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 THREAT TO LOCAL MARINE BIODIVERSITY: FILAMENTOUS MATS PROLIFERATING AT MESOPHOTIC DEPTHS OFF RAPA NUI
Review round: 1
Reviewer: 3

Output:
Basic reporting: It is my belief that the manuscript will benefit from revisions of the writing style and use of english. Wording is at times unclear and gramatical as well as spelling mistakes are occasionally present. I list some examples and suggestions below.

-----------
Abstract

Lines 28-30
We have sighted vast fields of filamentous cyanobacteria-like mats, covering sandy bottoms, as well as over dead corals of mesophotic reefs (mainly composed by Leptoseris sp.).

I suggest this could be re-written as follows to improve clarity and also quation if the authors refer to a single species of Leptoseries observed or if different species of the genera were observed in which case spp. should be used instead of sp.

We observed extensive fields of filamentous cyanobacteria-like mats covering sandy substrates and dead corals on mesophotic reefs dominated by Leptoseris spp.

lines 30- 32: “(up to ~100% of total seafloor surface)”
I suggest minor text change as brackets are not the most appropriate way to report this result which can be placed in the sentence and that approximately 100 % is not the most appropriate way to refer the coverage of nearly all of the substrate or at times all of the substrate. It may also be useful to specify the level of coral mortality here as a percentage. I  also suggest relying less on parenthesis throughout the manuscript and specifying results of information in the text. A suggestion is as follows:

“These mats cover up to 100% of the seafloor and also high mortality
of corals occurs between 70 and 95 m depth off Hanga Roa, the main village on the western side of the island.”

-----------
Introduction:

Lines 41 to 43:
I suggest revising
“Mesophotic coral reef ecosystems typically distribute from 30-40 m to over 150 m depth, they  occur in tropical and subtropical waters and are formed mainly by light-dependent corals, but also including other typical reef organisms such as sponge and macroalgae species (Baker et al., 2016).”

To

“Mesophotic coral reef ecosystems are typically distribute from 30-40 m to over 150 m depth, occur in tropical and subtropical waters and are formed mainly by light-dependent corals, but also including other typical reef organisms such as sponge and macroalgae species (Baker et al., 2016).”

Line 58
I suggest the terminology “anthopogenic issues” is inconsistent with the text above which refers to “anthropogenic stressors” at line 49.

Line 82
Typo in referencing - no space between “Nagle” and “&” in “(Nagle& Paul, 1999,…”

Line 82 and line 91
I suggest revising the English usage of “also” in the following sentences:

 “Cyanobacteria have been also directly linked…”
To
“Cyanobacteria have also been directly linked

 “It has been also…”
to
“It has also been…”

Lines 91 and 92
I suggest the usage of plural and singular is confusing in:
“… green algae (i.e., Chlorophytes) are one of the most
common indicator species of eutrophication…”
This could be revised to “… green algae (i.e., Chlorophytes) are
common indicators of eutrophication…”

English usage is incorrect and confusing on lines 94 -96
“…constituting an aggressive competitive species against sensible corals, and having sub-lethal effects over several biological functions of them…”

I suggest revising in a way similar to:

“Most of the species in this group proliferate due to increased nutrient inputs and tolerate a wide range of environmental conditions, aggressively competing with corals, and have sub-lethal effects over several biological functions of corals”

-----------

Results
Lines 146 to 147
It is unclear to me if a single species from each of the genera Pocillopora, Leptoseris and Cycloseris were observed repeatedly. If it is likely that more than one species was observed then spp. should be used instead of sp.

-----------

Discussion
Line 190
Confusing wording and spelling issues in : “…suggested that they are compossed of at least to cyanobacteria”

"compossed" should be spelt "composed"
"to" should be "two"

Line 191
I suggest deleting the word inhabitants in “…microbial mats are typical inhabitants of coral reef systems…”

Lines 193-194
I suggest revising
“…with anthropogenic disturbances that generate an increase in the nutrient supply to the marine environment…” to
“…with anthropogenic disturbances that increase nutrients in the marine environment…”

Line 212
It is unclear if the term “damage” is used to refer to “mortality” of corals

Lines 218 to 219
Wording is confusing in:
“At that opportunity, the tourist number reached ~20.000 visitants per year (Figueroa & Rotarou, 2016).

 I also suggest discussing this in the context of current numbers of tourists and clarifying how this is a proxy for the intensity of anthropogenic stressors. For example has the number of coastal residences increased and has the volume of sewage discharge increased?

-----------

Conclusion
Lines 228 -229
I suggest revising the sentance
“Catastrophic damage was observed at mesophotic Leptoseris sp. reefs off the main village of the island.”
To
“High mortality of Leptoseris sp. was observed on mesophotic reefs off the main village of the island.”
Experimental design: It is unclear from the manuscript if there is a specific design to the surveys. Rather I gain the impression that 56 opportunistic surveys of locations were undertaken as a result of vessels facilitating the surveys. The study therefore undertakes initial descriptions of mesophotic reefs and habitats. It is noteworthy that these surveys identify mats of filamentous algae uncharacteristic on the urbanised western side of the island. However, this is a correlational observation and causality cannot be determined. I suggest the authors can provide greater support for the argument that anthropogenic stressors have resulted in this observation by providing greater historical insight into the trend in  nature and intensity of anthropogenic stresses since 2000, the point in time that a previous observation is referred to at lines 214-218.
Validity of the findings: The study provides a useful description of rarely studied environments and is therefore novel. However, as a result of the nature of the study, the work is largely descriptive with limited insight to causes of mats of filamentous algae.
Additional comments: I suggest the manuscript can be improved with revisions of the text and encourage the authors to support the conclusions with historical insight to the anthropogenic stressors likely to have resulted in mats of filamentous algae and cyanobacteria.


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 THREAT TO LOCAL MARINE BIODIVERSITY: FILAMENTOUS MATS PROLIFERATING AT MESOPHOTIC DEPTHS OFF RAPA NUI
Review round: 2
Reviewer: 1

Output:
Basic reporting: Overall the manuscript has greatly improved. I have a just a couple of small comments on the reporting.
Line 158 - I think the authors can remove the reference to Ford et al. 2021 as the referenced study did not support the authors in identifying their taxa - there are plenty of other manuscripts that assist with identifying cyanobacteria that would be more appropriate to cite here, depending on what the authors used, or the reference could be removed entirely.
Line 198 - please correct spelling of Lyngbya.
Line 223 - I also have one small correction to an earlier comment from myself actually - the correct reference for the added statement on Lynbya sensu lato is Komárek 2016 [Komárek, J. A polyphasic approach for the taxonomy of cyanobacteria: principles and applications. European Journal of Phycology 51, 346–353 (2016)] NOT Duperron et al. 2020 - please ensure to correct this and apologies for this oversight.
Line 259-263 - I find these sentences a bit disjointed and suggest to rearrange (perhaps have all the statement on iron in one sentence, and the shifting N:P ratio is the other).
Line 278 - the study referred to here technically showed the cyanobacteria linked to increased organic matter (not nutrients - though of course the two often are hand in hand). Other references may be more appropriate for linking cyanobacteria to nutrient input.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Well done on the effort and time spent in improving this manuscript. I think this serves as a good preliminary study from which other work can build upon.


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 THREAT TO LOCAL MARINE BIODIVERSITY: FILAMENTOUS MATS PROLIFERATING AT MESOPHOTIC DEPTHS OFF RAPA NUI
Review round: 2
Reviewer: 2

Output:
Basic reporting: I congratulate the authors on revisions made to the manuscript which has been considerably improved since initial submission.
Experimental design: I continue to hold reservations regarding the nature of the study that remains primarily descriptive as a result of logistical limitations to the experimental design that result from the opportunistic video sampling from vessels.
Validity of the findings: The study provides a basic description of the mesophotic reefs of Rapa Nui and it is useful for this to be made available as a baseline reference. However, the strength of this is limited by the limitations to the experimental design and descriptive nature of the study
Additional comments: This manuscript has been well reviewed since it initial submission and has included a majority of the suggestions made by reviewers as well as revisions to read clearly. The study provides a description of habitats that have previously not been well studied and can provide a baseline reference for future work and I agree with the authors that this is valuable. I also agree that the authors have to some extent included quantitative approaches for the assessment of videos, despite limitation to these. My reservations with recommending publication of the work remain that this is primarily a descriptive study without a well defined experimental design and this limits the conclusions that can be made from the results. Therefore the manuscript may not represent the scientific rigour favoured by PeerJ.


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: FULL-LENGTH TRANSCRIPTOME ANALYSIS OF SPODOPTERA FRUGIPERDA LARVAL BRAIN REVEALS DETOXIFICATION GENES
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: As we all know that fall armyworm (FAW), Spodoptera frugiperda (J. E. Smith), is one of the most destructive agricultural pests worldwide. FAW has quickly invaded the Yunnan Province, southern China in December 2018 and causes huge economic losses to agriculture production. It has been classified as the top first destructive pest in China because of the gluttony, strong migration ability and high fertility of FAW. In the past several years, plenty of researches were focused on the toxicity of insecticides to this pest, the monitoring of population dynamic and insecticide resistance, and control strategies in the field in China, but little information of full-length transcriptome analysis of FAW revealed novel detoxification genes.

