
Reviewer: 1

Output:
Basic reporting: All my major concerns have been addressed by the author.
Experimental design: N/A
Validity of the findings: N/A
Additional comments: I'd like to thank and congratulate the author for thoroughly replying and improving the manuscript. I think the manuscript is in a better shape and ready to publish, provided the formatting is fixed by the journal.


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

Input:
Title: ALGORITHMS FOR EFFICIENTLY COLLAPSING READS WITH UNIQUE MOLECULAR IDENTIFIERS
Review round: 2
Reviewer: 2

Output:
Basic reporting: No comment
Experimental design: The author has clarified that the reason for the large number of UMIs at a single positions is that they simulate sequences from {A,T,G,C,N}. Could they common on how N bases are handled in edit distance calculations? Is N to any base seen as a match or a mismatch? They say that there bit-shifting edit distance method uses 3-bits per base, but this must be different for UMIs that contain Ns? Indeed, none of the discussion of methods seems to recognize that Ns might be included.
Validity of the findings: No comment
Additional comments: The author has addressed most or all of my previous comments. Apart from the minor quibble above I believe the manuscript is now ready to publish. If the above is clarified I do not envisage it being necessary to review the manuscript again.


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: LIMITED INITIAL IMPACTS OF BIOMASS HARVESTING ON COMPOSITION OF WOOD-INHABITING FUNGI WITHIN RESIDUAL STUMPS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The ms compares the effects of tree-length vs. full-tree harvesting on fungal diversity of the remaining stumps. I highly appreciate that although negative, the results are reported in detail. The text is well-written scientific English and easy to follow.

Introduction

An extensive number of references has been cited, and I was particularly delighted to see that the authors have found European literature (also in the Discussion part of the ms). However, despite the long and relevant list of references, some of the most recent publications seem to be missing, e.g. de Jong et al. 2017, and de Jong & Dahlberg 2017. Also, for the differences in the mycota of stumps and slash, you could check e.g. Suominen et al. 2018.

de Jong, J., Akselsson, C., Egnell, G., Löfgren, S., Olsson, B.A., 2017. Realizing the energy potential of forest biomass in Sweden - How much is environmentally sustainable?
Forest Ecology and Management 383: 3–16.
de Jong, J., Dahlberg, A., 2017. Impact on species of conservation interest of forest harvesting for bioenergy purposes. Forest Ecology and Management 383: 37–48.
Suominen, M., Junninen, K., Heikkala, O. and Kouki, J. 2018. Burning harvested sites enhances polypore diversity on stumps and slash. Forest Ecology and Management 414: 47-53.

Results

Although well-written, the Results chapter could be condensed a bit. Perhaps a bit too much emphasis is put to the taxonomic classification of the fungi. Considering the topic of the ms, ecological classification would be more relevant, because many ecological traits do not follow taxonomic classification on higher than the species level. I suggest that you delete the paragraph on lines 257-264. Also note the misclassification of Phanerochate sp. (which is a basidiomycete, not an ascomycete as you claim in table 1; see below), which may change the reported abundances. It seems clear that the great majority of the most abundant species are wood-decaying basidiomycetes.

The only figure in the ms, a map on the location of the study site, is clear and well-drawn. However, focusing the map still one step further to the block design and the selected stumps would make it more informative. Furthermore, consider adding a photo or two of the treatments, as they help readers a lot in understanding the local context. The supplementary figures are informative.

The only table is clear but it would be more informative, if there were two separate columns for abundances in each of the two treatments. Furthermore, the table contains at least one mistake. Phanerochate sp. is not an ascomycete but a wood-decaying basidiomycete in the class Polyporales. If the data has been analyzed with this misclassification, the numbers and shares of sequences reported on lines 255-264 in the text should also be checked (or rather, the paragraph on lines 257-264 could be deleted as I suggest above).

Discussion

In the Discussion I would like to see more ecological considerations and less discussion on the (molecular) methods. One perhaps the most likely reason behind the differences between the results of this study and the earlier European studies is that in this study only the stumps were sampled, whereas in European studies, the sampling usually includes also other logging residues (slash). If the main difference in between the treatments in your experiment is the presence/absence of slash, it surely affects the results whether you sample also the slash (where it is) or not. This point should be added in the Discussion. As sampling methodology was not the focus of this study, I suggest that you condense the paragraph on lines 341-368 considerably.

Minor details:

line 56 – fungi => fungal

line 57 – update the reference and citation: Rassi 2001 => Kotiranta 2019: “More than 40% of polypore fungi are redlisted.” Note that “endangered” (EN) is just one threat category among the IUCN Red List categories, the other threatened classes being critically endangered (CR), vulnerable (VU) and near-threatened (NT). (Kotiranta, H., Junninen, K., Halme, P., Kytövuori, I., von Bonsdorff, T., Niskanen, T. & Liimatainen, K. 2019. Kääväkkäät – Aphyllophoroid fungi. In: Hyvärinen, E., Juslén, A., Kemppainen, E., Uddström, A. & Liukko, U.-M. (toim.) 2019. The Red List of Finnish species 2019. Ministry of Environment & Finnish Environment Institute. Helsinki. p. 234-247.)

lines 102-103: H. annosum often spreads also vegetatively by mycelia via root contacts, although the primary mode of dispersal is via airborne spores.

lines 133-134, 146, 345: add space before the unit (m3ha-1 or mm or 2012)

lines 134-136: Change the sentence to past tense to make it clear that the values come from your experiment.

lines 248-249: delete “, although this difference was marginally significant” as this is stated at the beginning of the sentence.

line 263: format Perenniporia in italics

lines 264 and 267: Scytinostorma => Scytinostroma

lines 269-271: Add “Of” at the beginning of the sentence and change the whole sentence to past tense.

line 279: Erica => erica

line 285: What do you mean by “the other species”? Other than majority? Please clarify.

line 325: Why is Gilbertson referred here?

line 335: remove comma

line 355: Agaricles => Agaricales

lines 372-374: Delete one of the “five years after harvesting” to avoid repetition.
Experimental design: The methods are reported clearly and in detail. Unfortunately molecular methods are not my expertise.
Validity of the findings: No comment
Additional comments: No additional comments


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

Input:
Title: LIMITED INITIAL IMPACTS OF BIOMASS HARVESTING ON COMPOSITION OF WOOD-INHABITING FUNGI WITHIN RESIDUAL STUMPS
Review round: 1
Reviewer: 2

Output:
Basic reporting: no but see "general comments to the author"
Experimental design: no but see "general comments to the author"
Validity of the findings: The paper from Boué et al studies the effects two tree-harvesting methods on soil fungal communities in Ontario, Canada. The topic is interesting and timely, and answers important questions regarding the effects of forestry and removal of wood debris on soil fungal communities. The manuscript is also easy to understand and it is overall well-written. The experiment is also well-performed and replicated enough to provide solid conclusions. I am more skeptical about the hypotheses presented and the expected effects from the two treatments: for example, it is difficult for me to imagine any effects of the two treatments on fungal communities inhabiting stumps, since I would expect that communities inhabiting fine and coarse woody debris are different than the ones inhabiting the stumps. I would expect, for example, effects if most of the stumps would be removed. However, I still find these results interesting and they need to be reported.

