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: TRENDS IN FRAGMENTATION AND CONNECTIVITY OF PASPALUM QUADRIFARIUM GRASSLANDS IN THE BUENOS AIRES PROVINCE, ARGENTINA
Review round: 2
Reviewer: 1

Output:
Basic reporting: The manuscript describes trends in fragmentation and connectivity of grasslands in the Buenos Aires province, Argentina using 5 satellite images from 1974 to 2016. The introduction is well developed and sets the scene with adequate references. The revised Figures are relevant. I appreciate providing the raw data to the readers so they can replicate the steps described in the paper.
Experimental design: The design setup is within the scope of the journal, research questions are well defined and relevant. Methods are described sufficiently and reviewer comments were integrated.
Validity of the findings: The findings in the revised manuscript are sound and relevant.
Additional comments: Thank you for ingesting the comments on the original manuscript. The revised version reads much better. However, I still think the following minor changes should be inmplemented:
Line 55: In this way, and given the impact of

Line 116: .. (MSPA) and Network ...

Line 125: replace Vogt et al, 2017a with Saura & Torné (2009)
Saura, S. and Torné, J. (2009). Conefor Sensinode 2.2: a software package for quantifying the importance of habitat patches for landscape connectivity. Environmental Modelling & Software 24: 135-139.
DOI: 10.1016/j.envsoft.2008.05.005

Line 181: and the standard foreground connectivity of 8 was chosen.

Line 190: ECA was calculated via GTB. ECA measures the overall ...

Line 197: replace Vogt & Riitters, 2017 with Seebach et al. 2013
Seebach, L., Strobl, P., Vogt, P., Mehl, W., San-Miguel-Ayanz, J. (2013) Enhancing post-classification change detection through morphological post- processing – a sensitivity analysis. International Journal of Remote Sensing, 34 (20), 7145–7162. DOI: 10.1080/01431161.2013.815382

Line 199: The supplemental material provides a complete set of ...

Line 200: To simplify interpretation, only the start and end years of the observation time period (1974-2016) are shown in the main body of the text.

Line 209: ...around 25% between 1974 and 1988, and then kept oscillating between values that never reached the initial stage. A similar behaviour was found for medium cores, which dropped by approximately 50% in the same time frame. The main change was found for large cores with a decrease of more than 60% in 1988, followed by ....

Line 216: do you mean: Islets and perforation show a similar trend like cores.  ??

Line 225: although the percentage of relative ECA increased at a high rate ...

Line 234: and percentage of relative ECA after 2011.

Line 236: of relative ECA showed...

Line 237: .. of 28%, almost

In general: the term "relative ECA" already implies relative values (percentages) so there is no need to write "relative ECA%", just replace it by "relative ECA"

Line 276: With this definition, ECA provides an non species-specific measure...

Figure 2/3: the image shows 3 different types of Core area (dark/medium/light green - small, medium, large Core areas) but the legend does not reflect this distinction.
Morphological elements -> morphological pattern classes.

Figure 6, legend: .... 1.10575). Dark grey indicate zones with unchanged


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: REVISION OF THE AFRO-MADAGASCAN GENUS COSTULARIA (SCHOENEAE, CYPERACEAE): INFRAGENERIC RELATIONSHIPS AND SPECIES DELIMITATION
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript entitled “Revision of the Afro-Madagascan genus Costularia (Schoeneae, Cyperaceae): infrageneric reationships and species delimitation” is a quite complete and excellent revisión, using morphological and molecular tools, of Costularia, one of the least studied Cyperaceae genera until now.

English is correct and references are appropriate.

All figures and tables are an essential complement to understand the results and conclusions and the paper structure is also appropriate.

Introduction summarizes well the background of this taxonomically difficult group.

In most cases, morphological features match very well with molecular results and taxonomic conclusions are well supported.

I have indicate in the attached pdf file numerous corrections and comments that, in my opinión, could contribute to improve the manuscript. The most important weakness of the paper is relative to descriptions of the species, highly heterogeneous. Authors must systematize the lengh, order and content of them. Moreover, in several cases, monophyly of the species was not tested or scarcely supported, although I can understand that it is very difficult to get sufficient materials of several species and, anyway, I think that the taxonomic results would not be very different.
Experimental design: This article is suitable for publication in Peer Journal.

Conclusions are a very important contribution to the  systematics of the family Cyperaceae.

Methodology is detailed and appropriate, although nowadays there are molecular tools more conclusives as the employ of Next Generation Sequencing; nevertheless, as it is a scarcely collected genus, I can understand the difficulties to use those techniques.
Validity of the findings: This paper shows important novelties for researchers in systematics, mainly for cyperologists.

The statistical data (support of the clades) are generally highly robusts.

Conclusions are coherent with results.

Few important speculations are made.
Additional comments: No comment


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

Input:
Title: REVISION OF THE AFRO-MADAGASCAN GENUS COSTULARIA (SCHOENEAE, CYPERACEAE): INFRAGENERIC RELATIONSHIPS AND SPECIES DELIMITATION
Review round: 1
Reviewer: 2

Output:
Basic reporting: The present work presents a nice, clear and much needed revision of the problematic genus Costularia (Cyperaceae) in its entire distribution area.
According to the expertise in Cyperaceae of some of the authors, they present an accurate background of the topic, investigate the taxonomy of the group using accurate molecular and morphological approaches, and present the results and discussion in a perfectly clear manner. The English is professional and do not need correction, although I detected a few typos.
References, figures and tables are accurate (and very nice!). The new taxa they describe are prefectly justified. Even they contained the description of a clearly new species because of having too few material (although I would encourage the authors to do it in this paper).
Overall, it is a very nice piece of work, worth to be published in PeerJ.
Experimental design: The materials, tools, experimental approach and test designs are accurate for an extensive taxonomic study like the one that is presented here. The explanation of the methods is fine, although I suggest some minor improvements in the attached file. There is no doubt that the procedures have been performed with rigorous caution and ethics.
Validity of the findings: The authors present the taxonomic revision of a group that was needed of study. They present completely new data that accurately update with the use of modern tools other older works in the group. The work seems quite robust, their results sounds and the conclusions well stated.
Additional comments: I attach a commented file with additional suggestions that I consider that will improve the quality of the manuscript. Regarding the taxonomic treatments, the changes resquested in the first species must be performed over the entire set of synopses.


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: VALIDATION OF REFERENCE GENES FOR GENE EXPRESSION STUDIES IN TARTARY BUCKWHEAT (FAGOPYRUM TATARICUM GAERTN.) USING QUANTITATIVE REAL-TIME PCR
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript by Li et al described the validation of 7 genes from Tartary buckwheat for use as reference genes in quantitative realtime PCR for gene expression study under abiotic stress condition. Good reference genes for different stresses or tissue types were reported. The study design was fine as it followed similar standard article of this kind. The manuscript was well-written with clear and plain English.
Experimental design: The study design was fine as it followed similar standard article of this kind.
Validity of the findings: The manuscript could attract reader's interest upon publication and may provide a set of reference genes for gene expression study in Tartary buckwheat in the future which may help easier to compare cross-study.
Additional comments: However, before the manuscript can be accepted for publication, this reviewer would like to ask the authors to address the followings:
The authors should provide a rationale on their selections of these 7 genes for validation. If a good rationale cannot be provided, the conclusion can only be restricted to "the best reference gene among the 7 genes validated", in some cases, there are best/better reference genes out there but were not included in this validation study. One of the way to check if the seven genes selected were actualy the best candidates is to survey in publicly available expression data of tatary buckwheat for the top stably expressed genes and see if these 7 genes were among the list.
As geNorm has been known to rank co-regulated genes as the best reference genes, the authors should address this in the discussion. What the authors will suggest to avoid this.
About the 7 genes selected for validation, the authors should link the gene names into respective gene IDs, if available. As we know especially in plant, there always several highly homologous members in a gene family. In the case gene IDs are not yet available, a full sequence of each of the candidate gene must be supplied for future reference of the work.
Some minor comments:
- Scientific name of Tartary buckwheat should be provided in the title


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: VALIDATION OF REFERENCE GENES FOR GENE EXPRESSION STUDIES IN TARTARY BUCKWHEAT (FAGOPYRUM TATARICUM GAERTN.) USING QUANTITATIVE REAL-TIME PCR
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The authors present novel experimental study which results can be widely applied for gene expression studies in Fagopyrum. The manuscript is technically sound and I have only minor comments:

Lines 99-104 “Selection of candidate reference genes and design of qRT-PCR primers”
Could you provide here any rationale why exactly these genes were selected? Are the designed primers falls within single exon or not?

Line 117 - “Other statistical procedures were performed with SPSS v17.0. “ - please clarify what exactly was done;

Fig. 1 – please explain in the figure caption what the boxes and whiskers mean on the plot;

Fig. 3 and Fig. 4 - please explain in the figure caption what the whiskers mean;

Line 45 - add references for geNorm, normFinder and BestKeeper here (i.e. upon first mention).