In this study, the authors used PacBio single-molecule real-time (SMRT) sequencing to obtain the full-length transcriptome profile of FAW larval brain. They found that a total of 18,642 high-quality transcripts were observed with an average read length of 2371 bp, and 11,230 of which were successfully annotated in six public databases. Among these, 5692 alternative splicing events were identified. In addition, five main gene families associated with the detoxification function were classified, and they were 84 transcripts of cytochrome P450s including CYP4, CYP6, CYP9, and CYP12 clades, 54 transcripts of ATP-binding cassettes as A, B, E, F and G groups, 46 transcripts of carboxylesterases including A, B and C families, 33 transcripts of glutathione S-transferases from “Delta and Epsilon”, “Sigma” and “Omega”clades, and 31 transcripts of UDP-glucuronosyl transferases including 1 and 2 families. The methods and the results used in this study are scientifically sound. Their findings will greatly contribute to revealing the resistance mechanisms and further benefit the investigation of gene functions. However, this manuscript has few of errors as details in the lists of major and minor concerns as currently written. So I don’t think this manuscript is suitable for publication at present.

There are some deficiencies as illustrated in the following:
Major concerns:
1. As well-known that the detoxification sites are usually located in the midgut, fatbody and Malpighian tubules of insect, but not in the brain. Please explain why you select this tissue to analyze its transcriptome for revealing novel detoxification genes?
2. In Introduction (Page 3 Line 48), the reference described by “Wang et al. 2021” is not appropriate because it is not the original reference. Thus, I suggest the authors changed it into “Wu et al. 2019; Sun et al. 2021” following as:
Wu Q, Jiang Y and Wu K. 2019. Analysis of migration routes of the fall armyworm Spodoptera frugiperda (J. E. Smith) from Myanmar to China. Plant Protection 45:1-6.
Sun X-x, Hu C-x, Jia H-r, Wu Q-l, Shen X-j, Zhao S-y, Jiang Y-y, and Wu K-m. 2021. Case study on the first immigration of fall armyworm, Spodoptera frugiperda invading into China. Journal of Integrative Agriculture 20:664-672.

Minor Concerns:
1. In Abstract (Page 2 Line 37), the second “to” should be deleted.
2. In Page 4 Line 96, “14L:10D” should be changed into “14 h:10 h (L:D)”.
3. In Page 5 Lines 119 and 120, a space should be inserted in front of “1” and “0.9”, respectively. The same problem in Page 6 Lines 127 and 130 should be revised.
4. In Page 6 Line 142, please add developer information of “FigTree v1.4.3 software”.
5. In Figure 1 A, the Latin names of insect species shoule be in italics.

Recommendation:
I recommend the authors to further conduct the manuscript with Minor Revisions.


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: FULL-LENGTH TRANSCRIPTOME ANALYSIS OF SPODOPTERA FRUGIPERDA LARVAL BRAIN REVEALS DETOXIFICATION GENES
Review round: 1
Reviewer: 2

Output:
Basic reporting: Yang et al has carried out an interesting study to sequence the full-length transcriptome of the major insect pest, the fall armyworm (Spodoptera frugiperda). Through their methods they convincingly identify detoxification genes, although no discussion is given about their novelty. This manuscript utilizes modern fourth-generation long read sequencing to capture full length transcripts and I believe it will be of general interest to the PeerJ readership.

Concerns are as follows:
Title: I am not sure what context “novel” is being used in. I may have missed it (and I apologize if I did), but don’t recall new genes being identified.
Introduction: I suggest the authors separate detoxification enzymes into their metabolic phases in paragraph three. A naïve reader may assume they have similar functions. Enzymes are also listed from line 80 without order.
Line 109: I assume you mean DNA contamination? It would be worth specifying what kind of contamination you sought to identify.
Line 128: rephrase “hmmscan procedure” to include mention of the tool HMMER (if this is what was used) and cite the tool that was used.
Line 152: It would be useful to redefine FAW here as you are starting a new section.
Line 153: Can remove “as the results showed” and simply state the results.
Line 154: Specify the criteria used to determine “full length,” although the reader can guess how this was done (identifying poly-A tails), it is worth guiding the reader along a little more.
Line 155: This sentence is difficult to parse.
Line 160: “comprehensively” is a bit strong of a word to use, suggest softening or removing.
Line 162: should be “Swiss-Prot”
Line 164: Not exactly sure what “the homology distribution of these transcripts was revealed” means. I can take a guess that the authors are referring to the results in Figure 1A where the species designation of the closest BLAST hit for each transcript is plotted? The reasoning behind this analysis is not exactly clear. Why was this carried out?
Line 167: why were “6,715” classified? was this all the genes that had go terms annotated or a subset? From the wording of the sentence if seems as though this is a subset, but I don’t think that is the case.
Line 183: should be “A phylogenetic” rather than “The phylogenetic”

Discussion: The authors seem to make connections between detoxification enzymes based on their discovery dates. I believe it would serve the reader and the paper to have comparisons framed in terms of metabolic phase. This would help to highlight how dissimilar the mechanisms of these enzymes are and why it is important to have good gene models for each of them when it comes to understanding resistance mechanisms.
Line 254-5: “ABCs … act in neutralizing the toxicity of pesticides and metabolites.” Interpretation of this sentence is somewhat true, although misleading. ABCs are a part of the tertiary metabolism and primarily function as transporters, they do not actively “neutralize” substrates. In direct contrast to this statement, ABCC2 is largely responsible for transport of activated Cry toxins into the cell, resulting in cell death. Insecticide resistance is derived from loss of function in ABCC2.
Line 261: “screened out” implies they were removed.
Line 291: ABCs and UGTs are highly dissimilar. ABCs are involved in transport across the cell membrane (phase 3 metabolism) and UGTs conjugate UDP-GA to metabolites to decrease hydrophobicity (phase 2 metabolism).
Figure 1A: The 'species unknown' and S. litura colours are identical.
Experimental design: I see no major problems with the methodology underlying the manuscript, however the expression is severely lacking in the results (it was quite difficult to parse some sentences). Furthermore, I also ask the authors go through the manuscript, especially in the methods, to make sure tools are being cited as I noticed multiple chunks without proper citations or versions (eg. Databases used).
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: FULL-LENGTH TRANSCRIPTOME ANALYSIS OF SPODOPTERA FRUGIPERDA LARVAL BRAIN REVEALS DETOXIFICATION GENES
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: The authors have revised the manuscript according to the reviewer's suggestions and answered or explained the questions one by one. I’m satisfied with these revisions they have made. There are a few minor points that still need some attention in References. Please correct them.

1. The full-name of PNAS should be used as “Proceedings of the National Academy of Sciences of the United States of America”.
2. The “of” should not be capitalized the first letter in “Journal of Integrative Agriculture”.
3. Wu Q, Jiang Y, and Wu K. 2019. Analysis of migration routes of the fall armyworm Spodoptera frugiperda (J. E. Smith) from Myanmar to China. Plant Protection 45:1-6, 18.


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: PREDICTION OF THE MECHANISM OF MIRNAS IN LARYNGEAL SQUAMOUS CELL CARCINOMA BASED ON THE MIRNA-MRNA REGULATORY NETWORK
Review round: 1
Reviewer: 1