I have some minor comments and changes but also one major comment regarding one of the analyses performed by the authors. Overall, the authors did not find any effects of treatments on the fungal communities, but they claimed to detect several associations between specific fungal taxa and the intensity of residual biomass harvest, based on DESeq2 results. Given the high diversity obtained using high-throughput DNA sequencing (often more than 2000 OTUs detected), it is very likely that some specific species respond significantly to some treatment, as it usually happens with similar analyses like indicator species analysis. Especially the species more prone to be significantly affected by some treatment are the least abundant ones, but they may be significantly affected just by chance (i.e. they are found only in two samples from the same treatment by chance). A way to avoid this and report species is to analyses only species that have high occurrences. Therefore, I encourage the authors to set a threshold for occurrences (discard for example OTUs present at least in less than 4 samples). If still there are significant OTUs but they have low abundance, I would report this at the discussion (for example just mentioning that significant OTUs had low abundance).

In L353-363: I am not sure if this is really needed. I would remove from “Studies based” to “environmental samples”, since the authors did not perform any comparison or have data to really state these sentences. Many other studies already highlighted this and in this paper there are no new findings regarding these issues.
Additional comments: I have other minor comments:
L86: what do you mean with spreading? Perhaps influencing fungal networks would be better?
L87: Wouldn’t be “reduced decomposition rates” instead of “reduced rates decomposition”?
L89: There are several papers reporting distinct communities across wood debris sizes, therefore stumps are not only a long-term resource but also may harbor distinct communities… perhaps is good to mention this as well.
L100: “Oliva” instead of  “Olivia”
L106: Is there some new reference? These values were reported in 1998.
L150: It could be interesting some extra information about the sampling. For example, how many cm of sample? Did you sterilize the outer parts or removed from the analysis? This would prevent a lot of spores that may be present at the exposed part of the sample.
L168: Remove “which are the most frequently used in molecular studies of fungal communities” because these are no longer the best primers.
L180: Molarity is not provided for water. It can be mentioned “and water was added until 25 uL” or similar.
L190: Did you specify some threshold for single nucleotides or only mean quality?
L212: “…where biomass removal treatment was…”
L218: Since PERMANOVA is to statistically test the effects and NMDS is to visualize the community. I would rephrase: “A first fungal community analysis was performed using Permutational multivariate analysis of variance based on distance matrices. Matrices were calculated based on the Bray-Curtis dissimilarity index calculated from Hellinger transformed OTU data for communities rarefied to 1350 sequences per sample.
Subsequently, nonmetric multidimensional scaling (NMDS) was used to visualize community variation”
L230: Here I would not re-do the analysis but instead remove OTUs that have very frequency (i.e. <4 occurrences), perhaps it is necessary to include a column with their occurrences across the 50 samples
L236-L239: This part goes to the first paragraph of the data analysis section.
L257: change “agaricomycetes” instead of “agariomycetes”
L258-L264: Not really need to reported the sequences. I would leave only the %.
L265: Remove “ignoring identification and classification at higher taxonomic ranks”
L270: Strange to see Cenoccocum here since it is an ectomycorrhizal fungi. Perhaps it was at the part exposed..
L306-308: This is obvious, the other phylum have much lower abundant for most of the habitats. Perhaps I would remove it.
L309: how you identified the function or whether OTUs were white or brown rot fungi was not explained in M&M
L321: “not found to be abundant” or similar, instead of “were not found in large numbers”


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: CONSENSUSDE: AN R PACKAGE FOR ASSESSING CONSENSUS OF MULTIPLE RNA-SEQ ALGORITHMS WITH RUV CORRECTION
Review round: 1
Reviewer: 1

Output:
Basic reporting: Figure 3 is very misleading, please begin the y-axis at 0. By not beginning the y-axis of some plots at 0, bars look drastically different when they are really very similar. For example, consider Figure 3D: all bar heights are between 0.976 and 0.988, but some are only one tenth the height of another.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The paper describes a useful new package, consensusDE, which allows the user to perform differential expression analysis with 3 of the more popular methods in the literature, and compare the results. As well as providing a package to run multiple analyses with less work and easily compare the output (providing the p-values for each method is useful for users who may wish to add their own consensus method to combine the p-values), consensusDE also allows easy integration of RUV into the DE workflow.

Some specific comments:

1. One goal of the paper appears to be to demonstrate that a consensus approach improves the ability to detect DE genes. However, as the authors themselves note (lines 342-344), much of their consensus results are driven by voom, as genes called DE by voom essentially form a subset of those genes called DE by edgeR and DESeq2. Since this is the case, the results don't really show the benefits of using multiple RNA-Seq methods; just the performance of voom itself.

This does not mean that the consensus approach is a bad idea or that consensusDE is not useful (on the contrary, it is very helpful to be able to compare results easily across different methods), but that some conclusions in the paper are misleading. On lines 334-336, the authors write that "observing the lowest FDR for the intersect ... is consistent with a model where increased stringency through inclusion of multiple forms of evidence ... improves FDR". This is true, but the results are also consistent with a model where voom has the lowest FDR, and voom DE genes are the same set as the intersect, in which case including edgeR and DESeq2 don't really improve on voom. The argument for including multiple methods would be strengthened if the authors could provide a simulation in which DESeq2 or edgeR perform better than voom (see, for example, Figure 7 in Love et al.'s DESeq2 paper https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4302049/).

2. Because voom DE genes are basically a subset of edgeR and DESeq2 DE genes, it is hard to interpret the Jaccard similarity coefficients. As the JC is calculated in reference to the intersect, voom naturally has a JC very close to 1 (see lines 257-258, e.g.). However, in different cases a high JC to the intersect might be good or bad. If voom is too conservative, methods with a low JC might actually be better by capturing more DE genes, whereas if edgeR/DESeq2 are too liberal the a high JC might indicate better performance by voom. Therefore, when reporting the JCs for the different methods, please explain that context is necessary to determine whether we want a high or low JC. If additional performance metrics are known (precision, recall, etc.) then we know the context, but for real data where ground truth is unknown, the relative JC values are hard to interpret.