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: VALIDATION OF REFERENCE GENES FOR GENE EXPRESSION STUDIES IN TARTARY BUCKWHEAT (FAGOPYRUM TATARICUM GAERTN.) USING QUANTITATIVE REAL-TIME PCR
Review round: 1
Reviewer: 3

Output:
Basic reporting: - The manuscript "Validation of reference genes for gene expression studies in Tartary buckwheat using quantitative real-time PCR" described the validation of five ref genes for internal normalization to calculate the expression level of gene(s) in tartary buckwheat. The manuscript is suited to the PeerJ as per aims and scope.
- The Introduction (47 lines) has provided a comprehensive background of the necessary of ref gene(s) and the identification of the ref gene(s) in the kingdom.
- The figures and references were relevant and well-described.
Experimental design: - The materials and methods are suitable and were described carefully with right citations.
Validity of the findings: - The results and conclusion were fully stated and tightly linked with the research question.
Additional comments: - Comment #1: The authors should rephrase the sentence in L17-L20. The colon should not be used in the manuscript.
- Comment #2:  The authors should check the tense in the sentence in L20-L24.
- Comment #3:  Three statistical algorithms, including geNorm, normFinder and BestKeeper should be cited with right reference(s). (L45-46 and L.115-117).
- Comment #4:  The sentence in L47-48 should be cited.
- Comment #5: Need a space between "in the work,we aimded" (L.70).
- Comment #6: FtEF-1α and FtSAND should be italicized in L.138.
- Comment #7: The author should abbreviate the quantification cycle (Cq) at the first mention (not like in L.175). The 'aluminum' should be used as 'Al' in whole text after the first mention of the abbreviation.
- Comment #8: "t" in "buckwheat" should not be italicized in L.192.
- Comment #9: The author should check the style of citation in L. 206.
- Comment #10:  Please, check and put the style and word(s) of all references in the right form (REF. 4, 13, 15, 16, 17, 18, 19, 21, 22, 23, 24, 25, 27, 33, etc).
- Comment #11: The FtSTAR2 and FtDFR in the legend of Figure 3 and 4 should be italicized, respectively. All gene names in Figure 4 should be italicized.


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 NEW GLASSFROG (CENTROLENIDAE) FROM THE CHOCÓ-ANDEAN RÍO MANDURIACU RESERVE, ECUADOR, ENDANGERED BY MINING
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: This is a well-written and concise description of a new species of Nymphargus from Ecuador. I am not entirely convinced that the new species they describe is valid and I think more work is needed to explain how the new species differs from closely related species. I think that the new species is probably new (judging from branch lengths on the tree), but some key details are missing to convince me. I think that addressing these two issues would greatly improve the ms:

1)	The comparisons with other species seem to be under-developed and constrained to mostly color characters. It is unclear what species concept is being used here, and what evidence presented indicates the species is new. Genetic distances are not mentioned, which would be important to get a sense for how closely related the new species is to its sister species. Importantly, there is no comparison with N. balionotus in the ms, so it is difficult to know how the new species differs from its evolutionary sister species, which occurs syntopically. This seems like a key comparison and should be made directly in the “Comparison with similar species” section.
2)	The discussion of speciation patterns in Nymphargus in the Andes is too strongly stated given no biogeographic or other analyses, and should be framed as speculation. The conclusions here are drawn from observations on the phylogeny, with support coming from sister species being close geographically. However, there are many other Nymphargus that are not mentioned that could be contrary to these conclusions, and I think a future study would be necessary to figure out the drivers of speciation in this group. Additionally, many of the sister species pairs mentioned in the discussion occur syntopically, so it seems like the situation is fairly complex.
Additional comments: Minor corrections/suggestions:

Line 74: “patters” = “patterns”
Line 116: Could the specimens examined be moved to an appendix?
Line 160: can delete “between them”

Results:

Line 200: How many partitions did you use?
Line 247: The table does not show very clearly how the species all differ. Are there other characters that can be used to compare? It also seems like call characters could be used in this section as well. If there are no calls available for the other species, than that should be clarified.

Discussion:

The numerous headings and subheading here is confusing, with different formats for each (i.e. a new line for some, colon for others). Authors should make the heading format consistent between sections.

Figures and Tables:

Figure 1: the white mainland around Ecuador looks awkward. I would suggest either removing the blue ocean or giving the white mainland a grey or other coloration.

Figure 2: the blue tip coloration explanation was cut off in the figure caption?


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 NEW GLASSFROG (CENTROLENIDAE) FROM THE CHOCÓ-ANDEAN RÍO MANDURIACU RESERVE, ECUADOR, ENDANGERED BY MINING
Review round: 1
Reviewer: 2

Output:
Basic reporting: This is a well-done species description of a new glassfrog species from essentially a single locality in Ecuador. The authors went above and beyond in that they included sequence data from a number of Nymphargus species that had not been sequenced previously. The phylogeny is notable in that (1) it clearly supports the new species, (2) it resolves the status of "Cochranella" balionota as a member of Nymphargus, and (3) suggests an interesting biogeographic pattern of parapatric sister species, which the authors discuss.

The English is clear and unambiguous, references are apt, and the article structure is appropriate. The figures in particular are quite nice.

My major comment (aside from some minor stuff I've included as comments in the word file) is that the introduction is too short and doesn't do a great job at setting up the study. The intro has 6 sentences total, and one of the sentences is discussing species that are not even the focus of the paper. I would make the following suggestions: (1) elaborate paragraph 1 to include more background and history on Nymphargus, in particular N. balionota. (2) Elaborate on paragraph 3 so that rather than describing results from the study, the focus is shifted more towards biogeographic hypotheses (i.e. elaborate on dry river valleys in particular, give some possible examples, etc).
Experimental design: The paper meets all the relevant standards.
Validity of the findings: The validity of the new species is not in doubt and is supported by morphological and phylogenetic evidence. Aside from that the paper provides a substantial contribution to glassfrog systematics.
Additional comments: Most of my comments are given on the word document, which I'll be emailing to the editor.


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: TWO MEMBRANE-BOUND TRANSCRIPTION FACTORS REGULATE EXPRESSION OF VARIOUS TYPE-IV-PILI SURFACE STRUCTURES IN SULFOLOBUS ACIDOCALDARIUS
Review round: 1
Reviewer: 1

Output:
Basic reporting: See general comments
Experimental design: See general comments
Validity of the findings: See general comments
Additional comments: This study deals with two transcriptional regulators, ArnR and ArnR1 from the thermoacidophilic archaeon Sulfolobus acidocaldarius. They have been known to be involved in the transcriptional regulation of the archaellum operon by targeting the promoter of flaB that encodes the archaellum filament protein. ArnR and ArnR1 are capable of forming oligomers and are phosphorylated by the Ser/Thr kinase ArnC. Apart from binding to pflaB, ArnR but not ArnR1 bound to promoter sequences of aapF and upsX, which encode components of the adhesive (aap) and UV inducible pili (ups), demonstrating a regulatory connection between different surface appendages of S. acidocaldarius. The information provided is new, especially the fact that ArnR may be involved in the regulation of other surface appendages. The manuscript however needs some editing. In particular, the discussion should be trimmed and refined/clarified for the general reader. Perhaps a figure of the regulation network would make it much easier to understand. The conclusion section is not necessary unless this is the format of the journal. A list of specific comments, both major and minor is shown below.

2nd affiliation needs an address. Spemann Graduate School of Biology and Medicine (SGBM)

However, sigma factors are absent from archaea raising the question of how gene transcription is regulated. This sentence seems strange. If Archaea were to use bacterial transcriptional machinery, the absence of sigma factors would be intriguing, but this is not the case. I do not think the authors have to refer to the absence of sigma factors in order to raise the question as to how gene transcription is regulated.

While ArnR is conserved in Sulfolobales and Desulfurococcales, the ArnR paralog ArnR1 is exclusively found in S. acidocaldarius (Lassak et al., 2013). Is the distribution still the case taking into account the dramatic increase in genome sequences in recent years? I ask this as the citation is from five years ago.

Lines 95 and 101: Organisms in italic or is this the journal style for subtitles?

Line 147: DNA in italic?

Line 181: This was surprising. N-terminal His10-tag under control of an arabinose inducible promoter. How is the protein integrated into the membrane? Is there not a signal sequence at the N-terminus? A comment would help.
Related to this, although archaeal membrane proteins such as SecYEG have been known to assemble in bacterial membranes, is it not worth noting? Is the insertion machinery analogous between archaea and bacteria? Is the majority of protein localized in the membrane fraction? Some comments would help.

Change y32 to gamma (greek letter)32

Description of Figure 3 text lines 205-214: Why do the authors not mention the self phosphorylation of ArnD? Understanding the meaning of the bands takes a while and it would be helpful if this were written here.

Line 237: the first p in roman? Are paapF and pupsX designated somewhere?

Are there any comments or discussion on the quite large Kd values?

Line 262: mutant in italic?

since neither ArnR nor ArnR1 bound to the promoter sequence of the non-T4P related gene saci_2122 (encodes a putative sugar binding protein) it is tempting to speculate that rather the length of the DNA fragment and/or distance to core promoter elements such as the BRE, TATA-box and/or transcription start site are important for ArnR and ArnR1 function in regulating T4P surface structures of S. acidocaldarius. The logic is hard to understand here, please clarify.

Line 324: jannaschii

Line 325: M. maripaludis

Line 331: Is this e.g.?

Line 338: S. acidocaldariusis

Line 345: S. acidocaldarius in italic

Line 364: What exactly tempts the authors to speculate that phosphorylation of ArnR and ArnR1 defines their specificity to different T4P promoter sequences?

Line 372: ArnR/1?