Output:
Basic reporting: •	Several minor grammars and typing mistakes found. Kindly proofread the manuscript again during the correction.
•	Line 29-33: Suggest introducing HNSCC first, then only follow by the LSCC (the subtype)
•	Line 33-36: It ranks 22 or 23?
•	Line 37-42: The justification of the study is not clear. What is the standard treatment for LSCC? What are the prognosis and limitation? What is the diagnosis or biomarker for LSCC? This info will be helpful and link with the justification of the need for new molecular markers and therapeutic options.
•	Line 75-78: Please rephrase this sentence
•	Line 107: The P and Q should be lowercase.
•	Line 129: miRNA-140-3p, not miRNA-140
•	Line 129-130: Please put “-” between 5’ and 3’ with the sequence
•	Line 136: CCK-8, CCK 8 or CCK8?
•	Line 138: I don’t understand with this “60min was incubated at 37℃”. Incubated for 60 mins at 37℃?
•	Line 143: I don’t understand with this “cut with trypsin”? Are authors wash/rinse the cells with PBS before using the trypsin?
•	Line 171-172: Please standardize the naming for miRNA, either as hsa-mir-140-3p or miRNA-140-3p or miR-140-3p. I will suggest using “hsa-miR-140-3p”
•	Line 192-211: Please use the name of LSCC cell line, as LSC-1, not LSCC cells
•	Line 253: igenesis?
Experimental design: •	Line 71-74: Total 4 GEO data set were used, or just 2?
•	Line 81: What do you mean by “2 samples from normal group and the tumor group”?
•	Line 110-114: Culture condition for the cells?
•	Line 117: For transfection, the manufacturer will only suggest the range of concentration. Authors need to specify the details like plating cell number/conc, amount/conc of lipofectamine 2000 used, conc of miRNA mimic used, the transfection media, etc for a good reproducibility of the study.
•	Line 123: the reagent for cDNA synthesis is not mentioned. Please include a citation.
•	Line 125: Please double confirm again the cycle condition and mentioned/arrange in a correct order.
•	Line 128:  ΔΔCt need to superscript, or just mention “ΔΔCt” method is fine as well
•	Line 137: Please confirm that the 3*10^5 is 3x10^5 cells/mL or cells/well? Why this number is used? Please justify or support with citation.
Validity of the findings: •	Line 152-160: How about the GPL16770 and GPL6480 that mentioned in methodology earlier (line 72-73)?
•	Line 195-197: The current qPCR data only suggest the lower expression of miR-140-3p in LSC-1 cells. It is too early to conclude that it is involved in LSCC progression unless authors confirm this by using several LSCC cell lines with different stage/aggressiveness or using a xenograft model. Please correct accordingly.
•	Figure 1 & 2: Please correct the figure legend where N and T labels are not necessary.
•	Figure 3 & Line 237: I disagree with the authors where miR-140-3p is the most densely connected species. This reason is too weak to justify the selection of miR-140-3p in the subsequent experiment. How about miR-455-3p and miR-140-5p that also possess a similar density in the connection? The authors also mentioned that these miRNAs may influence the progression of LSCC (line 254-258) but why authors not consider these miRNAs but only focus on miR-140-3p? Besides, miR-140-5p also being reported involved in hypopharyngeal squamous cell carcinoma and tongue squamous cell carcinoma (https://pubmed.ncbi.nlm.nih.gov/26704053/ & https://pubmed.ncbi.nlm.nih.gov/24530397/), where miR-140-5p in fact attracting more attention from me. Suggestion: Authors need to perform additional experiment (qPCR, transfection, proliferation and apoptosis study) for miR-455-3p and miR-140-5p.
•	Figure 4 & 5: The X-axis label is fold change or gene number? I will suggest transforming these data into table formats with p, q, gene number and etc.
•	Figure 4 & 5: The GO and KEGG functional enrichment analysis did not contribute much to the current study. Authors tried to explain the involved pathway with LSCC progression (Line 259-274), however, undeniable, most of the pathway is not directly relate or totally irrelevant to LSCC. This is very strange as the signature KEGG pathways like “Pathways in cancer”, “viral carcinogenesis”, “p53 signalling pathway”, “cell-cycle” and other signalling pathway are not involved. I would suggest authors verify this GO and KEGG analysis with another set of GEO dataset.
•	Figure 7: How authors calculate cell viability?
•	Figure 7 & 8: What is the miR-140-3p status after transfection? At line 202-204, authors claimed that miR-140-3p is higher after transfection but no qPCR data to support.
•	Figure 8: there is no error bar for the bar graph. No axis labelling as well for flow dot-plot graph. How many sets of the experiment was conducted? What is the statistical analysis? How authors calculate the percentage of apoptosis?
Additional comments: •	There should have “space” between the in-text citations and text. For instance (line 33): squamous cell carcinoma (Lampri et al., 2015; Wei et al., 2018; Xiong et al., 2020)
•	Abbreviations: Authors need to define/describe the abbreviations before using them, for instance, QRT-PCR, CCK 8, CRC, PD-L1, etc. If one abbreviation was used, kindly be consistent and use it throughout the text, like laryngeal squamous cell carcinoma (line 18 &19, 34,174, 250, should be LSCC). Similar to miRNA (line 45). Please remove all the unnecessary abbreviation that only used once in the whole manuscript, like HNSCC, SMR,
•	Some of the facts/statement is lacking in-text citation, for instance: line 33-36, 216-217, etc. Please double-check again.
•	Please include the limitation of your study in the last paragraph of the discussion.


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

Input:
Title: PREDICTION OF THE MECHANISM OF MIRNAS IN LARYNGEAL SQUAMOUS CELL CARCINOMA BASED ON THE MIRNA-MRNA REGULATORY NETWORK
Review round: 1
Reviewer: 2

Output:
Basic reporting: English language should be improved. There still contained grammatical errors, typos, as well as jargons such as:
- MicroRNAs (miRNAs) is an endogenous single-stranded noncoding RNA ...
- Additionally, miR-140-3p inhibits the growth of colorectal cancer cells and promote apoptosis by ...
- "igenesis"
- ...

There must have more literature reviews on bioinformatics studies related to LSCC.

"Introduction" is verbose and unclear. For example, the authors started with LSCC, but then they moved to head and neck carcinoma, and then back to LSCC again. The logic is unreasonable and should be re-organized. Also, it is not clear about the rationale and which knowledge gap that the authors would like to fill in this study.

Quality of figures should be improved.

There must have space before reference cite.
Experimental design: The authors merged different datasets without concerning any batch effect removal.

GO database or analysis has been used in previous bioinformatics studies i.e., PMID: 31277574 and PMID: 31921391. Thus the authors are suggested to refer to more works in this description.

Source codes should be provided for replicating the methods.
Validity of the findings: The authors mentioned Fig. 2 is a heatmap, but I see a volcano plot.

The authors should have some validation data.

ROC curve and AUC analysis should be conducted.

The authors should compare the predictive performance to previous studies on the same problem/data.
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: PREDICTION OF THE MECHANISM OF MIRNAS IN LARYNGEAL SQUAMOUS CELL CARCINOMA BASED ON THE MIRNA-MRNA REGULATORY NETWORK
Review round: 2
Reviewer: 1

Output:
Basic reporting: Thank you authors for the correction and there is a tremendous improvement in the quality of the manuscript. Kindly refer to the several follow-up questions:

1) • Line 37-42: The justification of the study is not clear. What is the standard treatment for LSCC? What are the prognosis and limitation? What is the diagnosis or biomarker for LSCC? This info will be helpful and link with the justification of the need for new molecular markers and therapeutic options.

RESONSE: Thanks for the critic, and we concede the previous version is weak. We rewrote this sentence, trying to make it more meaningful. The sentence now is:

“At present, surgery is still the main treatment for LSCC, and the survival rate of LSCC has not improved significantly in recent years, which is caused by the lack of understanding of the mechanism of occurrence and progression of LSCC. Therefore, it is imperative to further explore and elucidate the pathogenesis of LSCC, identify new biomarkers, and study new effective therapeutic targets.”

Follow-up question: Thank you authors for the reply and correction. Kindly include the diagnosis and current biomarker for LSCC.

2) • Line 143: I don’t understand with this “cut with trypsin”? Are authors wash/rinse the cells with PBS before using the trypsin?

RESONSE: Yes, we washed the cells with PBS before trypsin was applied. To make it clearer, we have made the following changes:

    “After transfection with miR-140-3p mimics or control miRNA, LSC-1 cells were washed with cold PBS buffer. Then, LSC-1 cells were digested with trypsin and then washed twice with cold PBS buffer.”

Follow-up question: Thank you authors for the reply. Kindly use the term “detached” instead of “digested”.

3) • Line 117: For transfection, the manufacturer will only suggest the range of concentration. Authors need to specify the details like plating cell number/conc, amount/conc of lipofectamine 2000 used, conc of miRNA mimic used, the transfection media, etc for a good reproducibility of the study.

RESONSE: Thanks for the advice, we revised it with considerations of these recommendations.

“LSC-1 cells (3*105 cells per well) were cultured in 6-well plates overnight. Transfection was ready to begin when cell density reaches 60-70%. According to the instructions of Lipofectamine 2000 transfection reagent, 45 ng miR-140-3p miRNA mimics (miR-140-3p) or a miRNA mimic negative control (miR-NC) was transferred into LSC-1 cells, respectively. All cells were cultured in complete medium for at least 24 h before transfection and rinsed with phosphate-buffered saline (PBS, pH 7.4) before transient transfection. The generated cell clones were tested for stable miR-140-3p expression.”

Follow-up question: Thank you authors for the reply. Please confirm again the amount of miRNA mimics used. How about the amount of Lipofectamine used? Is antibiotics or OPTI-MEM used during the transfection? Please include the information for the manufacturer and catalogue number.

4) Figure 4 & 5: The X-axis label is fold change or gene number? I will suggest transforming these data into table formats with p, q, gene number and etc.

RESONSE: The X-axis label is gene number. Thanks for the advice, we revised it with considerations of these recommendations. In addition, we reperformed GO and KEGG analysis on differential mRNAs to obtain new results, which were modified in the original text.

“Functional enrichment analysis
We conducted GO and KEGG enrichment analyses of the 2758 differentially expressed mRNAs using p<0.05 and q<0.05 as screening conditions. The GO analysis includes three categories: molecular function (MF), cellular component (CC), and biological process (BP). These differentially expressed mRNAs promoted the occurrence and development of LSCC by participating in various BPs, CCs and MFs (Figure 4). Similarly, the results of KEGG enrichment analysis were also included Cell cycle (hsa04110), p53 signaling pathway (hsa04115), chemical carcinogenicity (hsa05204), etc (Figure 5, Table1).

Follow-up question: Thank you authors. Please include the whole result of GO/KEGG pathway analysis (completed with geneID, pathway, p-value, q-value and FC). It is good to have this as a supplementary file. Besides, you need to arrange the pathways based on q-value, not p-value.

5)  Figure 7: How authors calculate cell viability?

RESONSE: We used CCK8 to calculate cell viability.

    “We performed the CCK8 assay to measure the proliferation of LSC-1 cells. Cells were cultured in 96-well culture plate, and the inoculation density was 3*105/well. For cell transfection, cells were cultured overnight. After 48 hours of transfection, 10 mL of CCK8 solution was added to each well, and the cells were incubated at 37°C for another 60 min. The absorbance of the solution was measured at 490 nm by Smart Microplate Reader (SMR) 16.1.”

Follow-up question: Thank you authors for the reply. Please confirm again how you calculate the viability. By relative to the absorbance of control cells (miR-NC or untreated/non-transfected cells)?