Similarly, on lines 276-83 the authors describe how "RUV appeared to improve the overall concordance of different methods". The implication seems to be that this improvement is a good thing. However, this might not be the case. Mathematically, I can improve the JC for all methods by identifying an additional group of genes as DE in all methods. But without knowing whether this additional group is truly DE, we don't know whether actual performance has improved or not.

3. On lines 369-70, the authors write that "RUV improves the intersect FDR with smaller number of replicates but this effect diminishes with increased replication", and in the caption for Figure 3 the authors state that lower numbers are better for FDR. Please make it clear that a lower FDR is not always to be desired, if it leads to loss of power. For example, suppose we use BH to control FDR at level 0.05 for two different methods. Method 1 has an actual FDR of 0.01 and Method 2 has an actual FDR of 0.04. If both detect the same number of true positives, then we would prefer Method 1. If Method 1 detects many fewer true positives than Method 2, then we would prefer Method 2.

4. Accuracy is used to assess performance on the simulation. However, accuracy is known to be a poor metric when there are unbalanced classes, as is the case in the simulated data (5% to 95%). I recommend using pairs of precision (TP/(TP + FP) = 1 - FDR) and recall (TP/(TP + FN)) values instead.

5. In the introduction, the authors note that "there is no gold standard approach for the analysis of RNA-seq data" (line 49); this is a good point, and highlights the need for consensus-based approaches, or at least the need to compare results across methods. They later cite several papers (lines 227-230) to explain why they chose voom, edgeR, and DESeq2 as the three methods to compare in consensusDE. I believe the introduction would be strengthened by including these references in the introduction, to make the argument that while there is no gold-standard approach to DE analysis, some methods tend to perform better than others - hence we can (and should) restrict our attention to the better methods when looking for consensus.

6. There appears to be a small typo in the definition of the Jaccard similarity coefficient (Eq. 1). The JC is the ratio of the sizes of the sets, not the ratio of the sets themselves. I am also confused as to how the authors are summing the sets B, and what the sets B represent - please consider rephrasing the explanation on lines 122-4.

7. I commend the authors for making their code available and easily accessible on GitHub. This is very helpful and important for reproducibility.


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: CONSENSUSDE: AN R PACKAGE FOR ASSESSING CONSENSUS OF MULTIPLE RNA-SEQ ALGORITHMS WITH RUV CORRECTION
Review round: 1
Reviewer: 2

Output:
Basic reporting: The article is written well and the quality of the images and tables are sufficient to provide the necessary background, context and need for the software described in the text.

There are specific areas of the text that require some additional material to make the article self-contained:

1) a short description of the RUVr method
2) an exact description of the "modeled FC" (referenced in lines 178-179); does this come from a meta-analysis method? If not, how were the standard deviation calculated?
Experimental design: This is not entirely applicable. The text describes a meta-tool for the aggregation of the results of differential expression analysis using RNAseq. As a meta-tool the quality of the results critically depends on the selection of the algorithms used as targets for aggregation. 1) The major criticism in this section is why the authors chose to include only 3 algorithms for differential expression i.e. edgeR, voom and deSEQ2. I feel that the need is driven mostly by the availability of the relevant bioconductor packages (in my mind and research all 10-15 available algorithms for differential expression will fail spectacularly in specific datasets). I do ask the authors to provide a more vigorous justification (currently this is addressed in lines 227-230 of the text).

2) The analysis of the experimental airway data is not entirely convincing. The data do not include known positive and negative controls, so I do not really know what to make of the JC reported. Without a gold standard, one can assume that all algorithms are equally bad, so aggregating their results will not make anyone wiser. Do the authors have access to a dataset with positive (e.g. spiked in controls) controls that can be used? Alternative a bootstrap of the existing dataset in which the labels of the samples is permuted could be used

3) The R2 graphs are entirely inappropriate for the purpose of assessing agreement of the fold changes. The authors should switch to more appropriate methods for these analyses i.e. Blant Altman (or MA in the microarray world parlor) plots and statistics to establish the limits of agreement and bias.
Validity of the findings: The author's findings that voom is probably the best out of the methods there is well supported. The impact of replication, although highly not surprising, is welcome and enforces sound experimental design practice. The authors do not engage in wild speculation and one is comfortable that the results presented are aligned with the presentation in the text. I have no suggestion for this section, other than all the analyses and graphs reporting R2 should be redone with different methods as stated above.
Additional comments: No additional comments


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

Input:
Title: VEGETATION DYNAMIC ANALYSIS BASED ON MULTISOURCE REMOTE SENSING DATA IN THE EAST MARGIN OF THE QINGHAI-TIBET PLATEAU, CHINA
Review round: 1
Reviewer: 1

Output:
Basic reporting: This manuscript is far from well-writing and must be improved strongly. Some sentences are too long and complex, and with grammatical errors. Please carefully check and revise (I think the revision by native speakers is essential). Please refer to the specific comments (but not limited to).

I think the general field background is provided sufficiently, but could be improved according to the revision for research motivations (See the next section).

The article structure, figures, and tables could be improved. Please see the specific comments.
Experimental design: Generally I think this research is within the aims and scope of the journal, and the technique flow applied in this research is reasonable.

The motivation to study the scientific questions were explained inadequately, and I suggest to elaborate why the eastern edge of the Qinghai-Tibetan Plateau is important and valuable to be specially studied. Besides, the reason to study the consistency of the two datasets were not mentioned.

The description of materials & methods could be more concise but must be more exact. For example, the details for extracting the phenological variables (L175-L177) should be expounded (Also see the specific comments). Besides, I think the study area should be introduced with more detailed information on the geographic range and conditions, and how the representative sites chose should be explained in the section of methods.
Validity of the findings: This research is of less novelty as a case study and the authors should clarify the importance of this study area and what the difference varies from the related research.