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

Input:
Title: TWO MEMBRANE-BOUND TRANSCRIPTION FACTORS REGULATE EXPRESSION OF VARIOUS TYPE-IV-PILI SURFACE STRUCTURES IN SULFOLOBUS ACIDOCALDARIUS
Review round: 1
Reviewer: 2

Output:
Basic reporting: There were various inconsistencies throughout article that should be corrected prior to publication:

Line 70 – Spell out HAMP
Line 110 – Should list components of PBST
Line 149 – 16,000 instead of 16.000 rpm
Line 166 – “and” does not need to be italicized
Line 170 – aapf should be aapF
Line 171 – change “log2-folg” to “log2-fold”
Results
Figure 2 caption; second line - list separately as ArnR/ArnR1 for consistency
Lines 193 – 214 – The beginning of this paragraph contains a lot of information that seems like it would fit better in the Discussion section.
Figure 4 caption, fourth line – change pFlaB41 to pflaB41
Lines 237/238 – change paapF to paapF to stay consistent with italization between paapF and pupsX
Line 246 – change Arnr1 to ArnR1
Figure 6 caption, second line – italicize adh
Figure 6 caption, last line – delta symbol needs to be added instead of DarnR and darnR1.
Line 293 - list separately as ArnR/ArnR1 for consistency
Line 308 – add bp after 40
Line 345 – italicize S. acidocaldarius
Line 351 – change Arnr1 to ArnR1
Line 352 – remove “by on” replace maybe with “at”
Line 372 – change Arnr1 to ArnR1
Line 386 – change hosphorylation to phosphorylation
Experimental design: I think that the experimental design for the experiment were adequate, but a few sections in the Materials and Methods section need work. First, the phosphorylation studies were not even mentioned in the Materials and Methods. This should be added prior to publication of the article. This would also help in understanding Figures 3B and 3C which took a while to wade through because of the lack of information in M & M. Obviously not enough information to replicate this specific set of experiments.

The second issue was related to the qRT-PCR section in the M & M:

Lines 159 – 172 – qRT-PCR Section of Methods is confusing. On lines 165 and 166 the authors indicate they are targeting flab and arnR/arnR1, while in lines 169 and 170 they talk about normalization of expression of aapf and upsX. Since data was only included for aapf and upsX, I can assume that lines 165 and 166 should be corrected. They also listed Table S2 as listing primers; however, primers are listed in Table S3. Listing of primers in Table S3 is also not consistent. upsX primers are listed as qPCR primers, while the aapF and adh are listed as qRT-PCR primers. Italics are also used inconsistently in table. Please re-write section so that it is consistent with research task performed. Would also be worthwhile to state why adh was used as housekeeping gene.
Validity of the findings: The research was impactful and novel, showing that ArnR and ArnR are part of the regulatory network of S. acidocaldarius for activation of motility. Likewise, the work showed that there was regulation of other motility and surface structures.

The data presented is robust and the raw data and statistical analysis was sound and well controlled.

The conclusions of the research are succinctly stated and linked to the original question discussed in the introduction.
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: EFFECTS OF STIGMATA MAYDIS ON THE METHICILLIN RESISTANT STAPHYLOCOCUS AUREUS BIOFILM FORMATION
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: The authors have a good idea, but unfortunately, the introduction, the results and the discussion are complicated.

The introduction requires a careful review of the current  literature and the authors should clearly indicate the problem as well as the objectives. please delete the text on line number 90-96, the text was a results.

In the results, as reported by the authors, that the aqueous extract of stigmata maydis can inhibit the formation of biofilms of MRSA strains and does not affect the growth curves of MRSA strains and has no antibacterial activity against  MRSA strains, in this case how the aqueous extract could enhance the vancomycin susceptibility of the MRSA strains under biofilm-cultured conditions, and the plankton cells of MRSA strains has not been affected by the same of aqueous extract? The effects of the aqueous extract must be the same in  either free planktonic  cells or in biofilm formations, only the dose of antibacterial (if there is any) can present a significant difference in the formation of biofilms compared to free plankton cells.
In the next line, the text must delete, these are not results
Line 173-178
Line 190-195
Line 208-217

The discussion did not correspond to the objectives or the problem. Neither previous work nor real-time PCR results were well discussed. Most of the sentences were repeated from the results and methods. Finally, I think the authors should check the effects of Stigmata maydis on biofilm adhesion factors to MRSA


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: EFFECTS OF STIGMATA MAYDIS ON THE METHICILLIN RESISTANT STAPHYLOCOCUS AUREUS BIOFILM FORMATION
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Comments:
1. Keywords: I suggest adding stigmata maydis.
2. Lines 100-104: Please provide detailed description of MRSA strains SA2 and SA3. As a clinical isolated MRSA strain, which kind of antibiotic resistance dose it possess?
3. Line 115: “…diluted to a final concentration of OD600 0.05” change to: “diluted to a final optical density (600 nm) of 0.05”.
4. Line 120: Please provide specific calculation methods used in CFU assays.
5. Line 139: check the culture medium used here, TSB or TSB containing glucose?
6. Line 132, Materials Methods: Crystal violet is usually measured around 590 nm and not 560 nm. Please check.
7. The paper should be reviewed by an English native speaker reviewer. There are some sentences that are not clear.


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: EFFECTS OF STIGMATA MAYDIS ON THE METHICILLIN RESISTANT STAPHYLOCOCUS AUREUS BIOFILM FORMATION
Review round: 2
Reviewer: 1

Output:
Basic reporting: please correct all highlights text in PDF
Experimental design: no comment
Validity of the findings: no comment
Additional comments: please correct all highlights text in PDF


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

Input:
Title: EFFECTS OF STIGMATA MAYDIS ON THE METHICILLIN RESISTANT STAPHYLOCOCUS AUREUS BIOFILM FORMATION
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Thank you for responding to my original review comments and I do feel the manuscript now reads better. The paper is written within the Journal required style, It is suitable for publication in its present form.


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

Input:
Title: EFFECTS OF STIGMATA MAYDIS ON THE METHICILLIN RESISTANT STAPHYLOCOCUS AUREUS BIOFILM FORMATION
Review round: 3
Reviewer: 1

Output:
Basic reporting: nil
Experimental design: nil
Validity of the findings: nil
Additional comments: please see the notes in annotated PDF


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

Input:
Title: PSYCHOLOGICAL FLEXIBILITY AND ATTITUDES TOWARD EVIDENCE-BASED INTERVENTIONS BY AMYOTROPHIC LATERAL SCLEROSIS PATIENTS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The authors present an interesting, cross-sectional online study about the relationship between flexibility and the intention to adopt NIV and PEG. They also considered QOL, anxiety, and depression. The paper addresses a relevant clinical and scientific question, and I commend the authors for these efforts. However, before being considered for publication, there are some shortcomings and issues (including a major one) that should be addressed.
Experimental design: First, there is a problem with the title, which is misleading. The paper is not about the “adoption of”, but rather about the intentions. A title like the current one suggests a longitudinal (or, maybe, a retrospective) design, which is not the case. Please change it accordingly.

At the beginning of the introduction, please spell ALS, PEG, NIV, and RFT. Authors should re-write the beginning of the introduction with the idea that readers may not know what these concepts (especially RFT) are.
When discussing the use of NIV and PEG across different countries, please consider the role of welfare state support. For example, Italy has a very different percentage of NIV usage then the UK.

There are various theoretical frameworks around psychological flexibility, RFT is only one of these. Authors should tone down the emphasis on the theoretical model to remain more adherent to the data, which are interesting. However, the attempts to interpret everything on the light of this model reads somehow forced (it sounds like a spot or an advertisement of the model!). I suggest to re-write the aims section referring to the main construct that is explored, which is psychological flexibility.

In the methods section, authors write: “A sample size of 37 participants was recommended based on R2 = .15, power = .80, α = .05, Cohen’s f = 0.176, and six predictors.” – however, 37 was the number of participants who remained in the study. Please report this as a post-hoc power analysis (which is fine, just to be clear).

Now, the major issue. The assessment of NIV and PEG acceptance is problematic. The items have not been validated, and 37 participants are not many to validate the scale. Therefore, using an aggregated score is not the correct way to go, as there is no data that indicate the presence of a single factor. The alpha is a good indicator, but that does tell anything about the factor structure of the scale. The only way to handle this new instrument without validation is to use the items individually, with non-parametric statistics. Of course, that means to account for multiple analysis, using a Bonferroni correction or a similar technique. Most of the results section should therefore be re-written.
Validity of the findings: The reviewer will be happy to provide comments on discussion and conclusion sections, once proper analyses will be implemented.
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: PSYCHOLOGICAL FLEXIBILITY AND ATTITUDES TOWARD EVIDENCE-BASED INTERVENTIONS BY AMYOTROPHIC LATERAL SCLEROSIS PATIENTS
Review round: 1
Reviewer: 2

Output:
Basic reporting: Major comments:
1.	Discussion of RFT in the Introduction is confusing and needs clarifying further:
a.	Lines 53-54: What is RFT? A brief description needs to be given here… also please provide a reference for it.
b.	Lines 57-63: The study by Greenaway is mentioned in the second paragraph, but no attempt is made to link this to RFT, which is discussed in the same paragraph. Furthermore, on Line 108, readers should not have to navigate back to page 1 to see details about the Greenaway study. Please remove reference to this study on lines 57-63 and move it to Line 108 onwards.
c.	Line 130: What is a comparative frame?
d.	Lines 136-137: What is a frame of coordination between two relational networks?
e.	Lines 140-142: Please explain further.
2.	Lines 103-104: There might not be studies investigating the relationship between general psychological well-being and decision-making in people with ALS, but there are definitely studies in people with other chronic health conditions. These should be mentioned here rather than studies in health people.
3.	Line 146: What have studies of psychological flexibility in other chronic health conditions found? Again, there have been studies conducted in other relevant chronic health conditions (such as muscle disorders) that should be discussed.
a.	Graham CD, Gouick J, Ferreira N, Gillanders D. The influence of psychological flexibility on life satisfaction and mood in muscle disorders. Rehabil Psychol. 2016;61(2):210-7.