6) Figure 8: there is no error bar for the bar graph. No axis labelling as well for flow dot-plot graph. How many sets of the experiment was conducted? What is the statistical analysis? How authors calculate the percentage of apoptosis?

RESONSE: Thanks for the advice, we corrected the picture and completed the necessary information (Figure 9). Flow cytometer was performed to determine the percentage of apoptotic cells. The data were analyzed by FLOWJ software.

    “Figure 9 LSC-1 cells were transfected with miR-140-3p and miR-NC, respectively, and Annexin V-FITC and PI staining were performed to detect the percentage of cell apoptosis by flow cytometry.”

Follow-up question: Thank you authors for the reply. However, the error bar still missing from the bar graph. Any statistical comparison was made between miR-NC and miR-140-3p? Please confirm that the apoptosis rate is calculated by totalling the percentage from UR and LR of the dot-plat graph.

Besides, with merely a 4% increase of apoptosis, do you think it is still clinically relevant to develop miR-140-3p as the therapeutic target as what authors concluded?

7)  Please include the limitation of your study in the last paragraph of the discussion.

RESONSE: Thank you for your question.

    “However, there are still some deficiencies in this study, and further in vitro and in vivo experiments are needed to verify the specific regulatory mechanism of miR-140-3p in LSCC.”

Follow-up question: Thank you authors. Is your data answering the objectives of the study? What is the authors' opinion on its role as a new biomarker as it was not deeply discussed in the manuscript? Besides, from your result, ~4% of apoptosis induction (Fig 9) with ~10% of the decrease in viability (Fig 8) make miR-140-3p a potential therapeutic target?
Experimental design: No comment
Validity of the findings: No comment
Additional comments: No comment


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

Input:
Title: PREDICTION OF THE MECHANISM OF MIRNAS IN LARYNGEAL SQUAMOUS CELL CARCINOMA BASED ON THE MIRNA-MRNA REGULATORY NETWORK
Review round: 2
Reviewer: 2

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: My previous comments have been addressed well.


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

Input:
Title: STRAINFLAIR: STRAIN-LEVEL PROFILING OF METAGENOMIC SAMPLES USING VARIATION GRAPHS
Review round: 1
Reviewer: 1

Output:
Basic reporting: I enjoyed reading the manuscript. It is well written and easy to follow. I have a concern about the presentation of background/literature survey. Strain level abundance estimation is well studied problem and there are numerous algorithms that are designed to achieve this. I didn't see these studies being mentioned in the background. For eg. tools such as StrainPhlan and DESMAN provide strain level abundance. I encourage authors to provide detailed literature review that covers recent advances in the field.
Experimental design: The methods are described in detail.

Although authors have presented the results on simulated and synthetic datasets, the results on the real metagenomic datasets are not provided. Understood, that the composition of such samples would be unknown, but uncovering the relative abundance of known strains with StrainFlair would be very helpful.

As alluded before, authors need to compare their method with some of the existing tools for strain abundance estimation to show that their method provides value over the existing methods. It can be in terms of higher accuracy, lower computer resource requirements or lower runtime. I understand that the authors have done the comparison with Kraken2, but the strain abundance estimation is not the primary use case for Kraken2.
Validity of the findings: The discussion section is well written.

Due to the lack of comprehensive benchmarking, the novelty or the benefit of the proposed method can not be assessed.
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: STRAINFLAIR: STRAIN-LEVEL PROFILING OF METAGENOMIC SAMPLES USING VARIATION GRAPHS
Review round: 1
Reviewer: 2

Output:
Basic reporting: This manuscript presents StrainFLAIR, a promising approach to abundance estimation of bacterial strains in metagenomic samples. The article is well structured, with useful figures and interesting results. The introduction describes the relevance of the research, but a description of related work in the context of strain abundance estimation in metagenomics is missing. For example, I had expected methods like MetaPhiAn2, PanPhiAn, KrakenUniq and many more to be discussed. I was also surprised that the term “strain-level profiling”, which is part of the title of this manuscript, is not defined in the introduction (or anywhere else in the manuscript).

Some sections are well written, while others are rather vague or messy and require further clarification. To be more specific:
- The introduction starts with a discussion of linear reference genomes, stating that "the usage of reference

genomes represented as flat sequences reaches some limits". I'm not sure which limits the authors are referring to.
- The methods section has several subsections, describing the different steps in the workflow. Figure 1 illustrates this nicely, but the overall workflow is not discussed in the main text. I think the methods section would benefit greatly from a description of figure 1, the algorithmic overview, at the beginning of the section. Because this is currently missing, it was unclear what "reads attribution", "path attribution" and "colored path attribution" mean (these are subsections later on in the methods). Also the content of these sections is quite vague.
- The description of simulated datasets is very lengthy and not very clear; I think it would be easier to follow if this section was more concise.
- The last two paragraphs of the discussion were also very vague and require further attention. I think the message can be very strong, but in its current form that is not clear.

I found the figures in this manuscript very helpful, in particular figure 3a. Also fig 3b was useful, but the caption requires some extra attention: the description is not very clear and I think the figure misses an illustration of the second step of the algorithm (last sentence of the caption).

The software and the simulated datasets are provided online, on a well-organized git repository. The only thing that I could not find were the exact commands to reproduce the results presented in the manuscript.
Experimental design: The methods are described in sufficient detail, but some parts of this section are too vague; please see my previous comment for further details and examples.

I have some concerns regarding the design of benchmarking datasets. The strains from which reads are simulated are fully identical to the references used, which is a highly unrealistic scenario. To truly evaluate performance of the proposed algorithm, one needs a dataset that is independent of the reference set used. If I understand correctly, the authors try to tackle this problem by excluding one of the strains from the reference set, but as a result, the algorithm predicts a different strain to be present. To me this seems like a wrong, misleading prediction. I would be very interested to see how the algorithm performs in a setting where the reference set was chosen independently from the simulated data, because this is closer to reality.
Validity of the findings: The results show that on simulated data the proposed algorithm outperforms Kraken2 in many aspects. On real data, however, this advantage disappears. I agree with the authors that it is difficult to evaluate performance on real data, as it does not come with a hard ground truth. But I'm not convinced by the simulated data sets for two reasons: first, because the references used are identical to the strains present, while it is reasonable to expect some mutations here; and second, because a threshold on the minimal number of genes to be present for a strain to have a prediction is set to 50%, but this seems to be based on results in Figure 4. So it looks like the method has been overfitted to this specific data set, making the results less convincing.

The authors compare their methods against one other method, Kraken2, which is among the state-of-the-art methods in metagenomic analysis. However, there may be better candidates as well: KrakenUniq, MetaPhiAn2, or DiTASiC, to name a few. I don't expect the authors to run every available method, but I'm not sure that Kraken2 is the best choice to compare against.

I appreciate the supplementary material, in particular the performance analysis (Table S1). I think this analysis should be discussed in the main paper, also including these statistics for other methods, as this is a crucial aspect of any bioinformatics workflow.
Additional comments: Some remaining comments/questions:

The manuscript uses both the terms indexation and indexing. I'm not familiar with indexation, but I guess the authors mean indexing? Since this is the common term in the field I would suggest to replace indexation by indexing throughout the manuscript.

L115: "minimap2 is used to generate a multiple sequence alignment". As far as I know, minimap2 does not perform multiple sequence alignment. I guess this sentence actually refers to all-versus-one alignment, from which a multiple sequence alignment is inferred?

L134: "continuous paths (successive nodes joined by only one edge)". This definition is not clear, it looks like you define either just paths in the graph, or simple paths.

L162: "the abundance is distributed to each matching colored path relatively to the ratio between them". Intuitively this does not make sense to me: for example, if the ambiguous alignments involve several colored paths, with some of them hardly any unique mappings because the paths are so similar, then the resulting coverages could be very misleading. Could you explain why this approach makes sense?

L179: "the gene abundance is estimated by the mean of the nodes abundance". Do you take node lengths into account by taking a weighted average here?

L180-182: "In order to not underestimate ... without the nodes of the path never covered by a read is also outputted". Could you also address this problem by looking at the median node abundance instead of excluding certain nodes?

L335: "a key resolution, for instance opening the field of precision medicine". Very vague statement, could you be more precise?

L363: "seems more adequate and closer to what is expected in reality" -> Compared to what?


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: STRAINFLAIR: STRAIN-LEVEL PROFILING OF METAGENOMIC SAMPLES USING VARIATION GRAPHS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have addressed many of my concerns satisfactorily: the manuscript reads very well, the background and aims are clear and the methods are well described. Supplementary results now include benchmarking results on other tools, which is very informative. However, I still have some major concerns regarding the experimental design and conclusions, which I will explain below.
Experimental design: In metagenomic profiling, the sample composition is unknown. Hence, the reference set cannot be the same as the queried genomes, it has to be much larger. In line 319, regarding the experiments on real data, the authors write that all known 91 strains were included "to mimic a classical StrainFLAIR use case where the queried data is mainly unknown". But this still does not reflect a realistic scenario, because one would not know the selection of 91 strains beforehand. In general, the databases used for such analysis are huge. Would StrainFLAIR be able to index and make predictions for thousands of genomes, how does it scale? And what do the predictions look like if one uses a larger reference set?

L341: "Kraken2 affected non-null abundances to all the references and thus could not be used to definitely conclude on presence/absence of strains in this sample". Also a similar statement in line 389-390. However, commonly, one applies a minimal abundance threshold to filter out such false positive calls, so these statements are not entirely fair. Moreover, I could not find a description of how the relative abundances are computed from the read counts. Are read counts normalized by genome size or aligned length?