I think data applied in this research is robust. My main concern is that the interannual analysis should be carried out in the same valid (with vegetation) pixels for all the years. But it seems the authors directly conducted the variation and trend analysis at all pixels including non-vegetation pixels and didn’t consider the changes of land cover.
Additional comments: More specific comments:
L31 and L40. “with…was…” please rephrase.
L38. “Ruergai” should be “Ruoergai”?
L43-L44. Rephrase.
L47-48. Rephrase.
L49. “the”
L50. “vital” ->” vitally”.
L55-58. I don’t think this conclusion (GIMMS-NDVI and SPOT-VGT are the best NDVI products) could be summarized from the cited two papers.
L62-L67. I think the methods or specific indicators applied in these cited papers are less important here than the research objects, study area or results related to the Qinghai-Tibetan Plateau. And the related research backgrounds about the Qinghai-Tibetan Plateau were not summarized enough.
L68. Is there any reason for “EMQTP is an important epitome of QTP”?
L85. “long-time series” -> “long-term time series”.
L98. Please add a reference for the GIMMS dataset.
I think the numbers of the scenes of satellite images are unnecessary to be shown.
L116. Rephrase.
L115-117. I think such pre-processing, which is record in the user manual of datasets, is unnecessary to be shown.
L120. Please add references and general information (e.g. version and producer) of the software tools.
L127. Reference.
L146. “remains unchanged after interchange”. Please rephrase.
L156. Please clarify how the seasons determined in this study.
L197. Are all the pixels valid in all the years?
L206 and L209. Please add references.
L212-L215. What the variables in the wavelet variance represent in this research should be clarified.
L219-L222. Rephrase.
L277-L282. Move to the section of methods.
The section of results. I think mixing the interannual analysis and the comparison between GIMMS-NDVI and SPOT-VGT is confusing. I suggest to describe the results separately, especially there could be one specific section for data comparison.
L309. What does “case study” mean here? I think the cited papers are for the whole TP, except for the cited paper Zhang et al. (2013) which is not in the list of reference.
L310-L311. I think it’s too casual to attribute the difference to this.
L352-L353. Please add references.
Where is Fig.2 described in the manuscript?
Figure 3. What these curves represent for should be clarified, the value of one pixel or average of the study area, and one certain year or the multi-year mean. Besides, I think such comparison is not informative enough and unnecessary.
Figure 7. The caption is incomplete. The caption of Figure 8 should be rephrased.


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: VEGETATION DYNAMIC ANALYSIS BASED ON MULTISOURCE REMOTE SENSING DATA IN THE EAST MARGIN OF THE QINGHAI-TIBET PLATEAU, CHINA
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript as presented is generally sound, using good English. There were some typos which should be double-checked for the entire text.
For example:
Line 57: VGEATION -> VEGETATION
Lines 167-169: Eq. (4) should be written as a mathematical equation or programming code. "v" should be NDVI (italic). "i" did not exist in Eq. (4).
Lines 221-: "p" should be italic.

Please use consistently only one abbreviation for each term such as "SPOT VGT" or "VGT" or "VGT NDVI"; "VGTPROBA-V" or "VGT\PROBA-V NDVI"; "GIMMS NDVI" or "GIMMS 3g NDVI".
Please check all figure titles by a native English speaker.

Fig. 1: Add scale bar and north arrow; Place legend of B inside its box.
Fig. 2: Add explanation for GIMMS 3g NDVI in the title.
Fig. 4: Not professional, especially the position of the text.
Fig. 6: Title was not clear. Why were c, d placed after e,f? What was each box represented? Ecosystems (If yes, please add their names)? Why 2017-2018 was not included?
Fig. 7: Add e-h into the title. Add scale bar.
Fig. 8: Add B-D into the title.
Fig. 9: "No change" in the legend but "weak change" in the title. Add scale bar.
Fig. 10: Add legend title, the x-axis, and y-axis labels.
Experimental design: The results are somehow valuable and the methodological approach will be of interest to some readers. However, I must be honest that this paper did not bring something which was really new in term of the research topic, data, the method, and even study area.
This research was very similar to Peng et al. (Ecological Indicators, 2012). Peng et al. analyzed vegetation dynamic trend during 1982–2003 by using GIMMS NDVI data for the whole area of Qinghai–Tibet Plateau which was three times as big as the study area of this manuscript. Basically, the findings might be an updated version. However, we have little idea about the differences between the two studies because the authors did not describe or compare deeply their project and Peng's project in the introduction, result, and discussion.

The good point of this research that VGT\PROBA-V NDVI was used. Because two types of NDVI data were different in term of spatial resolution and sensors, their generated results should be analyzed independently before comparison for the same period. For example, the structure of results can be presented as below.
1. GIMMS NDVI-based vegetation dynamic trend from 1982-2015
2. VGT\PROBA-V NDVI-based vegetation dynamic trend from 1998-2018
3. Comparison of GIMMS NDVI and VGT\PROBA-V NDVI data during 1998-2015
In comparison, it is great if the advance of VGT\PROBA-V NDVI data could be clarified because of the finer resolution (1km against 8km).

Some minor suggestions:
- Please clarify SPOT VEGETATION 1 or 2 was used.
- Which UTM zone was projected?
- Lines 115-117: Valid range of DN for NDVI must be 3-255. Please put the formula of DN and NDVI as a mathematical equation with cited reference.
- A Bayesian test should be used instead of p-value and t-Text because the p-value was very easy to pass. Please check [doi:10.1038/519009f] and newer related publication.
Validity of the findings: Because the r-squared was very low in the slope analysis, was vegetation trends validity and believable? It is great if the reason for vegetation dynamics could be somehow clarified. We can only guess based on the information presented here. Were vegetation dynamics linked to climate change or deforestation or land-use change?
Additional comments: Please consider my comments and suggestions to improve the quality of the manuscript.


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

Input:
Title: VEGETATION DYNAMIC ANALYSIS BASED ON MULTISOURCE REMOTE SENSING DATA IN THE EAST MARGIN OF THE QINGHAI-TIBET PLATEAU, CHINA
Review round: 2
Reviewer: 1

Output:
Basic reporting: 'no comment'
Experimental design: 'no comment'
Validity of the findings: 'no comment'
Additional comments: where is the Bayesian 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: VEGETATION DYNAMIC ANALYSIS BASED ON MULTISOURCE REMOTE SENSING DATA IN THE EAST MARGIN OF THE QINGHAI-TIBET PLATEAU, CHINA
Review round: 2
Reviewer: 2

Output:
Basic reporting: The manuscript was much improved; however, it needs to be checked by a native English speaker. There are still grammar mistakes, for example, L50-55, L285-286, etc.

Figure 10 should be introduced earlier, in the section of image sources.
Revise the captions of Figs.10 and 11 to help readers understand the contents of these figures. Many readers will read the figures separately from the text.

Table 1: I can't distinguish the dates of each sensor.
Experimental design: Because EMQTP is part of that of Peng’s work and Peng also identified the vegetation dynamic within the area, the originality of the research should be clarified more clearly in the introduction section. The author mentioned in L64 that similar works were already done, so why does this research need to be conducted? Please give readers strong reasons and new points.

Because four types of remote sensing images were used, an image coregistration technique should be added in the method section.
Validity of the findings: Again, the authors should compare their results to previous works. Are there any different phenomena?

L267: the authors conclude that the result of GIMMS 3g NDVI-based was basically consistent with that of VGT\PROBA-V NDVI-based. What does this mean? Does higher resolution NDVI data not bring any benefits? Please add more discussion.
Additional comments: Other considerations:
- What is "global change" in the first line of the abstract? Please check your scientific terms throughout the text.
- L219: should be in the method section.
-L324: Landsat was used for validation, not for mapping by combination with other data.