Minor comments:
1.	Line 81: Why is there a difference in the take up rates of PEG feeding in the US vs. UK and Australia?
2.	Line 91: Please spell out BiPAP abbreviation in full and abbreviate thereafter.
3.	Lines 108-112: A 5-line sentence is taxing for the reader. Please separate long sentences such as these into shorter sentences.
4.	Figure 1 is unnecessary and therefore should be dropped. It is a simple model that does not add much to the paper beyond the description that is already given in the text.
5.	The Abstract fails to mention the lack of findings for NIV.
Experimental design: Major comments:
1.	Line 279: The authors should correct for the number of statistical calculations that are conducted (e.g. by adjusting the alpha level)… if you perform enough calculations then you’re going to find a statistically significant result somewhere.
2.	Line 245: Was the Understanding and Acceptance of Interventions scale that was designed for the purpose of the study piloted or validated outside of the study? This scale should be presented in the Appendix so that readers can actually see what the 9 items are…

Minor comments:
1.	Line 186: What criteria were used to diagnose ALS (e.g. El Escorial criteria) and who made the diagnosis? Did the authors ask participants what type of MND they had? Different types of MND vary in disease progression rates (with some being much more rapid and others being much slower), which may affect the relationship between psychological flexibility and acceptance of life-prolonging interventions.
Validity of the findings: Major comments:
1.	Lines 287-289: How many of the sample had actually accepted/rejected PEG feeding? And how many had actually accepted/rejected NIV?
2.	Lines 298-300: Interpretation of the results should occur in the Discussion and not the Results. Also, no reference was made to the fact that the lack of internal reliability for willingness in the current study (Cronbach’s alpha = 0.47) is probably the biggest factor here, rather than “willingness… may not be an influential variable”.
3.	Lines 358-364: There is not enough discussion of the lack of findings for NIV with respect to psychological flexibility. This needs to be discussed more.
4.	Line 330: There are a number of issues that need discussing further:
a.	The impact of type of MND on psychological flexibility and acceptance of life-prolonging interventions. The authors noted that psychological flexibility (action) was associated with time since diagnosis (r = .450, p < .01, two-tailed) and time since first symptoms (r = .361, p < .05, two-tailed), but failed to discuss this further.
b.	Whether participants had actually accepted/rejected PEG feeding or NIV.
c.	The effect of cognition on decision-making in ALS (mild cognitive changes are common, with mild deficits being typically seen in executive functioning, attention, social cognition and language).
5.	Lines 366-378: Please discuss the limitation that this is a cross-sectional and not longitudinal study and the implications of this with respect to the findings. Please also discuss the limitation that N=10 participants per predictor variable is typically recommended, making this study underpowered given that there 6 variables.

Minor comments:
1.	Lines 290-291: Please give an indication of what the correlation actually means for those who don’t know the AAQ e.g. “However, psychological flexibility (action) was associated with time since diagnosis (r = .450, p < .01, two-tailed) and time since first symptoms (r = .361, p < .05, two-tailed). That is, the greater X, the greater Y.”
2.	Lines 315-317: No mention is made of the fact that the overall model was not significant (p > 0.05).
3.	Line 338: Please make reference to Acceptance and Commitment Therapy (ACT) when discussing what might be helpful for clients with ALS. Please also mention the ongoing studies that are looking at ACT in people with ALS and their caregivers.
4.	Line 372: Please discuss other studies that have identified problems with the AAQ… there’s a reason why the AAQ-2 was developed!
Additional comments: This was a small cross-sectional study (N=35) that examined the relationship between psychological flexibility, psychological wellbeing and understanding and acceptance of life-prolonging interventions in people with ALS. This is an interesting topic in an under-researched area, and so represents a novel study. The paper was generally well-written, but requires major amendment in light of the specific 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: PSYCHOLOGICAL FLEXIBILITY AND ATTITUDES TOWARD EVIDENCE-BASED INTERVENTIONS BY AMYOTROPHIC LATERAL SCLEROSIS PATIENTS
Review round: 1
Reviewer: 3

Output:
Basic reporting: Thank you to the authors for this enjoyable and interesting manuscript. It will add to our understanding of the difficulties patients face when considering NIV.

My comments on the reporting of the study focus on terminology, and better explaining the concepts raised to a non-psychology audience.

In the first paragraph of the introduction, please expand all the acronyms used. Although they are given in the abstract, they should also be present in teh manuscript body

RTF is raised in the second paragraph of the introduction, but no explanation is given for the acronym, or the theory at this point. I suggest you give an explanation of RTF at this point. Your manuscript requires that readers understand this theory very early.
Experimental design: The details of the method are all included and reported sufficiently. The significance of the work is stated and justified sufficiently.
I am unable to comment on the statistical analysis.
Validity of the findings: The Discussion is generally well stated and referenced.
Even so, the expression of last statement of the discussion might need reconsideration -the complexity of PEG decision-making definitely differs from that of NIV.

The Limitations section covers a good range of points. Also consider any possible cultural differences/attitudes towards NIV between participant groups.(See Andersen et al (2018)Therapeutic decisions in ALS patients: cross-cultural differences and clinical implications.)
Additional comments: This manuscript forms a basis for future work, both qualitative and quantitative.


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: PSYCHOLOGICAL FLEXIBILITY AND ATTITUDES TOWARD EVIDENCE-BASED INTERVENTIONS BY AMYOTROPHIC LATERAL SCLEROSIS PATIENTS
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: Despite several improvements in the text, following the reviewers' suggestions, the authors have failed to address the methodological shortcomings that were previously highlighted. In my opinion, the methodology does not meet the standards for a scientific publication.
Validity of the findings: Given the methodological issues that have remained unaddressed, the findings are not valid. There are great risks in publishing a flawed study, including the fact that readers may overlook the methodological shortcomings, and just go straight to the results and implications.
Additional comments: The authors, despite improving several sections of the paper, have not addressed the core methodological issues.


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: PSYCHOLOGICAL FLEXIBILITY AND ATTITUDES TOWARD EVIDENCE-BASED INTERVENTIONS BY AMYOTROPHIC LATERAL SCLEROSIS PATIENTS
Review round: 2
Reviewer: 2

Output:
Basic reporting: See below
Experimental design: See below
Validity of the findings: See below
Additional comments: The authors have addressed the majority of my comments well. However, there are a number of comments that still need to be addressed or require further clarification, as noted below:

1) Line 81: Why is there a difference in the take up rates of PEG feeding in the US vs. UK and Australia?
REPLY: It is presumed that the reasons for variations in take-up rates is similar to the story with NIV (as described in line 95 – 97).
RESPONSE: Then this presumption should be noted in the manuscript.

2) Line 279: The authors should correct for the number of statistical calculations that are conducted (e.g. by adjusting the alpha level)… if you perform enough calculations then you’re going to find a statistically significant result somewhere.
REPLY:  Statistical analyses testing the a priori hypotheses are not adjust. However, adjustments should be made for ‘fishing expeditions’ such as might happen in tables reporting a correlation matrix. That said, we are not engaging in ‘fishing expeditions’ in our correlation tables and have now added a warning in the note section of each table regarding this matter. It is also important to focus on effect size criterion for such expeditions as p values are a simple function of effect size thus not a reliable criterion from one study to the next.
RESPONSE: This warning in the note section of each table needs to be added to Table S1.

3) Line 245: Was the Understanding and Acceptance of Interventions scale that was designed for the purpose of the study piloted or validated outside of the study? This scale should be presented in the Appendix so that readers can actually see what the 9 items are…
REPLY: The questionnaires have now been added as requested.
RESPONSE: First, the authors have not addressed my original question “Was the Understanding and Acceptance of Interventions scale that was designed for the purpose of the study piloted or validated outside of the study?”. Second, I can’t seem to find the questionnaires – they have not been uploaded as part of the manuscript or as supplemental documents.

4) Line 186: What criteria were used to diagnose ALS (e.g. El Escorial criteria) and who made the diagnosis? Did the authors ask participants what type of MND they had? Different types of MND vary in disease progression rates (with some being much more rapid and others being much slower), which may affect the relationship between psychological flexibility and acceptance of life-prolonging interventions.
REPLY: As per Line 186, verification of diagnosis was not requested. The participants weren’t asked what type of MND they had. It was considered at the time of constructing the questionnaire. In an effort to minimise participant fatigue, the value of this question in the context of this research was seen as limited.
RESPONSE: The issue of not verifying diagnosis and not asking participants what type of MND they had needs to be discussed as a limitation of the study. The issue of acceptance in different types of MND that vary with respect to rate of disease progression also needs to be discussed. It may be more difficult for participants to accept interventions when the disease is rapidly progressive as they are constantly having to adjust to physical changes and associated losses. Anecdotally, clinicians report that patients struggle more with acceptance issues if the disease is rapidly progressive as they do not have sufficient time to adjust to or come to terms with the physical changes (or they just about come to terms with one physical change and the associated decisions in relation to interventions and then something else changes, which renders the previous decision no longer applicable).