This also relates to the colored path attribution step in StrainFLAIR: shouldn't unique read counts be normalized by alignment length here, before computing the ratio between colored paths? Or is it the ratio of alignment paths? It would be helpful to distinguish more clearly between alignment paths and reference paths, instead of just "paths" which can mean either.
Validity of the findings: Although the conclusions and discussion have improved considerably, there is one claim (made at several points in the manuscript) with which I strongly disagree:
L87-88: "Notably, we could also identify close strains not present in the reference"
L293: "Labelled as K-12, it also gave close estimations for BL21"
L300-301: "the proportion of detected specific genes for each strain seems to highlight a pattern allowing to distinguish present strains absent strains and likely new strains close to the reference"
L377-379: "StrainFLAIR was able to highlight the presence and also to estimate a relative abundance for a strain similar to existing references, but absent from these references"

I see that in these specific data sets, the proportion of detected genes provides an indication of the similarity of the true strain to the predicted strain. But what if the (falsely) predicted strain (in this setting K-12) is also present? And what if there are multiple unknown strains? Or if the unknown strain is equally similar to multiple references? If I understand the approach correctly, you would not be able to tell the difference. In reality there will be many genomes that are not present in your reference set, which would affect the predicted abundances substantially, and I don't see how the proportion of detected genes will help with this. I find a statement like "StrainFLAIR can identify strains not present in the reference" misleading and it seems that using an incomplete reference set can lead to highly skewed abundances due to false positives, like the K-12 predictions in the absence of any K-12 in the sample.

What I also don't understand is why the authors do not include DiTaSic results (from the supplement) in the manuscript. Table S5 shows that these results are far better than Kraken2 and also better than StrainFLAIR. What is the reasoning for not including these results?
Additional comments: Some minor textual things:

L30: "later" -> latter

L72: "... are based on de novo approaches using known core genes and a single reference genome" - I'm confused as to why you call this de novo of it's using a reference genome?

L98: I think it would be helpful to specify here that you mean strain-specific reference genomes

L163: "In the meanwhile, ..." -> Meanwhile, ...

In many places in the manuscript you write about "specific genes". But what do you mean by "specific"?


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: STRAINFLAIR: STRAIN-LEVEL PROFILING OF METAGENOMIC SAMPLES USING VARIATION GRAPHS
Review round: 3
Reviewer: 1

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: Thank you for addressing my questions and concerns, I have no further comments.


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

Input:
Title: REEF MANTA RAYS FORAGE ON TIDALLY DRIVEN, HIGH DENSITY ZOOPLANKTON PATCHES IN HANIFARU BAY, MALDIVES
Review round: 1
Reviewer: 1

Output:
Basic reporting: As below.
Experimental design: As below.
Validity of the findings: As below.
Additional comments: I very much enjoyed reading this study on what is clearly an incredible natural event. It's an excellent addition to the general literature on manta ray ecology and behaviour, as well as improving understanding of the drivers of this economically important feeding aggregation. The study is well-written and clearly presented.

As someone coming to this 'fresh', without any personal knowledge of Hanifaru, I do have a few general suggestions on additional context for the broader readership.

While this was a short-term study, it'd be helpful to have more background on why this specific timing was chosen. Do these conditions only take place on a seasonal basis at a specific moon phase? That would be useful ancillary information for interpreting manta residency and movements with regards to the site. Guy's thesis is cited for more information, but it would still be good to include summary information within this study.

Manta metabolic requirements are an important discussion point (and in Fig. 4), but the assumptions underlying the 2016 paper's calculations do not appear to have been critically re-examined here for this new geographical area and study population. For instance, I note that the 2016 estimate included assumptions on manta sizes, body mass, and swim speeds while feeding. I realise this wasn't a key part of this study itself, but it would be worth at least mentioning this in the Discussion, or preferably adding a Methods subsection that demonstrates the applicability of this value to Hanifaru mantas.

I like the use of hand-towed nets! However, it'd be good to include a quick mention somewhere of whether this may affect the results in any way – how fast are Undinula? Can they evade a small net? Does their behaviour influence manta feeding tactics at different densities? The aims don't explain 'why' you were anticipating why a change in manta tactics might occur.

The Results summary is good, but it would be useful to include more specific values, potentially on Fig. 3 and in the text: what was the lowest biomass at which mantas were observed feeding? The highest biomass at which mantas were absent, or present but not feeding? It's hard to pull this from Fig. 4. Similarly, I don't see non-response-scale data on manta abundance. It's all in the Supplemental data of course, but adding these data to the main MS would be helpful.

The importance of these results to the management of the site is mentioned in the Conclusions (L416–17). My understanding is that Hanifaru is an important area for tourism, and that access and activity restrictions are in place. It would be good to be more explicit with recommendations on how this new understanding of plankton and manta ecology could inform management and tourism activities going forward.

Specific comments:

L37 – I'd reverse this to say that high zooplankton biomass is a significant predictor of feeding events (i.e. L257–258).

L46 – This sentence could be broken up for clarity.

L94 - Prebble et al. (2016) is a conference abstract. Better to use a published work here.

L111 – Given that Hanifaru is an important tourism area, it'd be good to include more specifics on both the situation and aims in terms of local conservation and management.

Figure 3: Surely manta behaviour is correlated with zooplankton biomass, not a 'predictor' in real terms?

Figure 3b: This seems like a very confusing way to present these data – with the biomass on the vertical axis it looks like 'real' data, i.e. Feeding at ~100–~310 mg.m3, but this is actually showing the confidence intervals? A figure that accurately summarises the raw data would be preferable for interpretation. I have the same basic comment about Fig. 5.

I hope this is generally helpful.
Simon Pierce.


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: REEF MANTA RAYS FORAGE ON TIDALLY DRIVEN, HIGH DENSITY ZOOPLANKTON PATCHES IN HANIFARU BAY, MALDIVES
Review round: 1
Reviewer: 2

Output:
Basic reporting: General comments
This study analyses zooplankton samples taken in the presence and absence of feeding/non-feeding and solitary/grouped reef manta rays (Manta alfredi) in the Maldives to achieve the stated aim to improve understanding of the feeding dynamics of the species. Zooplankton samples were taken in an semi-enclosed bay over a week or so when mantas were present and conducting various behaviours in addition to when they were absent. The zooplankton biomass, size structure and community composition were determined to establish critical prey density thresholds, whether prey density influences feeding strategy, and, lastly, whether behaviour changes with zooplankton characteristics.

This is a well written paper that presents results of value to the field. The design of the study is good, not least in trying to mitigate the logistic difficulties of working on wild foraging ecology of large planktivores. The methods used are appropriate to this high degree of difficulty. The zooplankton analysis methods in particular are comprehensive and open up new questions and potential comparisons with other plantkivores that progresses the field. I recommend the paper for publication after some minor additions and points of clarification.

Specific comments (by line number)
L38. Abstract. Need to state that the threshold is expressed in mg DW m-3.
L59. Need to clarify that it is “often difficult for researchers..”.
L60-61. Need a review reference or two to substantiate this statement given it refers to planktivores generally.
L75-76. Need to reference other studies on planktivores where this has been done to support your view that this is a valid approach.
L143. What was the tow speed? Even an estimate would be useful because it may be useful to readers to be able to assess the possibility of zooplankton escaping small-diameter nets at slow tow speeds. Check the net diameter is given too.
L143-145. Need to give the numbers of replicates taken per feeding/non-feeding event and for each behaviour type if this was possible, or state why it wasn’t and what the limitations might be as a consequence (can add to Discussion of limitations). Important for assessing the representativeness of a sample assigned to a feeding/non-feeding event or behaviour type.
L159. The source of tide data is given as “..from a local government representative”. However this gives no opportunity for precise replication. Need to give the government department name at the least.
L181. Why was a cactus spine used? Is this important in some way to the analysis? Please clarify or remove.
L267-271. Need to discuss or at the very least acknowledge the sampling limitations. For example, that zooplankton biomass was not different for feeding strategies or solo or group behaviour may be down to too few samples and lack of replicates for particular behaviour events. Add sentences to Discussion to acknowledge potention limitations.
The zooplankton size structure and community composition results are strong elements of the paper. These are not easy to quantify but this is done very well; well done.
L359. Usually this net energy gain/loss relates to “daily energetic requirements”.
L375. To help future research, it would be useful to suggest what you think would be a suitable sample size to test the hypothesis.
L379-396. The Discussion would benefit from broadening to include a few sentences to compare what was found with other large planktivores for which copepods have been identified as being important identifiers of feeding conditions and for which critical swithing thresholds have been determined. For example, did those swarms also comprise of larger numbers of copepods with greater body sizes in the mid-range of the size distribution? Could there be general rules across planktivores?
L.400 onwards. There is no critical discussion of the study’s limitations. This should be added with a few sentences, particularly on sample size and replicates. It is mentioned that hand-towing of a plankton net is not mentioned in the records, but it isn’t discussed why this might be? It is possible hand-towing isn’t used because too many large zooplankton escape as tow speeds are slow. So it would be useful to provide your view on the limitations and why in your situation it was a valid technique for sites not allowing engines. Also, it may also help other researchers to know how in your experience vertical samples could be taken at such sites.
Fig. 1. Adding to the map a schematic of the main water movement directions/currents occurring in the study site (Hanifaru Bay) would help readers to understand the complexity of the site.
Fig. 7. Explain in the legend the term biovolume and its units, that is, how specifically it is different from body volume.
Experimental design: See previous section
Validity of the findings: See previous section
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: FIRST REPORT OF LEOPARD FOSSILS FROM A LIMESTONE CAVE IN KENTING AREA, SOUTHERN TAIWAN
Review round: 1
Reviewer: 1

Output:
Basic reporting: 1) Language: the paper must undergo professional editing for English.
2) Literature cited: the manuscript requires many more references, especially in the methodological section, the taphonomic perspective in the discussion, and biogeography.
3) Field background: a plan and a section of the cave is required.
4) Why has the sediment not been dated, using 14C (for the overlying deposits), U-Th, or OSL?
5) Data summary and presentation is often faulty, see comments on pdf
Experimental design: The research is meaningful, as it potentially describes a new top predator taxon in a major location in Asia where it was not known before.
The investigation is not rigorous: I'm not sure that the GMM analysis (or the metric one) provide unequivocal taxonomic evidence that the teeth have belonged to a leopard. Consider using LDA or at least anova to separate groups. Also, zooMS and aDNA are basic in identifying new taxa today, especially when the morphological analysis is not equivocal.