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

Input:
Title: DEVELOPMENT AND CHARACTERIZATION OF A MULTIPLEX PANEL OF MICROSATELLITE MARKERS FOR THE REUNION FREE-TAILED BAT MORMOPTERUS FRANCOISMOUTOUI
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: In the current study the authors develop novel microsatellites for the tropical bat (Mormopterus francoismoutoui) endemic to the island of La Réunion. M. francoismoutoui was only elevated to species status in 20081 and little work has been conducted on it since. Therefore, the development of this genetic resource is an important and very welcome advance.

The paper is written in a very clear and engaging style and is without any major flaws.
That is, it fulfils the BASIC REPORTING, EXPERIMENTAL DESIGN and VALIDITY OF THE FINDINGS criteria required by PeerJ.

GENERAL COMMENTS

Below are some comments, suggestions and minor typos from the text.

INTRODUCTION

This includes a very good introduction to island bats, to La Reunion and to the species. The goal of the study is clearly stated and some of the many uses of this novel resource are detailed.

Suggestions
Line 57: “However, mitochondrial markers have a much slower mutation rate than nuclear markers,” Rephrase needed: “mitochondrial markers have a much slower mutation rate than nuclear microsatellite markers”. Generally of course the mitochondrial genome has a much higher mutation rate than the nuclear genome. Microsatellites are the exception to this.

Typos/Minor text errors
Line 55:  “28 haplotypes and 2.82% sequence..” 2.91% within La Réunion 1

MATERIALS AND METHODS
Typos/Minor text errors
Line 76: “were collected in two colonies in the frame of a”.. Suggested rewrite: “..two colonies as part of a..”
Line 82: “ethic committee”.. ethics committee (add ‘s’)
Line 93: “adapters/vectors removal,” adapter/vector removal, (remove ‘s’)
Lines 102-106: please also detail which Taq you used, how much buffer was added and which PCR machine was used in the amplification.
Line 109: “Then, fluorescent labeled forward primers and unlabeled reverse primers were synthetized”… Probably the reverse primers had already been used at this point in the unlabelled testing of primers? “Then, fluorescent labeled forward primers were synthetized and…”

RESULTS AND DISCUSSION
Suggestions
Line 141-143: LD. What statistical cut off was used to infer there was not LD? Was a correction made for multiple testing (e.g. Bonferroni?) The lines are a little confusing at present as it looks like there is LD between two pairs of loci.
Line 153-154 “These results are in line with the findings from Goodman et al. (2008) (Goodman et al., 2008) who used mitochondrial DNA and found elevated genetic diversity.” Careful here. On the one hand we use the diversity at genetic markers as a proxy for the whole genomic diversity of an organism. Microsatellite loci are a bit of a special case though in that we specifically select the most variable amongst the hundreds of thousands of microsatellite repeat regions in the genome. Thus, when we chose only the most variable, it follows that we should find high variation when using such loci. If we would select microsatellite loci at random across the genome (whether polymorphic or not) then this could be used as an unbiased estimate of genetic diversity and could eventually be used in comparisons.
For the control region, it is hard to say if a certain value is ‘elevated’ or not, as the meaning of this can be vague. Elevated as it is above a certain cut-off value for this most variable part of mtDNA? Elevated compared to other bats, or island bat species for this same region?
Comment:
Line 144-146. It is quite interesting that you tested HWE at the species-level and found almost no deviation. This suggests that not much population structure will be found, and that the species is a single panmictic population.

Typos/Minor text errors
Line 132: “After QDD process”.. “After the QDD process” (add ‘the’)
Line 153-154 The Goodman et al. 2008 reference appears twice.

CONCLUSIONS
Suggestions
Line 159-160: This line returns to the potential uses of this new resource (also outlined nicely at the end of the introduction). All of these things are quite true. However, I am not aware of microsatellite loci availability for this genus at all. If that is so, this could also be mentioned as it is quite a plus.
It is quite likely that these loci will cross-amplify well in closely related taxa (e.g. M. acetabulosus from Mauritius, M. jugularis from Madagascar) as the currently available data suggest these three are very closely related indeed.

These microsatellite loci would be very useful to clarify the biogeography of this genus for the Afro-Malagasy species, their colonisation histories and taxonomy.

Typos/Minor text errors
Line 160: “to help developing conservation” to help to develop conservation

FIGURES & TABLES
Figure 1 is really lovely. Please do add a scale bar for Reunion though.

ANIMAL ETHICS
Typos
Line 187: “ant reference numbers):” ‘and’ reference numbers
Line 188: “ethic” ‘ethics’

SUPPLEMENTAL INFORMATION
-Please also submit the genotypes for the 31 individuals (e.g. as an excel attachment, or genepop file or similar).
-Submitting the sequences of the microsatellites to GenBank will make it much easier for other researchers to find them and eventually reuse them. Also consider submitting the whole illumina run unless there are good reasons not to do so.

Submitting microsatellite sequences in GenBank: https://www.ncbi.nlm.nih.gov/WebSub/
You will need a fasta file containing the sequences and a features file. The feature for your first sequence would be something like this:
>Feature MF_loc03
179  220	repeat_region
          rpt_type	tandem
          rpt_unit_seq	"gt"
          satellite	<microsatellite>

Supplementary table 2: usually the ‘motif’ should match the sequence and not its complement. A mixture of the two seems to be used in this table. Please clarify this.

References
1	Goodman, S. M., Jansen van Vuuren, B., Ratrimomanarivo, F., Probst, J.-M. & Bowie, R. C. K. Specific status of populations in the Mascarene Islands referred to Mormopterus acetabulosus (Chiroptera: Molossidae), with description of a new species. Journal of Mammalogy 89, 1316-1327 (2008).