5) Lines 287-289: How many of the sample had actually accepted/rejected PEG feeding? And how many had actually accepted/rejected NIV?
REPLY: This data was not collected. The capturing of actual acceptance and rejection status was strongly considered at the time of designing the research, in fact it was the starting point to work from this position. However, we opted not to ask this question for a number of reasons. We expected that most participants may not yet be at the point where they had to accept once of these interventions. Given expectations on the number of participants, we believed setting the dependent variable at this point could leave the study grossly under-powered. Therefore, the study focussed on intentions rather that behaviour. The survey was designed so that all participants could provide answers reflective of their position, irrespective of acceptance or rejection status.
RESPONSE: Then this needs to be discussed as a limitation of the study. The fact that the authors cannot comment on whether intentions actually affect behaviour is something that should be considered further.

6) The impact of type of MND on psychological flexibility and acceptance of life-prolonging interventions. The authors noted that psychological flexibility (action) was associated with time since diagnosis (r = .450, p < .01, two-tailed) and time since first symptoms (r = .361, p < .05, two-tailed), but failed to discuss this further.
REPLY: Point acknowledged. The following has been added to the discussion sections…
“The data also suggested that a patient’s ability to take action in line with chosen goals is positively related to the time they have had ALS. This relationship may be somewhat intuitive. As time passes, patients develop a better understanding of the nature of their disease as well as the options available to them that can support their goals. Consequently, they may become better equipped to evaluate options and make decisions, as time progresses.”
RESPONSE: The issue of type of MND and rate of disease progression also needs to be discussed here. It may be more difficult for participants to accept interventions when the disease is rapidly progressive, for reasons as noted above.

7) The effect of cognition on decision-making in ALS (mild cognitive changes are common, with mild deficits being typically seen in executive functioning, attention, social cognition and language).
REPLY: The influence of Fronto Temporal Dementia (FTD) was not included in the scope of this study. Consequently, it was not included in the discussion.
RESPONSE: The issue here is not FTD, but the mild cognitive changes that are seen in executive functioning, attention, social cognition and language in 30-50% of people with MND (Massman et al., 1996; Ringholz et al., 2005). The potential impact of these deficits on decision making with respect to health interventions should be discussed further.

8) Lines 290-291: Please give an indication of what the correlation actually means for those who don’t know the AAQ e.g. “However, psychological flexibility (action) was associated with time since diagnosis (r = .450, p < .01, two-tailed) and time since first symptoms (r = .361, p < .05, two-tailed). That is, the greater X, the greater Y.”
REPLY: This should now be covered as a result of changes made in response to point 4a.
RESPONSE: I can’t seem to find this in the manuscript as the authors have not indicated where they have made the changes in the document.

9) Line 338: Please make reference to Acceptance and Commitment Therapy (ACT) when discussing what might be helpful for clients with ALS. Please also mention the ongoing studies that are looking at ACT in people with ALS and their caregivers.
REPLY: I have used the term “applied RFT” throughout the paper. This is interchangeable with ACT. Given the heavy usage of acronyms I was reluctant to introduce another.
RESPONSE: Whether or not the authors use ACT or RFT, the authors should mention ongoing studies that are looking at ACT in people with ALS and their caregivers (such as the COMMEND study in the UK).


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: PSYCHOLOGICAL FLEXIBILITY AND ATTITUDES TOWARD EVIDENCE-BASED INTERVENTIONS BY AMYOTROPHIC LATERAL SCLEROSIS PATIENTS
Review round: 2
Reviewer: 3

Output:
Basic reporting: The authors have addressed the concerns raised in my previous review. Thank you.
Experimental design: Unable to comment on design.
Validity of the findings: Unable to comment on validity.
Additional comments: From my perspective, the authors have suitably addressed the concerns raised. I look forward to seeing this manuscript as a 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: PSYCHOLOGICAL FLEXIBILITY AND ATTITUDES TOWARD EVIDENCE-BASED INTERVENTIONS BY AMYOTROPHIC LATERAL SCLEROSIS PATIENTS
Review round: 3
Reviewer: 1

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: PSYCHOLOGICAL FLEXIBILITY AND ATTITUDES TOWARD EVIDENCE-BASED INTERVENTIONS BY AMYOTROPHIC LATERAL SCLEROSIS PATIENTS
Review round: 3
Reviewer: 2

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: See below
Additional comments: The authors have addressed all of my comments well. However, I have one point that needs to be addressed in relation to their response to one of my questions:

1) Line 245: Was the Understanding and Acceptance of Interventions scale that was designed for the purpose of the study piloted or validated outside of the study? This scale should be presented in the Appendix so that readers can actually see what the 9 items are…

REPLY: The questionnaires have now been added as requested.

RESPONSE: First, the authors have not addressed my original question “Was the Understanding and Acceptance of Interventions scale that was designed for the purpose of the study piloted or validated outside of the study?”. Second, I can’t seem to find the questionnaires – they have not been uploaded as part of the manuscript or as supplemental documents.

REPLY R2: It was validated for the purpose of the present study this is stated in the method section but the wording has now been made clearer “Understanding and Acceptance of Interventions. For the purpose of the present study, a 9-item scale measuring understanding and acceptance of PEGs and a 4-item scale measuring understanding and acceptance of NIV were designed.” Also, the scale should now definitely be included as a supplementary material.

RESPONSE 2: The authors note that the questionnaire was not piloted or validated outside of the study, and was designed specifically for the study. The fact that the scale is unvalidated and the resulting impact on study findings needs to be mentioned as a limitation in the discussion. This may explain why the internal reliability of the measure of psychological flexibility was relatively low in the current study. The study findings and implications need to be couched in much more tentative terms throughout the whole paper (i.e. not just in the Limitations section) given that the questionnaire is not validated outside of the study. This may go some way towards responding to Reviewer 1's concerns, who does not think the paper is of publishable quality at present, and whose comments have not been properly addressed.


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

Input:
Title: GENETIC DIVERSITY OF SIMAO PINE IN CHINA REVEALED BY SRAP MARKERS
Review round: 1
Reviewer: 1

Output:
Basic reporting: This manuscript investigated the genetic diversity of Simao pine based on the SRAP markers. The authors found that this important conifer species have the high level of genetic diversity and low level of genetic differentiation among populations. They also proposed the some conservation and management strategies. Actually, the manuscript is interesting for the general reader of Peer J.
There are some questions which need to address before acceptance in the Journal.
Line 32, “the genetic diversity of the entire remaining natural populations…”, what is mean of the entire remaining natural populations? It seems that the means of the sentence is unclear, which should need to revise.
Line 39-40, Results. The 11 populations reveal a relatively low level of genetic diversity in the species. Results show a high level of genetic diversity (PPB = 94.45%, H = 0.4567, I = 0.6484). The means of these two sentences are confused. Please revise it carefully.
Line 42, AMOVA analysis changed to AMOVA.
The cited literatures style, e.g., Line 59 (Wang et al., 2012) and others. The comma should be body not italic, please carefully revise the whole manuscript.

In the whole manuscript, there are some mistakes and errors of the grammars and sentences. I think the authors could thoroughly revise the manuscript in the help of a native English speaker. Some spaces should be noted. E.g., Longitude(° E) Latitude(° N) in Table 1.

In addition, I suggest that the authors could run the STRUCTURE analysis for their datasets and they could discuss the population structure furtherly.
Experimental design: useful.
Validity of the findings: Interesting and useful.
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: GENETIC DIVERSITY OF SIMAO PINE IN CHINA REVEALED BY SRAP MARKERS
Review round: 1
Reviewer: 2

Output:
Basic reporting: Table 2
Please note the direction of the primers' sequences

Figure 2
Please note the four groups mentioned in the manuscript.

Figure 9
Adding a Chinese map will make clear about the locations of sampling in this study.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: 1 Authors may discuss about how ecological factors influence the level of genetic diversity of Simao pine populations in nature.
2 Please give some possible reasons why Gst of Simao pine is different from some species or similar with some species.


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

Input:
Title: GENETIC DIVERSITY OF SIMAO PINE IN CHINA REVEALED BY SRAP MARKERS
Review round: 1
Reviewer: 3

Output:
Basic reporting: In general, the English needs to be improved. There are many places with items such as missing punctuation and  use of the term "use". These are not major problems, but need attention to improve the readability of the manuscript. Please see other specific comments on the attached manuscript.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Thank you for your contribution. In general,the manuscript is clearly written in professional, unambiguous language. If there is a weakness, it is in the missing punctuation and  use of the term "use". (as I have noted above) which should be improved upon before Accepted. What environment were these Simao pine populations? Full sun or shade? Did this affected genetic clustering? And I think some original electrophoresis map is needed to public.