The digitization process of the GMM is not clear: add a figure.

The pervalence of morphological criteria in each species is not clear.
Validity of the findings: Statistics are far from being state-of-the-art. The authors would benefit from a more thorough treatment of both their metric and GMM data.
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: FIRST REPORT OF LEOPARD FOSSILS FROM A LIMESTONE CAVE IN KENTING AREA, SOUTHERN TAIWAN
Review round: 1
Reviewer: 2

Output:
Basic reporting: The present manuscript tried to identify the species of the fossil felid teeth from Kenting area. The morphological features were well described and combined tests including linear measurements and geometric morphometric methods were well done. I believe this manuscript is worth publishing after minor revision for several points.
Experimental design: There may be other ways that provide more robust evidence (such using discriminant analysis or comparison of Procrustes distances etc.), but the study design is enough to the conclusion of the present manuscript.
Validity of the findings: The authors used several different methods to distinguish the taxon of the fossils. Accordingly, I consider the result is valid enough.
Additional comments: Major points:
The term “geomorphometry” possesses several different meanings. I suggest using “geometric morphometrics” which is the special term indicating landmark-based morphometrics.

When comparing extant and fossil specimens (or bite marks in fossil bones), it might be cautious that clouded leopard teeth (especially canine) have been evolving at a very fast rate (Harano and Kutsukake, 2018). However, I feel a discussion about it is not essential.

Minor points:

Line 50: I am afraid that “Ai Kawamura” is a mistake of “Yoshinari Kawamura”. Ai did not belong to Aichi University of Education (She belonged at Osaka City University during her PhD course and moved to Toyama University). Otherwise, affiliation and prefix may be changed.

Line 93: I think this sentence needs a citation.

Line 135: “TPS software” should be clearly explained as TPS Dig etc.

Line 150-151: This explanation seems unclear. It might be better to explain it clearly.


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: FIRST REPORT OF LEOPARD FOSSILS FROM A LIMESTONE CAVE IN KENTING AREA, SOUTHERN TAIWAN
Review round: 1
Reviewer: 3

Output:
Basic reporting: -
Experimental design: -
Validity of the findings: -
Additional comments: The statistical analysis of the study is appropriate however some points need to be clarified and clearly described.
1.	The authors indicated that the all PCA in this study were performed using R (line 149-150) thus it is not necessary to mention about R in line 157, 163, 165 with similar sentences.

2.	A brief of the advantage of using two rounds of PCA should be described in term of statistical concepts as it will help some audiences who are not familiar with such technique to understand.

3.	Line 181. Do not need to address Fig. 6 as this figure is the result.

4.	Line 230. It is not necessary to address “see Material and methods”. This term should be removed.

5.	The author showed Fig 9 but no description for the construction of bivariate plots and also the regression analysis. These analyses should be described in MM. Results from analyses should be included in the results sections.


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: FIRST REPORT OF LEOPARD FOSSILS FROM A LIMESTONE CAVE IN KENTING AREA, SOUTHERN TAIWAN
Review round: 1
Reviewer: 4

Output:
Basic reporting: The submitted manuscript yields very important data on the occurrence of leopards in Taiwan during the Late Pleistocene, probably during the Last Glacial Period, when Taiwan was connected with the continental Asian part. Although it is so far an isolated fossil record of juvenile individual, it is a clear evidence for migration routes of these felis cats and their biogeographical distribution in the past. From this point of view, the presented data are very fundamental for our knowledge of the Quaternary megafauna.
Since I am not native speaker, I am not able to consider the level of used professional English, but I think it is clear, unambiguous, and technically correct, even though there are a few imperfections (I think) in some places (see attachment).
The used literature references are relevant and appropriately reffered in the vast majority of cases. I only found cited works of Pocock (1917) and Oken (1816) in the "systematic part" but not listed in the "References" section, whereas other two works (Fischer de Waldheim, 1817 and Linnaeus, 1758) are reffered there... (see attachment).
The article structure, figures, and tables are professional and relevant, meeting the standard for this type of publication. Raw data are shared and their analyses support the obtained results. Based on these, two hypotheses are propounded. For comments to their relevance, see attachment.
Experimental design: The manuscript presents an original primary research. The main research questions are well defined, with the relevant introduction to the problem of study. The investigation meets all scientific and ethical standards. The analythic methods described are appropriate, producing relevant proof data and results that are also useful for another investigators.
Validity of the findings: All obtained data and results of all used statistic analyses support the attribution of found felid teeth to Panthera pardus. Based on that, I do not understand why authors in some place abandon this correct assignation and write "we are only able to assign them to the subfamily of Pantherinae", when the determination of fossil record under study is unambiguously supported by metrics, morphology, and statistics.
The conclusions, in my opinion, need major revision. The hypothesis (1) could be focused rather on the individual variability than on the ontogenetic one and the hypothesis (2) is rather speculation, since uncovered teeth are only so far isolated fossil record, too small for the thinking about a dwarfism, especially when Taiwan was probably a part of continental Asia minimally during the Last Glacial ... In spite of that, I think the article should be accepted.
Additional comments: Although isolated, but very important fossil record of leopard in Pleistocene of Taiwan is supported by well realized morphometric and statistic analyses. However, hypothetical impact of this find should be re-interpreted. All my comments and remarks are highlighted directly in the attached text of manuscript.


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

Input:
Title: FIRST REPORT OF LEOPARD FOSSILS FROM A LIMESTONE CAVE IN KENTING AREA, SOUTHERN TAIWAN
Review round: 2
Reviewer: 1

Output:
Basic reporting: I feel all concerns I had pointed out were corrected. I have no further comments.
Experimental design: The design is enough to the present study.
Validity of the findings: I feel the result is valid enough.
Additional comments: I have 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: FIRST REPORT OF LEOPARD FOSSILS FROM A LIMESTONE CAVE IN KENTING AREA, SOUTHERN TAIWAN
Review round: 2
Reviewer: 2

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


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

Input:
Title: FIRST REPORT OF LEOPARD FOSSILS FROM A LIMESTONE CAVE IN KENTING AREA, SOUTHERN TAIWAN
Review round: 2
Reviewer: 3

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comments
Additional comments: I found only a few failures in this re-worked manuscript, all highlighted in the reviewed file.


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: COVID19.EXPLORER: A WEB APPLICATION AND R PACKAGE TO EXPLORE UNITED STATES COVID-19 DATA
Review round: 1
Reviewer: 1

Output:
Basic reporting: Abstract:
The first sentence could be rewritten. ‘Beginning at the end’ reads a little oddly for the intro of the abstract. Maybe something like “Appearing at the end of 2019…”
Remove ‘I Hope’ from last sentence. Make the abstract confident of the work presented.

Introduction:
Paragraph 2: R is also a programming language and not just an environment. - Insignificant point
Introduction is too short.
Can you please provide information on other examples of similar systems and how this improves upon those such as: https://coronavirus.jhu.edu/map.html / https://covid19.who.int/ / https://www.worldometers.info/coronavirus
Can you expand the aim of this project/paper?
Experimental design: Methods:
1.3: You are repeating some points from above. I understand why but the use of the same citations makes it too repetitive on the same page. Can you remove or shorten the first mention of case underreporting?

"As such, I consider confirmed COVID-19 deaths to be a much more reliable indicator of disease burden than 94 confirmed cases” : I imagine there are others which agree here so could you cite something such as  https://www.nature.com/articles/s41586-020-2918-0

I understand these are estimations or examples but using an estimated ‘IFR’ ratio in the middle of another estimation function could be clarified further. Maybe detail IFR first?

“covid19.Explorer permits the user to specify a time-varying IFR by fixing the IFR at each quarter” - This could be linked to outbreaks and stress on local health services. IFR are likely to be demographically dependant. For example, some states have older and poorer populations but if a single IFR is used across the US, can this have influence?

Line 129:  “Since the true number of new infections cannot exceed the number of confirmed cases, logic dictates that this ratio must have a value between 0 and 1.” I do not understand. Please clarify.

Line: 154: “.... simply assumed that the population of NYC 155 has changed between 2015 and 2020 in proportion to the rest of the state.” I understand the reasoning but NYC is a very different place with very different attractions as compared to NY-State. Is there anything to back this up?