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: DEVELOPMENT AND CHARACTERIZATION OF A MULTIPLEX PANEL OF MICROSATELLITE MARKERS FOR THE REUNION FREE-TAILED BAT MORMOPTERUS FRANCOISMOUTOUI
Review round: 1
Reviewer: 2

Output:
Basic reporting: In general, the article has clear and professional English, structured according to the author guidelines and presents relevant information related to a newly registered endemic bat species in Reunion Island. However, there are some points that should be addressed here, related to a critical lack of references in the introduction and methods section. In the introduction, some lines lack references (lines 32, 35, 40, 45) and should be provided. However, from line 55 up to 75, the structure of the text is pretty similar to Cleary et al. (2016) without mention to this study. Thus, I suggest great modifications in these paragraphs, and proper reference to the authors should be made.
Tables are informative, as well as supplementary materials. The map from sampling areas, on the other hand, is not well descriptive and should be improved, adding elements required in a map. Suggestions are highlighted in the text body.
Experimental design: The study fulfilled the gap in relation to the absence of molecular markers to this species and presented effective and reliable methodology. However, I haven't found GenBank number of sequences and this should be provided according to the journal rules. Few details such as DNA quantification and annealing temperature should also be written in the text. Lastly, sampling coordinates should be provided in supplementary material 1.
Validity of the findings: No comment
Additional comments: No additional comments


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

Input:
Title: BODY HEIGHT AND WAIST CIRCUMFERENCE OF YOUNG SWISS MEN AS ASSESSED BY 3D LASER-BASED PHOTONIC SCANS AND BY MANUAL ANTHROPOMETRIC MEASUREMENTS
Review round: 1
Reviewer: 1

Output:
Basic reporting: .
Experimental design: .
Validity of the findings: .
Additional comments: Introduction: An over-the-top introduction is not the focus of the research problem.

The work is over-extensive on the basis of the objectives presented.

We should know which method of reference is: What is the anthropometry or the scanner?

As always in comparative studies, correlation coefficients are not sufficient or adequate. In this case only with the concordance study of Bland Altman sufficient, contributing the differences and its 95% CI.

I honestly think there are minimal differences between anthropometry and scanning, you don't think they have much influence on BMI.


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: BODY HEIGHT AND WAIST CIRCUMFERENCE OF YOUNG SWISS MEN AS ASSESSED BY 3D LASER-BASED PHOTONIC SCANS AND BY MANUAL ANTHROPOMETRIC MEASUREMENTS
Review round: 1
Reviewer: 2

Output:
Basic reporting: Some improvements in grammar needed, particularly in the introduction.

I would suggest the addition of discussion into other 3D-based scanning techniques such as medical imaging and infra-red. Some suggested papers are listed below:

-Norton et al. (2002), 3D whole body scanning to determine mass properties of legs
-Smith & Bull (2018), Rapid calculation of bespoke body segment parameters using 3D infra-red scanning
-Cheng et al (2000), Segment inertial properties of Chinese adults determined from magnetic resonance imaging
-Durkin & Dowling (2003), Analysis of body segment parameter differences between four human populations and the estimation errors of four popular mathematical models
Experimental design: Line 99 - please add a reference for the larger research project if possible

line 102 - would it still not be possible to do a power analysis? You mention previous studies similar to yours which could provide the basis for one

line 112-115 - you could provide some justification as to the advantages or this equipment over other, smaller machines.

line 284-287 - if possible differences exist between WC measurements due to arm positions, why did you not place the subjects in the same position for both? The 3D scanner may required a specific position, so simply use this consistently.
Validity of the findings: Authors are thorough in addressing differences between manual and scanned data, suggesting possible reasons such as patient movements, breathing in during manual measurements etc ...
Additional comments: I would like to thank the authors for submitting a straight forward validation of using 3D scanners to produce height and WC measurements. The technology sector for anthropometrics is fast-growing and provides a rapid method for obtaining good data without causing discomfort to the subject.

In addition to the comments above, I would like to make some general observations:

- the use of the bioelectric impedance machine was only mentioned in the methods, but should appear in the introduction to justify its importance and necessity.
- you make mention to the fat mass results, but do not discuss at all. Again, there is mention in the abstract, but your discussion purely mentions WC and BMI.
- please add age group (not just range) of participants in Table 2.
- I think it would be interesting to observe the clinical significance between your calculated and manually measured WC and BMI values. Do the statistical differences impact on the categorisation of WC and BMI?
- Discussion does not place the work into any type of clinical 'bigger picture'. Authors make mention to the comfort of the patient, but nothing further is added in the discussion.


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: BODY HEIGHT AND WAIST CIRCUMFERENCE OF YOUNG SWISS MEN AS ASSESSED BY 3D LASER-BASED PHOTONIC SCANS AND BY MANUAL ANTHROPOMETRIC MEASUREMENTS
Review round: 1
Reviewer: 3

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The authors present novel 3d-body scanner data to validate automated body height and width circumference measurement in comparison with gold-standard manual measurement. They found very good correspondence, accompanied by systematic biases discussed appropriately.

In general, the manuscript is sound and statistical analysis straight forward. Despite relatively low sample size, which the authors also acknowledge as a limitation of the study, the data is worth publishing and results underline reliability of 3d-body scanning in health research.

I, however, have few comments and questions which the authors should address:

1) I suggest to rephrase "visual association" as e.g. used in line 232. Something like "inspection of figure xy revealed...."

2) Left panels in Figures 1-3: Please add a description to figure legend what the lines in the plots represent (x=y? linear regression?)

3) Also figure 4 needs a legend for the three line types used.

4) I suggest to blur the participant's face in Supplementary figure 1 to avoid irritations concerning privacy.

5) in line 181 it is stated that weight was obtained from BIA. Do you mean the internal scale of the BIA instrument? Please clarify.


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: MOLECULAR PHYLOGENY OF MULBERRIES RECONSTRUCTED FROM ITS AND TWO CPDNA SEQUENCES
Review round: 1
Reviewer: 1

Output:
Basic reporting: The first word in the title is “Classification” but there is no clear presentation of any taxonomy based on the results. Results are only communicable via scientific names and this has not been done in any clear way. The authors show no knowledge of standard taxonomic practices when presenting their results, they would do well to find a taxonomist to be a collaborator.  As it is, it is quite difficult to find out the actual taxonomically significant results, particularly how to apply them without access to a molecular laboratory.
Experimental design: Need to clarify what is meant by ‘sample’, are these single living trees or groups of trees? what are the provenances of these ‘samples’?  are there permanently preserved vouchers?  The authors state that 7-20 clones sequenced from each sample, are these from different trees or different parts of a single tree? were these vegetatively propagated?
Validity of the findings: The conclusions are totally inadequate as there is no clear link to the actual classification of Morus.
Additional comments: line 72: consistent with the morphological classification, not ‘consisting’ with …
line 75: Nepal et al. recognised 13 species, not … ‘classified’ 13 species (or accepted 13 …)
Nepal et al. provided evidence that Morus is paraphyletic because two species of Trophis were embedded within it in their analysis but there is no follow up on this even though this account is supposed to be a review of the genus.
line 182: this section needs to be rewritten so as to clarify the identities of the various clades - at present there is nothing connecting the initial list of taxa and the clades / groups, especially as it listed 9 taxa and then refers to seven out of eight clades.
line 246: there are no details of the morphological polymorphisms mentioned, these need to be described / tabulated in some detail and their possible origins discussed. There can be significant differences between juvenile and adult leaves, how relevant is this?
line 254: Is not the practical need to find which morphological features correlate best with the groupings based on the molecular data and chromosome numbers? That way the results can be used outside of a molecular lab.
line 298: what taxa correspond with these ‘molecular species’?
line 329: Fig. 6 needs to be linked to names, how does it relate to the M. alba / M. rubra hybridization discussed in the previous paragraph?
line 347: The sentence starting ‘However’ needs rewriting, mulberries are normally propagated vegetatively thus giving long generation times. ‘sticking’ is not a good translation (too ambiguous), better ‘propagated via cuttings’ perhaps “However, the cultivated examples of Mulberries have been propagated vegetatively via grafting and cuttings, giving long generation times, which may . . .”