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

Input:
Title: GENETIC DIVERSITY OF SIMAO PINE IN CHINA REVEALED BY SRAP MARKERS
Review round: 2
Reviewer: 1

Output:
Basic reporting: Clearly.
Experimental design: Reasonable.
Validity of the findings: accurate and interesting.
Additional comments: I have checked the manuscript carefully again and found it thoroughly revised the questions that I have suggested previously. So I recommend accept the manuscript after a minor question addressed: L 120, please authors add some parameters of STRUCTURE analysis for the datasets.


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: OXIDATIVE STRESS AND THE PRESENCE OF BACTERIA INCREASE GENE EXPRESSION OF THE ANTIMICROBIAL PEPTIDE ACLASIN, A FUNGAL CSΑΒ DEFENSIN IN ASPERGILLUS CLAVATUS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The authors in the submitted manuscript titled “Oxidative stress and the presence of bacteria increase gene expression of the antimicrobial peptide aclasin, a fungal CSαβ defensin in Aspergillus clavatus” presents information which I believe will be of interest to many researchers in the field of microbiology, especially those with interest in the area of anti-microbial peptides. I would like to commend the authors for their work. However, there are several shortcomings which needs to be addressed as outlined below:
1.	While I appreciate the general background information provided in the introduction section, it lacks details in some sections. Most of the citations in this section are from review articles, book chapters etc. While reviews and book chapters are good source of general information, lack of direct inputs from original research papers makes this section of the manuscript very vague in some places. For example, in lines 46-47 the authors have merely mentioned how presence of pathogen or abiotic stress factors induce defensin gene expression without elaborating the significance of this information. Explaining the link between abiotic stress and need for expression of anti-microbial defensins would allow the readers to better understand the physiological significance of such anti-microbial peptide production. It is also true for lines 43-46. Additionally, in some places like lines 46-47 and 56-58, it is not clear whether the statements are true for all organisms within the group mentioned (plants, fungi etc.) or, are only true for some organisms within the group.
2.	In the last paragraph of the introduction section, the authors have summarized their statements from the previous paragraphs of the section and have attempted to rationalize their endeavor for the experiments undertaken. However, a compelling statement to justify the motivation of the proposed study is lacking. The authors should consider modifying this part to summarize their motivation, findings and significance of their study in broader context more effectively and clearly.
3.	In lines 43-46, it will be better to rephrase the sentence “…microorganisms can induce defensin gene expression through bacterial recognition through receptors…” to convey the message more clearly.
4.	Line 18: The abbreviation “(CSαβ)” should be introduced at the end of the sentence “… cysteine-stabilised α-helical and β-sheet motif” before it is mentioned stand alone in the next sentence.
5.	Line 41: “…structure composed by an α-helix…” should be “…structure composed of an α-helix…”
6.	Line 48: “however, few fungal” could be better written as “however, only few fungal”.
7.	Line 120: “checked through an agarose gel” could be better written as “checked by agarose gel electrophoresis”
8.	Lines 134-135: it could be better written as “…upstream of the translation start site were…”
9.	Line 165: I believe the authors meant “aclasin” instead of “afusin”.
10.	Line 187: it could be better written as “…both in the presence of viable and…”
11.	Some minor formatting issues with the references like incorrect or missing page numbers in lines 306, 338, 351 and 358.
Experimental design: 1.	The authors have studied the effect of oxidative stress on aclasin expression using 5 or 10 mM H2O2 (line 172). It is not clear why such concentrations were chosen? Is it known previously that such concentrations induce oxidative stress? It is particularly important because the authors also used a lower concentration of 2 mM later (in lines 178-179). The motivation for checking the lower concentration is not clear.
2.	In addition to the live culture of B. megaterium, the authors have also used heat-killed bacteria. The inspiration of checking the effect of heat-killed bacteria on aclasin expression is not clear.
Validity of the findings: 1.	Lines 210-214: the authors have mentioned that they also found STRE sequences in the 5’ UTR of the act1 gene which was included in the study as a negative control. The authors should reconcile how the presence of this sequence in the 5’UTR of the act1 gene affects their observation in this regard. The authors have themselves written in lines 212-213 that act1 is a house keeping gene whose levels are stable under stressful conditions. If this is true, then should the presence of STRE sequences alone be interpreted as a signature of stress induced regulation of gene expression? Should we call act1 as a “negative control”? Does it invalidate the findings by the authors in this regard?
2.	In Figure 1, if we look at the supplemental excel file provided by the authors, the glucose concentration at 24h time point is recorded as “0”. This is bit surprising that the glucose levels would be zero at that time point whereas it is present in the previous and succeeding time points. Is it an experimental error? Or, the glucose concentration was not determined at this time point?
3.	In the lines 150-151, the authors claim that the glucose reduction does not influence aclasin expression. Based on the data from Figure 1, I think this conclusion is a bit inaccurate. The technique used in this study (qPCR) only measures the steady state levels of mRNA present at that point and not transcriptional control. Hence the conclusion should be modified to state that the “steady state levels of aclasin does not change with glucose depletion”.
4.	In Figure 2A, aclasin expression at 47 C from 30 min to 60 min reduce by ~50%, whereas the same for 37 C reduces by ~3-4 fold. The authors need to discuss the significance and/or explanation of this observation in their results section.
5.	Figure 2C: the authors do not provide any possible explanation for the observed reduction in the levels of cat2 from 5 mM to 10 mM H2O2. Ideally, we would expect stronger induction of cat2 levels in higher concentrations of the oxidant.
6.	The authors need to reinforce the discussion section by mentioning how their study fits into the broader picture in the field. Does their finding lead to further questions which should be addressed in future?
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: OXIDATIVE STRESS AND THE PRESENCE OF BACTERIA INCREASE GENE EXPRESSION OF THE ANTIMICROBIAL PEPTIDE ACLASIN, A FUNGAL CSΑΒ DEFENSIN IN ASPERGILLUS CLAVATUS
Review round: 1
Reviewer: 2

Output:
Basic reporting: •	The manuscript is professionally written and the reading seems fluid for not expert in microbial molecular biology also. It conforms to journal standards. The language is unambiguous at all.
•	The literature is sufficient referenced.
•	Fig.1 _ What’s the experimental condition that was set as control and allowed to calculate and compare the levels of the relative expression of aclasin (2-ΔCt method) at the different time points? This is not clear. If the plot represents instead an absolute quantification among all the samples, please correct the graph axes and figure 1 title.
•	Fig. 1 Legend _ Even though there are not significant differences, the performed statistic method is not indicated. It should be mentioned together with the p value threshold, as the authors already did for the rest of the figures.
•	Figure 2 panel A _ aclasin graph, please indicate that the black bar of the Control (28 °C) used for relative quantification is =1 for the 60 minutes’ condition. I guess so because the black bar average at 30 minutes is higher than 1.0 in y-axis
•	Line 165 _ ‘afusin’ level is not present in the figure. I guess it was written by mistake instead of ‘aclasin’. In fact, there are not following comments on afusin results or primers listed in the submitted table. Please correct it.
Experimental design: •	17 hrs for A. clavatus is the time of culture before treating with different kind of stress.  For co-incubation experiment the authors used the bacteria after 16 hrs + 30 minutes. Are the bacteria in exponential phase growth at that time? Please comment it
•	About heat-killing of B. megaterium, the authors should provide a brief explanation or a reference or previous experimental data that assure there were not bacterial spores at the time of killing at 60°C.
•	It would be nice if the authors confirm in the main text that 2 mM-10mM concentrations are subletal (or including previous citotoxicity exclusion data as supplementary info). This to exclude a decrease in mRNA expression due to cell death.
•	Regarding the co-incubation experiment, did the investigators separate bacteria from fungi before extracting RNA? Or bacterial RNA from the eukaryotic RNA? If yes, please write how in material and method (strainer? Filter paper cut off? other?), on the contrary it would be necessary to describe or to show supplementary data about the specificity of the primers used for the PCR starting from mixed RNA sample.
Validity of the findings: •	The paper is technically sound. Results are expressed clearly. the study design is organically structured and the sections are well linked each other.
•	As the authors highlighted the fungal defensin transcriptional regulation during bacterial interaction is poorly understood, thus the research question is interesting.
•	Data are well presented. Statistics is convincing. Speculation and conclusion are properly confined within the results presented.
Additional comments: It is a paper with basic RNA-only focus, thus the results are not soon useful to the scientific community. On the other hand, the manuscript is well-written and the topic of the research is very attractive for future biotechnological applications.
The data are robust. The methods are good. The limits of the study are clearly described by the authors, but the results are promising and encouraging downstream projects.


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: OXIDATIVE STRESS AND THE PRESENCE OF BACTERIA INCREASE GENE EXPRESSION OF THE ANTIMICROBIAL PEPTIDE ACLASIN, A FUNGAL CSΑΒ DEFENSIN IN ASPERGILLUS CLAVATUS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The revised manuscript is very well written with appropriate references, figures and logical conclusions.
Experimental design: The authors have successfully addressed all the concerns.
Validity of the findings: The authors have successfully addressed all the concerns.
Additional comments: I would like to convey my best regards to the authors for their effort and work.