Section 1.3/1.4:
Did you look at other types of regression now that we have so much data over the past year?

The paper states that small or arbitrary changes in IFR have major unfluence on the model output but the system entirely hands over the responsibility of selecting an appropriate IFR without much guidance or example (Specifically for the US). I think IFR selection could do with some defaults computed from some previous studies, some already reported in the paper. The website is presented to a user with defaults but they are not described.

 Line 179 - There have been a number of studies into the under or over reporting of COVID-19 related deaths - There are many points in the methods which require citation (example https://jamanetwork.com/journals/jamainternalmedicine/fullarticle/2767980)
Listed are a number of assumptions are why they are mostly incorrect or incomplete. Can you comment more on the impact of these assumptions?

Section 1.6:
Noted again are how some assumptions are likely not adequate but not backed up or taken further. Please either rewrite or state evidence for their continued use after discreditation.

Section 1.7:
If you take into account the jurisdiction population for computing excess deaths, you may need to take into account state-wise migration events throughout the pandemic. Cities and some regions have seen large levels of migration: https://academic.oup.com/jn/article/150/11/2855/5896932?login=true
Validity of the findings: Results:
Section 2.1:
Herd Immunity often does not take into consideration loss of antibodies and T-cells over time and therefore this 67% population figure is not the full picture: https://www.nature.com/articles/s41591-020-0965-6

Section 2.2:
Figure 7.a: Can you expand on why the IFR does not correlate well to the second and third death ‘spikes’?

Section 2.4:
Deaths of young adults 15-24 peaking while out of session in the summer have the influence of more outdoor activities and therefore more ‘risky’ situations. This same age group is seen to be little medically affected by SARS-2, therefore a better analysis would be of other age groups as the 15-24 age group would act as a vector of transmission to these older groups.

Discussion:
Line 317: Minor point - The pandemic is ongoing and likely to persist past 2021.

Line 320: Please elaborate why “understanding the temporal and geographical dynamics of SARS-CoV-2 320 infections and COVID-19 deaths remains a critically important endeavor”? (I agree but why?)

Line 335 “(based a hypothesis or external information)” - Do you mean “based on”?

Line 336: Assumptions are also parameters in this context?
Additional comments: Final Points:
Especially for a web-service, there should be an option for colour-blind friendly colours.

Please give some detail for the default parameters presented to the user on the website.


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

Input:
Title: COVID19.EXPLORER: A WEB APPLICATION AND R PACKAGE TO EXPLORE UNITED STATES COVID-19 DATA
Review round: 1
Reviewer: 2

Output:
Basic reporting: The research article is well structured and written in easy to understand language for international readers.
The author has provided all the supplemental materials (including codes).
The figures are legible and of good quality for publishing.

One very minor edit to suggest is:
In some sections the author has addressed the steps as 'I have done this....' while in the other section he has addressed as 'We have done this....' - its better to be consistent.
Experimental design: The research, code and web product are very useful and definitely adds up a lot to the ongoing research in this field.

Author has laid the whole paper in a manner that is easy to understand and implement, with flexibility to customize the parameters and build your own insights.

Various graphical outputs of different conditions gives an outlook based on current data and future scenario - which is very useful from a research point as well as from decision making aspect.
Validity of the findings: The results, discussions and overall findings have a good basis to support with proper references (including recent ones).

The tool will be very useful to build new insights and initiate future advanced research.
Additional comments: Great work - looking forward to see your published articles.


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: COVID19.EXPLORER: A WEB APPLICATION AND R PACKAGE TO EXPLORE UNITED STATES COVID-19 DATA
Review round: 1
Reviewer: 3

Output:
Basic reporting: 1. What are the advantages of covid.Explorer over the existing R packages that also aimed to provide analysis and visualization of the COVID-19 data?  What has been missing in those R packages that the author identified and filled in this covid.Explorer. I would suggest the author to provide sufficient literature review of the existing packages and explicitly identify the knowledge gap in the field in the introduction.
Experimental design: 2. Line 129 “Since the true number of new infections cannot exceed the number of confirmed cases…” Does the author mean the opposite?

3. Could the author explain in Figure 1 a) and Figure 2 a), what are the models for the blue lines.

4. For the latest k days estimates of new infections, has the author considered, on top the estimated ratio of confirmed cases to new infections from the sigmoid shape, any other factors that may change that ratio? And how would that affect the accuracy of the estimates.

5. Could the author give more details on estimating the confidence interval of IFR?
Validity of the findings: 6. Could the author give a detailed description of the U.S. data? What are the descriptive statistics of the characteristics of the data that has been used to illustrate the point?
Additional comments: In this manuscript, Revell developed a flexible R package and web portal to analyze and visualize the COVID 19 data of the United States. It has the ability to estimate the “true” cases on a certain date based on the number of deaths from a date that is k days before.


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: COVID19.EXPLORER: A WEB APPLICATION AND R PACKAGE TO EXPLORE UNITED STATES COVID-19 DATA
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The corrections and changes made in accordance with the first review have been much appreciated.
I am very happy with the current manuscript and have no further comments to make.


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: COVID19.EXPLORER: A WEB APPLICATION AND R PACKAGE TO EXPLORE UNITED STATES COVID-19 DATA
Review round: 2
Reviewer: 2

Output:
Basic reporting: 1. Line 141 no need to spell out the full name of IFR

2. Line 178 “Figure 1)” -> Figure 1 b).

3. The caption of figure 1 b) where the author described the CDR, “Ratio of daily
estimated infections over confirmed SARS-CoV-2 infections (grey points … (CDR)”, I think the author really means the opposite?

4. Line 187-189, “…shows daily estimated infections (under our model) / confirmed cases for all U.S. data…”, I guess the author would want to say “…confirmed cases / estimated infections (under our model)…”?

5. Line 194, “…a constant IFR value or function.” I don’t follow what is the relationship between CDR and IFR, could the author explain?

6. Figure 4 and 5, depending on how difficult it is, could the author make the color of states by the # of estimated infections, rather than by the spatial relationship to the state of Florida? It’s not intuitive to understand the color when it comes to the severity of the pandemic by states.

7. Line 376 add “at time t” for definition of R_t
Experimental design: 1. I am still having trouble understanding figure 1 a). What does the author want to illustrate with this figure? E.g: is the author saying that the IFR is changing over time or changing over # of deaths? Is this trend over all the U.S. or specific to any region? How about population structure? Where does this user defined IFR come from? Has it been suggested from literature? Has the author estimated it from data? I would suggest the author to be more specific. Also the label of y axis and legend needs to be corrected accordingly.

2. Line 181-186, a suggestion that would make the argument stronger, that the author could provide a goodness-of-fit statistics on how good that sigmoid function fits the observed CDR over time. And it will be helpful to understand the sigmoid relationship if the author could give the details of the model, for example, the covariates and coefficients.
Validity of the findings: No comment
Additional comments: I think the author has responded to my comments well. The manuscript is in good shape, with sufficient background, assumption and description of the results. Only some minor revisions is needed. I am looking forward to the publication of this manuscript.


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

Input:
Title: A MOLECULAR PHYLOGENY OF THE CHINESE SINOPODA SPIDERS (SPARASSIDAE, HETEROPODINAE): IMPLICATIONS FOR TAXONOMY
Review round: 1
Reviewer: 1

Output:
Basic reporting: I indicated in the pdf file some unclear statement or too complex sentences which can be split to improve the flow of the text

Otherwise, no principal comments
Experimental design: I highly recommend to use IQtree for ML analysis, please add a comment on bootstrap parameters

You are not allowed to designate a new HOLOTYPE
Validity of the findings: The study has not clearly separated results and discussion. As a result Conclusions replace discussion (too long, partly detailed, no clear, strong summary)
Additional comments: Congratulation to valuable contribution which makes classification much stronger.
Please, consider the comments in enclosed pdf file


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 MOLECULAR PHYLOGENY OF THE CHINESE SINOPODA SPIDERS (SPARASSIDAE, HETEROPODINAE): IMPLICATIONS FOR TAXONOMY
Review round: 1
Reviewer: 2

Output:
Basic reporting: 1. Just a few typos and using terms errors which are mentioned in the text directly
2. one reference missing
3. structure and figures have high quality
4. the taxa names in the Phylogenetic tree should be edited and fonts enlarged for better tracing by the reader, one suggestion is to split the tree into two pages
Experimental design: good but would be better if they add data of Sinopoda species from outside China indeed. But still useful and informative for the development of the study area
Validity of the findings: worth publishing, but consider facts below please:
1. using the term "first study" is not necessary since Sinopoda was treated in major works before
2. Your finding suggests polyphyly of okinawana-group, you may remove those species which you find out of the clade and propose new species group names for them. But you can not reject or do any kind of nomenclatural act for species group name. I suggest let the species-group name okinawana remain for those species from Japan (where the original sp-group name proposed).
more info inside the text's comments.
Additional comments: as bove


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 MOLECULAR PHYLOGENY OF THE CHINESE SINOPODA SPIDERS (SPARASSIDAE, HETEROPODINAE): IMPLICATIONS FOR TAXONOMY
Review round: 1
Reviewer: 3

Output:
Basic reporting: Review for peerj-57907

In this manuscript, the authors study the spider genus Sinopoda with a focus on Chinese species. Using a Sanger-based molecular phylogeny, they propose a first molecular phylogeny for the group. This phylogeny allows the authors to revise the taxonomy and propose taxonomic changes or create new species groups.