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: MOLECULAR PHYLOGENY OF MULBERRIES RECONSTRUCTED FROM ITS AND TWO CPDNA SEQUENCES
Review round: 1
Reviewer: 2

Output:
Basic reporting: The English used here are not very clear and I encountered many occasions where the presentation are ambiguous. Although the authors provide many literature references, I cannot guarantee that they have enough background on evolutionary biology, which is essential to this article. The article do not have a very good structure to broadcast their ideas to the readers, since they have not distilled their idea carefully and thoroughly. I also do not see a clear hypothesis that the authors have clearly presented.
Experimental design: Methods described with sufficient details and information to replicate. However, the research question may need to be further distilled.
Validity of the findings: All underlying data have been provided and they are robust.
Additional comments: Mulberry is one of the most improtant economic crop in ancient China, where the economic chain of silk road rest on. Doubtless, there are numerous times that the cultivated populations of mulberry in China were domesticated from wild populations, or be crossed between cultivated populations or between cultivated and wild populations. In this manuscript, the authors did a good job to infer hybridization between different evolutionary lineages in the light of ITS sequences, pseudogenes of ITS sequences and cpDNA sequences from 33 samples in China, in the help that cpDNA and pseudogenes of ITS sequences are inherited form maternal progenitor. However, I found that the manuscript await substantial revision before being considered for publication.

First of all, I think the author may need to distill the key questions they want to address in this manuscript, and then restructure the introduction, as well as the discussion section. Although I found Fig. 6 very attractive, it seems that the authors do not tell a story on this topic, they start their manuscript with introducing of ITS! Mulberry and their evolutionary history are much more attracting! It would be much more interesting to start with an brief of mulberry history in China, and point out the important questions that still await answers, then tell the readers that ITS are very good tool to use. Finally, tell clearly to the readers what questions you are going to address.

Second, I found it is very odd that the authors avoid to discuss about morphological species all over the manuscript, and the 33 samples are always named after sampled sites or simply pronunciations of Chinese names. I suppose that the authors did it like this because the molecular data are not consistent with morphological species. Nevertheless, this is one finding of your work, and it should be discussed carefully and logically. It is common that only ITS and cpDNA cannot be able to solve the evolutionary history of cultivated popuations of mulberry, then propose that next generation sequencing data will be a good tool to do that.

Thirdly, according to my experience, the authors are not very familiar with species concept. I recommend the authors read more references about species concept as well as variety and cultivars, e.g. De Queiroz (2007) and references therein, as well as its citations (https://doi.org/10.1080/10635150701701083), and then reconsider their forging of the concept ‘molecular species’.

At last, there are numerous typos and awkwardly presented sentences, I recommend that the authors ask a native English researcher to co-work on this manuscript to improve not only the language, but also the logic and structure of this manuscript that holds considerable potential.

I will be happy to see a revised version of this manuscript, and I am confident that the authors will improve their manuscript to present their findings better.


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: MOLECULAR PHYLOGENY OF MULBERRIES RECONSTRUCTED FROM ITS AND TWO CPDNA SEQUENCES
Review round: 1
Reviewer: 3

Output:
Basic reporting: Line 73-76 The authors introduced former study of classification of Morus with molecular markers, which helped the readers to understand the background. However, the sampling density and the marker used may influence the phylogenetic reconstruction and the classification in the former studies. Thus, it would be helpful for readers to understand the background if the authors mention the number of sampled species in Zhao et al. 2005 and the marker used in Nepal and Ferguson 2012.

Line 79 “grouped into the clade B…”. It would clearer if the authors can add a citation for this “clade B” here, as this classification system has not been introduced in the text above. The authors mainly compared with this classification system (clade B) instead of those listed in line 62-67 in the following discussion. Thus, it is worth to write at least one sentence to summary this system in the introduction part.

I did not find any information about how to access those new sequences in this study. The figures and tables in this article are clear and informative for me, except the tip names of the phylogenetic trees. They are too long to follow except those with scientific names. It would be better if change the code names of tips into M. alba1,2,….
Experimental design: It would be easier for readers to trace the logic of this article if the authors claim their hypotheses or key questions clearly before the brief summary of their results. For now, it is, at least for me, confusing what is the main purpose for this study. I suppose it could be:1) improve the impact of polymorphism of ITS on the phylogenetic reconstruction in Morus; 2) investigating a potential evolutionary pattern (hybridization history) in Morus.

If proposing a new phylogenetic relationship of Morus is one of the key questions of this study, I have one question for the authors: would you please justify the reason for using ITS and two chloroplast markers to reconstruct the phylogenetic relationships? On one hand, the bait set for species level phylogenetic reconstruction in Moraceae has been designed by Gardner et al. (2016 App.PlantSci., see also in Zerega and Gardner 2019 Phytotaxa)., It is worth to try this bait set in Morus, considering the fact that Morus nuclear genome was used as a conference during the design. On the other hand, low-copy nuclear markers have been suggested to be ideal markers for phylogenetic reconstruction in angiosperms (Zhang et al. 2012 NewPhyto). ITS has been proved to show polymorphisms and may mislead the phylogenetic reconstruction in this study, which exactly suggested that ITS is not an ideal marker to do so.