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

Input:
Title: OXIDATIVE STRESS AND THE PRESENCE OF BACTERIA INCREASE GENE EXPRESSION OF THE ANTIMICROBIAL PEPTIDE ACLASIN, A FUNGAL CSΑΒ DEFENSIN IN ASPERGILLUS CLAVATUS
Review round: 2
Reviewer: 2

Output:
Basic reporting: The additional references now included in the manuscript makes the background and the whole paper more thorough than the previous version.
The figures have been improved and corrected following the reviewers' suggestions.
Experimental design: Methods section is now accurate and unambiguous.
Validity of the findings: The results and the discussion have been improved as well.
Additional comments: The authors were able to reply critically the reviewers and all their comments were addressed.


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

Input:
Title: SOME METHODS TO IMPROVE THE UTILITY OF CONDITIONED LATIN HYPERCUBE SAMPLING
Review round: 1
Reviewer: 1

Output:
Basic reporting: It is clear.
Experimental design: It is clear.
Validity of the findings: It is clear.
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: SOME METHODS TO IMPROVE THE UTILITY OF CONDITIONED LATIN HYPERCUBE SAMPLING
Review round: 1
Reviewer: 2

Output:
Basic reporting: The English of the manuscript is clear to the readers and sufficient references are provided.
The article structure meets the standard and R scripts of the study are provided.
The results confirm the aims/hypotheses of the paper.
Experimental design: The article is novel because it provides modifications to a previous widely used sampling algorithm proposed by one of the authors.

The research questions are well defined and the three problems related to sampling identified in the article are also widely faced in other field studies.

The use of various statistical indices are rigorous and provides useful insights into the validity of the new algorithms the author proposed.

The R scripts provided by the authors enable others to repeat the results.
Validity of the findings: The data, methods and results and robust for the first two algorithms and case studies.

Additional statistical metrics should be provided for the third case study.
Additional comments: In this study, the authors collated, summarized, and extended existing solutions to problems that field scientists face when using cLHS sampling. They provided solutions to optimize the sample size, re-locate sites when an original site is inaccessible, and to account for existing sample data when additional samples are to be taken. The authors also provided the R scripts to facilitate other researchers with the sampling design in the field.

In general, the manuscript was well written and the ideas are novel to the audience in the related fields. I believe that the manuscript will interest a large number of researchers who face similar problems in using the popular cLHS sampling in the field and provide guidance for people who attempt to improve the existing random stratified sampling algorithms.

I have a few minor comments on the manuscript.

Lines 134-135: Why the penalty of missing a sampling from the tail of a distribution is larger than that close to the mode? When the sample has a small distribution, O_i should be smaller, isn’t it?

Figure 1: the legends are too small to read.

Figure 3: Please provide a new subfigure that shows the additional sample data coverage across the study area.

Table 2: Please provide the proportions of the existing sample data coverage. Please also provide the mean values of the ancillary data calculated from the existing samples, additional samples, and total samples as well as the mean values of the ancillary data across the whole study area.

It will be better for the authors to include three separate flowcharts for algorithms 1–3. This way, the readers can understand the algorithms more easily.


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: SOME METHODS TO IMPROVE THE UTILITY OF CONDITIONED LATIN HYPERCUBE SAMPLING
Review round: 1
Reviewer: 3

Output:
Basic reporting: The basic pieces of this manuscript are in place and flow together nicely. There are some grammatical errors (e.g. lines 377-378) that need to be fixed. Another example is on line 403: soil samples are not being re-located, the sampling location is. I recommend one more proof-reading to find these errors. They are small but jarring.

The background on KL divergence was helpful. I suggesting linking it to the concept of entropy if possible in 1-2 sentences and suitable citation. Reviewing the literature on entropy helped me understand how the authors are using KL divergence.

I think that it is important to elaborate on the fact that KL divergence is based on probability distributions--which have to be estimated by histogram / binning when applied to continuous variables. Number of bins, bin size, and interval selection are critical aspects of this step and need to be described. A couple of sentences with a concrete example would likely bring more readers closer to the methods.

It is not clear to me if the statement on line 141 is correct: "...bin size of 25 (effectively 25 quantiles)...". 25 equally-spaces bins dividing a distribution are not the same as the locations of 25 quantiles estimated from said distribution. Is there an assumption that has been left out of the description whereby the bin width is not equal? Please elaborate.

Lines 192--194 are confusing: Gower's (generalized) distance metric applies to collections of nominal, ordinal, and ratio data. What are the differences between "numerical and continuous variables" (line 194)?

The topic of sampling and sample location selection has been heavily influenced by the work of de Gruijter, J., Brus, D.J., Bierkens, M.F.P., Knotters, M. -- consider mentioning some of this work and citing either their book or specific papers.

Tables need to have units listed for each column: Table 2 is very hard to understand due to this omission.

Figure 1 would benefit greatly from labels near color legends. What do they mean and what are the units (if appropriate). Figures 1 [a,b,c] could benefit from a line denoting the 500m buffer. It was not clear at first as to what the non-circular shape represented.

Figure 2, consider changing "sample number" to "Number of Samples".
Experimental design: Commenting on specific methods here since there is no experimental design.

Lines 216-222: Why not just use distance? Converting to a similarity score seems like further abstraction of an otherwise abstract notion. Please elaborate.

Line: 224 What is the significance of the selected threshold? Why is a threshold needed when a ranked list of alternatives is the goal?

Lines 309-322:

This is an interesting idea although without tremendous computing power not likely the be of practical use to the intended audience. Imagine a situation where the scientist has 6 10,000 x 10,000 pixel maps of interest--that is a lot work for a single (?) maximum distance. Given the spatial correlation in most environmental data, 1/10th -- 1/100th of the  original pixels could probably be used to compute "magpd".

Step 2 in this section, is this computed per pixel or over all pixels?

I expect dd to have a very large range and deviate from a normal distribution. Would the equation on line 317 be overly biased by the distribution of distances?
Validity of the findings: Overall this is a nice synthesis of what has been done, what is possible with current software (cLHS package for R), existing shortcomings, and some reasonable improvements.

The paper is written as a technical note but provides very little in terms of concrete example and viable implementation.

A worked example with real data near a cLHS-proposed sampling site would be a nice addition. Ultimately someone using cLHS and related methods should have a practical understanding of the results. Referencing the Brungard and Johnanson paper is one avenue but an additional figure and commentary would be more useful. This example would also provide an opportunity to inject some reality into the discussion: distances a fine way to rank pixels but fail to accommodate our understanding of the world. We do not go out into the wild blindly searching for sampling locations.

As for implementation: would the authors consider submitting material to the cited cLHS package for R? This would be a fine way to make the theoretical available to the target audience. Typically, methods papers cite an implementation that is already been made available or is available post-publication.
Additional comments: I think that the methods outlined in this paper will be of great interest to those in the fields of Earth and environmental sciences. The popularity of the cLHS algorithm is evidence enough.

In addition to my comments above I would like to add one more thing, more of a philosophical comment. Anyone who has worked in the field understands that pre-specified sampling locations are more useful as suggestions. Access, local (unexpected) disturbances, etc. all contribute to re-evaluation of what otherwise seemed like a great place to dig a hole. Furthermore, we never have a complete "stack" of covariates that describe the processes we are trying to wrangle. Therefore, it might be helpful to include a a reminder that cLHS and related sample-optimization strategies leave room for _more_ thought about how limited resources should be spent, and should not replace subject expertise or experience when theory meets practice. I am sure you all can make this point more effectively than I just did here.


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: SOME METHODS TO IMPROVE THE UTILITY OF CONDITIONED LATIN HYPERCUBE SAMPLING
Review round: 2
Reviewer: 1

Output:
Basic reporting: The English of the manuscript is clear to the readers and sufficient references are provided.
The article structure meets the standard and R scripts of the study are provided.
The results confirm the aims/hypotheses of the paper.
Experimental design: The article is novel as it provides modifications to a previous widely used sampling algorithm proposed by one of the authors.
The research questions are well defined and the three problems related to sampling identified in the article are also widely faced in other field studies.
The use of various statistical indices are rigorous and provides useful insights into the validity of the new algorithms the author proposed.
The R scripts provided by the authors enable others to repeat the results.
Validity of the findings: The data, methods, and results are robust for the algorithms and case studies.
Additional comments: The authors have provided detailed responses to comments raised by all the reviewers. The manuscript has been materially improved and become much clearer to the readers after extra figures and tables have been inserted.


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: ASSOCIATION BETWEEN MATERNAL PRE-DELIVERY BODY MASS INDEX AND OFFSPRING OVERWEIGHT/OBESITY AT 1 AND 2 YEARS OF AGE AMONG RESIDENTS OF A SUBURB IN TAIWAN
Review round: 1
Reviewer: 1

Output:
Basic reporting: 1) Abstract: (line 3-42), and in the reporting of the results please consider reporting the adjusted differences in the BMIs and BMI percentiles, rather than just the p-values.

2) Introduction: (line 63), this sentence needs rewording as it is not written in clear English. You cite a recent paper (2016) and suggest that the developmental origins of health and disease has been used "for the past few years". This hypothesis was established by Barker over 30 years ago. You need to make it clear whether you mean the application of this hypothesis to obesity is more recent, or are referring to the original hypothesis.
This article may be useful:  Wadhwa PD, Buss C, Entringer S, Swanson JM. Developmental Origins of Health and Disease: Brief History of the Approach and Current Focus on Epigenetic Mechanisms. Seminars in reproductive medicine. 2009;27(5):358-368. doi:10.1055/s-0029-1237424.