For the “phylogenetic analysis” part in Materials & Methods, I think the authors may have several points to be improved:
1.	Outgroups: two Broussonetia species were selected as outgroups. However, Broussonetia and Morus are in different tribes in Moraceae according to the species level phylogenetic reconstruction in the former studies (Clement & Weiblen 2009 Syst. Bot.; Zhang et al. 2019 Ann. Bot.). It would be better to choose phylogenetically closer outgroups to avoid potential long-branch-attraction (Bergsten 2005 Cladistics) and more than one genera would be better.
B. papyrifera alone was used as outgroup in the reconstruction with trnL-trnF and trnT-trnL. However, two species of Broussonetia were selected as outgroups in the reconstruction with ITS. This is not a serious problem, but commonly same species of outgroups will be used in this comparing analyses. Please justify the difference of outgroup selection.
2.	Reconstruction approaches: the authors used maximum-likelihood (ML) approach alone to reconstruct the phylogenetic tree, which is the cornerstone of the following discussion of this study. It would be better to apply both Bayesian and ML approach to reconstruct the phylogenetic tree, then compare and confirm the phylogenetic relationship. It is because the advantage and shortcomings of both approaches that we have to reconstruct in this way. In brief, Bayesian approach take into account the uncertainty such as branch length in phylogenetic reconstruction, while applying wrong prior probabilities in Bayesian approach will lead to poor reconstruction (Nei and Kumar, 2000, Molecular evolution and phylogenetics).
3.	Substitution model: Kimura 2-parameter and Tamura 3-parameter model were selected to reconstruct the phylogenetic tree. However, the authors did not mention how they make this choice? Which criterion did they use? How many potential models had they compared? Please give more details for model selection.
Line 134 “…two categories (+G,…)” To increase the accuracy of the estimate, the more categories of gamma distribution the better. However, considering the computational burden and the decreasing of accuracy by increasing the number of categories, it is commonly use four or five categories (Nei and Kumar, 2000, Molecular evolution and phylogenetics). Please justify the reason for using two categories.
Another potential problem is reconstruction without considering the presence of invariable sites (+I). Large amount of invariable sites was mentioned in line 246-247 (“The 29 samples with morphological polymorphisms clustered together with a low phylogenetic resoluiton and had similar or identical ITS sequences”). Commonly the presence of invariable sites can be overlooked only when the it is low in dataset. Please justify this point.
Validity of the findings: The authors did not give the detail information such as access number of those sequences they cited from previous studies.
Additional comments: This study provided a phylogenetic analysis of Morus with multiple sampling in four species and single sampling in other twelve species in Morus with two main goals: 1) to attract the attention to the influence of polymorphism in ITS on phylogenetic reconstruction; 2) to investigate the potential evolution processes of Morus. The sampling strategy is a significant improvement over previous studies in Morus. Although several points needed to be improved and confirmed in the phylogenetic reconstruction part, the speculation of the potential evolutionary pattern of Morus is clear and reasonable, which help to extend the understanding of mulberry evolution.

Hereby, I listed some general comments.

Line 75-76 Please keep consistence of the citation. For my understanding, “et al.” is used when the number of authors is more than three and “&” is used when there are only two authors. I suspect “Nepal et al. (line 75)” is the same citation as “Nepal & Ferguson, 2012” in the next line. To keep consistence, it would be better to change the former one into “Nepal and Ferguson”.
Line 80 “among the materials”. I do not understand the meaning of “materials”, do you mean specimens?
Line 82-83 If this “previous study” has been published, please cite it here.
Line 84 It would be better to mention how many species these 33 mulberry samples represented.
Line 151 Loci is different from sites. A locus is a fixed position on a chromosome. A (structure) site is a nucleotide base on a DNA strand. I think “sites” is more accurate in this context.
Line 241 It would be easier to follow if this “clade 3” (including clade 1 and 2) was labelled in Figure 3.
Line 254-256 Phylogenetic reconstruction in genomic scale is also a promising solution for systematic classification of mulberry. It would be a reasonable point in the perspectives in an age of high-throughput sequencing.
Line 284-286 Comparing with which markers trnL-trnF and trnT-trnL are the most variable regions?
Line 303-312 I think there are probably some typos. If I understand well, it must be “pseudogroup 2” in line 303 and 308, and “pseudogroup 1” in line 306. Please capitalize the first letter in “morus” in line 312.


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: MOLECULAR PHYLOGENY OF MULBERRIES RECONSTRUCTED FROM ITS AND TWO CPDNA SEQUENCES
Review round: 2
Reviewer: 1

Output:
Basic reporting: My conclusion is that this work is just an analysis of cultivated mulberries that has very little practical application to our understanding of the systematics and nomenclature of the genus as a whole.  I have no doubt that this is a valuable reference for those working on the breeding of cultivated mulberries and I believe that it would be best to reflect this in the title of the paper where ‘Morus’ should be replaced by ‘cultivated mulberries’.  If the presentation could be reorganized to more clearly reflect this, it would then merit publication.
Experimental design: no comment
Validity of the findings: I am still puzzling over how existing names should be applied to the hierarchy of clades presented in this paper.  The conclusion states that ‘clade B was separated into two species’ but does not say what these species should be called. This is further confused by Figure 3 which shows 3, not 2, clades within Clade B.  Various names are presented at the start of the section on ITS phylogenetic analysis, M. alba, M. wittiorum, M. australis, M. macroura and M. mongolica but these names do not appear within any phylogeny so is their status?
There is also the problem of how to name the two ‘pseudogroups’ in Fig. 3, can these only be referred to as hybrid swarms?
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: MOLECULAR PHYLOGENY OF MULBERRIES RECONSTRUCTED FROM ITS AND TWO CPDNA SEQUENCES
Review round: 2
Reviewer: 2

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: This is a manuscript that I reviewed before. I am satisfied with most revision of the authors, which have, together with other referees' comments, improve the clarity and integrity of the story.
Now, two major concern still remains:
(a) The abstract is very difficult to follow by readers who are not familiar with the background, I therefore suggest the authors to further improve to make it more reader-friendly.
(b) Since one of the highlight of this work is the further exploration of Clade B, I suggest the authors to connect Clade B with geographic distribution, key morphology traits or taxonomy, among other factors, if there is any, to make the findings easier to be understood.
Minor concerns:
L39-40: Concerted evolution only act on nrITS sequences, since nrITS is a multiple-copy gene region. It is not proper to say that concerted evolution have played roles in the evlution of mulberry, although it is proper to say that concerted evolution have played roles in the evlution of nrITS in mulberry.
L114: I still do not know what species the authors have sampled for the 33 mulberry accessions. Although the authors said there are seven species in the last part of introduction. Please also give the detail in L114.
L251-L260: I would like to see the morphological species of each accessions, this information is important since accession names does not correlate with any geographical or morphological information!
L271-272: How many accessions of mulberry did the authors find ITS pseudogenes? What is the difference between long/short or polymorphic ITS sequences (14 accessions, L191) and ITS pseudogenes (11 accessions)?
L251: Please use words like "diverged first" among the 33 mulberry accessions. "were the most ancient" is not quite proper in a phylogenetic background, since each accessions kept evolving after divergence.
L434-435: see comments on L39-40 about "concerted evolution".
Table 1: In my opinion, the accessions are not listed in a very reasonable order, I suggest to sort it out following taxa, then the name of accessions alphabetically.