3) Introduction (line 65), remove the word "hypothetical", as a theory is a hypothesis.

4) Introduction (line 69/71), reword this as it doesn't read quite right. Try something like "As there may be a connection between maternal BMI and offspring health, this is a potentially useful means of predicting and preventing child overweight and obesity."

5) Introduction (line 81/83), change to  "For these women, and their future children, it became a priority to identify an alternate predictor of overweight/obesity in their offspring."

6) Materials and methods: Consider putting the information on participants first and measures second. Line 110/112 Consider including numbers and percentages, also please change to "Mothers who were diabetic...... ".

7) Results (line 167/169), for all other variables you report the rates, please report the percentage overweight and obese by maternal BMI grouping as you do for cesarean section delivery.

8) Discussion (line 238),  when you say previously do you mean in Taiwan or do you mean historically? It is unclear.

9) Discussion (line 246), body habitus is jargon, please clarify.
Experimental design: 1) It is not clear that an aim of the paper is to determine a pre-delivery BMI cut point for estimating the risk of overweight or obesity in offspring. Therefore the suggested BMI cut point of 27.15 kg/m2 is surprising. You need to set this up as an aim of the paper more clearly. Lines 90/91 of the introduction I think are suggesting you will try and find a BMI cut off value, but it is unclear.

2) This is the most important comment to be addressed. Out of 1753 records, you only include 101 records in your analysis. Many of these records are cut because you have conducted a complete case analysis. In the text you write that 1331 are excluded because they are missing information on any variable required for analysis. There is insufficient recognition of this fact in your discussion of the results, and the conclusions that you draw. Of those with incomplete data who were excluded, how many had some information, so that you could compare their characteristics with those included in the final analysis. If those who are excluded differ in important ways than those who are included, then your findings may not be generalisable. I think it is highly likely that the sample you analyse is not representative of the group you describe.

3) Expanding on the point above, please provide a short rationale for why you only wanted to look at term children with appropriate weight for GA.
Validity of the findings: The conclusion is overstated and the authors need to be more cautious with their interpretation. The small group of people you end up analysing is likely not representative of the 'healthy mothers who give birth at term in a given hospital to children of appropriate weight for gestational age' you try and study. There is a huge problem with missing data in this study that is not discussed in the strengths and limitations section of the discussion, and that is not addressed in the analysis. Please provide a discussion of the large number of records that were excluded due to missing data and the potential impact this has on the results. If these mothers and their children were more likely to be overweight for example, could it mean the BMI cut off value that you propose may change? Just how sensitive is the BMI cut off value? If possible provide some analysis to show how those included in the final analysis compare to those excluded (you may have complete information for mothers where the child information is missing for example).
Additional comments: I think this is a really novel and important piece of work.  A lot of the comments I have given suggest minor changes.  However, I have major concerns about the large number of records that were excluded due to missing data on all variables. The article would be strengthened substantially by discussing the potential implications of this, and if possible, by comparing those excluded because of missing information on all variables with those included.


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: ASSOCIATION BETWEEN MATERNAL PRE-DELIVERY BODY MASS INDEX AND OFFSPRING OVERWEIGHT/OBESITY AT 1 AND 2 YEARS OF AGE AMONG RESIDENTS OF A SUBURB IN TAIWAN
Review round: 1
Reviewer: 2

Output:
Basic reporting: 1.	Abstract; I suggest authors to include point estimates (beta coefficients) with 95%CI
2.	Weight gain calculation should be reported in the methodology section.
3.	Results; Most of statements form the first paragraph are either duplicates of the methods section or should have been taken to the methods section. For instance, line 154-155, 157-158.
4.	At times authors confuse key the exposure and confounders adjusted for. For instance, line 214-217 “We found that the maternal pre-delivery BMI was linearly associated with the BMI of children aged 1-2 years even after adjustment for birth weight, sex, mode of delivery and maternal pre-delivery BMI.”
Experimental design: 1.	There is minimal information about the infant BMI calculations and classifications. BMI is in children is highly sex and age specific. Thus, it is not clear whether the infant BMI take into account sex and age; whether the CDC or WHO child growth chart was used.  How were children categorised into overweight or obese?
2.	Authors need to justify use of WHO BMI cut-offs for pregnant women. BMI calculated at the end of pregnancy does not show the level of adiposity.
3.	It is unclear how confounders or covariates were chosen. For instance, birthweight may be on the causal pathway between pre-delivery BMI and the infant BMI. Adjusting for mediator variable is not appropriate analysis.
Validity of the findings: 1.	I agree with authors that gathering pre-delivery weight may be feasible in most settings, but it has limited implications: it is too late to prevent maternal obesity and the possible intrauterine effects.
2.	The conclusion is drawn from a small highly selective sample. Only a small proportion of the original sample of children was included.
Additional comments: This study has examined the association between maternal pre-delivery body mass index and offspring overweight/obesity at 1 and 2 years of age among residents of a suburb in Taiwan. As childhood obesity is a serious public health concern, the analysis may provide some evidence for obesity prevention in children.


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: ASSOCIATION BETWEEN MATERNAL PRE-DELIVERY BODY MASS INDEX AND OFFSPRING OVERWEIGHT/OBESITY AT 1 AND 2 YEARS OF AGE AMONG RESIDENTS OF A SUBURB IN TAIWAN
Review round: 2
Reviewer: 1

Output:
Basic reporting: Abstract line 25: change "weight gain in pregnant women" to "weight gain for women"

Abstract line 35: The abstract still reads 101 mother-child pairs, instead of 204.

Discussion line 236: change "that have evaluate" to "that have evaluated"

Discussion line 295: You refer to breastfeeding as a confounder, would it not be a mediator? Overweight mothers are less likely to breastfeed and cease breastfeeding sooner than non-overweight mothers for a variety of social and physical reasons - this in turn can increase the probability that the child will have a higher BMI.
Maternal overweight-->breastfeeding-->child BMI. Care needs to be taken when deciding what is classified as a confounder and what is classified as a mediator - this is needed throughout the manuscript.

Discussion line 297: This sentence is difficult to understand. I think you want to say there was a large amount of missing data, or that a large number of cases were excluded due to incomplete data.
Experimental design: I still have two major concerns with this paper, which should be straightforward to address.

Major concern 1)
I understand that you have restricted the data to create a cohort of "healthy children". However, there are still 102 children excluded because they were lost to follow up after discharge, 64 children excluded because of incomplete medical records, and 7 children excluded because they were missing some maternal information. Some of these children would have contributed to the sample of "healthy children" if there were not missing data. You have some data on these children. It would be interesting to know how the excluded children differ in terms of the characteristics that you do have data on, compared to the children included in the sample. At the very least you need this information for the discussion to make a case as to why we should believe that the findings from your 204 children represent the group they are supposed to.

Major concern 2)
It is not clear what the purpose of the multiple regression model is and you need to state the purpose clearly in the text.

If the purpose is to assess the association between maternal BMI and child BMI/weight, then birth weight is not a confounder - it is a mediator. Mediators and confounders should be handled differently. You will underestimate the total effect of maternal BMI on child BMI if you control for variables on the causal pathway.

 If the purpose is to get the best possible prediction of child BMI/weight, then none of the variables are 'confounders' they are all predictors contributing to the prediction.

I suspect the purpose is to understand the association between the exposure (maternal BMI) and the outcome (child BMI/weight). Therefore you need to identify confounders and control for these only. Anything on the causal pathway - mediators - should only be controlled for if assessing the mediating pathway or trying to estimate the 'direct' effect. If you are controlling for mediators, then delivery mode (caesarean delivery) also needs to be considered as you have it in your data set and you falg it int he discussion as an important variable.

reporting of the 27.15 cut point
Given the positive predictive value of 20% and the specificity of 58%, I believe you should include a more thorough discussion of the merits of using this as a cut point in your discussion section. The majority of children born to mothers with a BMI greater than 27.15 are not obese. But the prevalence of obesity is 5-fold higher among this group. I think there is scope here to add more useful information that just the cut point. Resources might be better targeted to towards women who have a BMI > 27.15 and other characteristics, such as a child with a large birth weight, caesarean delivery and so forth....
Validity of the findings: no comment
Additional comments: Hello,

Thank you for the effort that has gone in to making the changes to the manuscript. As you will see, I still have some comments/suggestions to improve this manuscript. My comment about the characteristics of the children excluded due to incomplete information was not sufficiently addressed in the revision. Thank you again for the opportunity to review this manuscript. I believe it is an interesting piece of research.


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: ASSOCIATION BETWEEN MATERNAL PRE-DELIVERY BODY MASS INDEX AND OFFSPRING OVERWEIGHT/OBESITY AT 1 AND 2 YEARS OF AGE AMONG RESIDENTS OF A SUBURB IN TAIWAN
Review round: 3
Reviewer: 1

Output:
Basic reporting: The reporting is clear and sufficient context is provided. Figure 1 is very helpful for determining who is included/excluded and why. Overall, the manuscript is much clearer now.
Experimental design: The research question is well defined and the analyses are appropriate. The methods are described in sufficient detail for replication.
Validity of the findings: The expanded discussion on the limitations of this study has improved this manuscript. The findings, and the more cautious conclusions drawn from these findings are appropriate.
Additional comments: I have reviewed this manuscript twice already.  The authors' have been very responsiveness to previous comments made and I have no further comments to make.