The study has many merits as it represents a first attempt to fill in the knowledge gap of spider phylogenetics and taxonomy, especially for a complex group like this one. I have learnt a lot because I’m not a spider specialist. So I think the paper has a great potential for a large audience since it addresses the difficult topic of classifying / ranking specimens and species with morphology only. Molecular data should be helpful and is apparently helpful here. I have very little to say but I have been a bit disappointed by the low quantity of details for the phylogenetic analyses.
Experimental design: The experimental design is totally appropriate for the study. In short, the authors have extensively sampled across China and even Southeast Asia to cover a large range of the Sinopoda species. Although the sampling has many gaps, this work represents a first attempt, and will probably be complemented in a near future. They have sequenced a number of nuclear and mitochondrial markers, which are useful for reconstructing molecular phylogenies of spiders. The morphological study of the group is thorough. High-quality pictures of different anatomical parts are clear and allow diagnostic identification for the readers.
Validity of the findings: Given the taxon sampling and molecular data, the phylogeny is quite robust and well resolved. Such a phylogenetic framework can allow stressing general conclusions and making taxonomic changes and revisions. I have some comments below to improve the presentation and robustness of the phylogenetic analyses.
However, as it seems obvious, I am not a spider specialist and I have not been able to assess this part of the study. I hope the other(s) reviewer(s) have been able to evaluate the taxonomic implications of the results.
Additional comments: Major comments

(1) All the steps to prepare the phylogenetic dataset are good to me. The maximum likelihood analyses are particularly reduced to a single sentence. There is much more to do here, and I would encourage the authors to look at IQ-TREE for this type of analysis. It’s very easy and fast. It also provides interesting results with the SH test for topological hypotheses.

(2) What is the global level of support for the phylogeny of Sinopoda? I mean what is the average or median node support with posterior probabilities and bootstrap values?

(3) Why not providing a dated phylogeny? I understand the focus is on the taxonomy and a first molecular phylogeny, but it could be an additional source of information to see the divergence times. They can further help to revise the systematics.

(4) Although the work is good, I am wondering how much these conclusions can change when more species are added. You have sampled 29% of all the species known to date, but we don’t know where the missing species will be in the phylogeny. So, are they going to change your conclusions? Perhaps you should include a cautionary note toward the end of the manuscript to state that incorporating new species can alter some of the conclusions.

(5) The molecular dataset is available, which is great. However, I would recommend depositing the MrBayes nexus file instead for both better reproducibility of the analyses and better data presentation.


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 MOLECULAR PHYLOGENY OF THE CHINESE SINOPODA SPIDERS (SPARASSIDAE, HETEROPODINAE): IMPLICATIONS FOR TAXONOMY
Review round: 2
Reviewer: 1

Output:
Basic reporting: Comments accepted. I have read again the study and made some highlights in case of formal mistakes. Italics should not be used for taxonomic authorities throughout the manuscript.
Experimental design: OK
Validity of the findings: OK
Additional comments: read very carefully for small errors again


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

Input:
Title: A MOLECULAR PHYLOGENY OF THE CHINESE SINOPODA SPIDERS (SPARASSIDAE, HETEROPODINAE): IMPLICATIONS FOR TAXONOMY
Review round: 2
Reviewer: 2

Output:
Basic reporting: good
Experimental design: sufficient
Validity of the findings: enough
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: MEASUREMENT ERROR USING A SEEMALAB STRUCTURED LIGHT 3D SCANNER AGAINST A MICROSCRIBE 3D DIGITIZER
Review round: 1
Reviewer: 1

Output:
Basic reporting: 1) clear and unambiguous, professional English:
- Overall well written with just some minor words/phrases where clarification would improve the reading.
- lines 24/25: Please rephrase to stress that each operator annotated the landmarks twice each on the skulls and the 3D models.
- lines 64/65: Please rephrase to make the point clearer and not to give the impression that Bookstein compared precisions. Maybe split the sentence in the 3 types of landmarks with the Bookstein reference and then go into the testing of precision.
- line 183: You refer to Figure 1. Do you mean Figure 2? From my point of view referring to the landmark set is confusing at this point.
- line 251: Maybe change …(Fig. 2)… into …(c.f. Fig. 2) to make clearer that not the PCA is shown in Fig. 2 but the setup of the data set.
- lines 304/311: Please consider addressing the landmarks like number 22 differently. Addressing them as teeth landmarks gives the impression that the landmarks are actually located on the teeth and not on the alveolar process of the maxilla as they are shown in Fig. 1.
- lines 318: The wording “most popular choice” is maybe not a as clear as you intend it to be in this context. It seems really to depend on the (sub-) field if the Microscribe is still used today or already abandoned. If you want to focus on your specific field please stress it in this sentence or phrase it more openly to fit a wider audience.

2) Literature references, sufficient field background/context provided:
- lines 48-49: In the first sentence of the introduction you name a few studies. However, none of the old publications about the main advances in the field is mentioned and some references to underline the importance and the versatile use of it as toolkit might be useful as your manuscript can be of interest also in other fields where geometric morphometrics is used (e.g. Bookstein, Rholf, Gunz, Slice, Zelditsh).
- lines 92f.: This very much depends on the used scanner (medical, industrial, synchroton, neutron). For materials in which the radiation exposure is not of importance also rather big structures can be scanned in very high resolution (a few microns). Please rephrase and/or add references.
- line 105: The reconstruction of teeth can also be tricky in cases of surface scanning. You even mention potential reflections in the dental area further down in the manuscript. Based on experience its usually the very smooth and sometimes polished dental surfaces that create the issues. Please consider rewriting to show that this disadvantage is not only limited to photogrammetry.

3) professional article structure, figures, tables. Raw data shared:
- figure 4: Please clarify in the legend what the colors stand for and chose color-blind friendly colors (easy to check for compatibility for example in Photoshop).

- table S1: Would it be possible to provide sex and clarify that all individuals are adult? I assume this information is available and kind of relevant in getting an understanding in how homogenous the shape (cf. sexual dimorphism, ontogeny) is for landmark collection.

- table S3: If you provide a more detailed table in the supplements than please really include all the information like in Table 2.

4) Self-contained with relevant results to hypotheses:
- fulfilled
Experimental design: 1) original primary research within Aims and Scope of the journal:
- fulfilled

2) research question well defined, relevant & meaningful. It is stated how research fills an identified knowledge gap.
- fulfilled

3) Rigorous investigation performed to a high technical & ethical standard:
- Overall yes, only a few points that need consideration or maybe just clarification.
- lines 31f; 115f.; 205ff.; 374; 383; 399ff.; 460ff.; : Landmarks used and omitting landmarks
-- I am not sure I get your point of highlighting the omission of landmarks in the abstract. To my understanding you either exclude those landmarks very early in your study (or even pre-study) and mention it in the methods or when testing sources of error make a point of seeing how much error such problematic landmarks may introduce.
-- In addition, in this case you have to balance on how to phrase it very carefully because you used published landmark sets. In other words, by not being able to measure those landmarks, you indirectly question the reproducibility and reliability of the results in the published papers. Therefore, please consider something like “non-reproducible here” or “non-reproducible in this context” in line 205.
-- Further, please clarify why you could not measure landmark 19. Very hidden in the discussion (line 448) you state that some of the measurements were collected with a temporal gap and thereby presumably before the beginning of the here described study. Is this the reason why you stuck to that measurement protocol for the Microscribe? If yes please mention this in the method section (see also my other comment about this).
-- Nevertheless, it would be advantageous to explain in one sentence why you did not make use of taking landmarks from two different perspectives with the Microscribe. It is rather common to take landmarks in at least 2 views and to combine them mathematically into one coordinate system based on reference points (e.g. via the DVLR software). This concerns especially landmark 19, 17/31 and 18
- lines 190 & 448: number of repetitions per operator
-- Please describe when the data was collected in the method section. Were the measurements collected close in time or was there a deliberate break between the two measurement rounds? If you measure only twice super close in time to each other the results might be more similar to each other than with temporal gaps.
-- Furthermore, please provide a reason why each operator measured twice. Multiple measurements (3-5 rounds) might give more robust results in terms of inter- vs. intra- operator errors.
Validity of the findings: 1) Impact and novelty not assessed. Negative/inconclusive results accepted. Meaningful replication encouraged where rationale & benefit to literature is clearly stated:
- Yes it is meaningful and clearly states where it adds to still existing gaps in the published studies.

2) All underlying data have been provided; they are robust, statistically sound, & controlled:
- All data necessary has been provided. Nevertheless, there is one aspect that could be added to strengthen the manuscript.
- lines 256ff.; 282ff. & Figure 4: You really nicely discuss the results in terms of pairwise Procrustes distances and how much of the observed variation can be attributed to the different kinds of error. In addition to this, it would have been nice to see the overall distribution of pairwise Procrustes distances in regard to error measurements vs. interindividual. This could be visualized for example as histogram or box plots. What I am aiming at is to see whether the greatest error in all tested categories is smaller than the smallest pairwise Procrustes distance between individuals (here the different seals measured) in the sample. I think it would strengthen your manuscript as it would underline that yes there are sources of error but if being aware of them and controlling them as good as possible that the obtained results will still be meaningful.

3) Conclusions are well stated, linked to original research question & limited to supporting results:
- Over all yes, only one conclusion needs some clarification.
- lines 41f.; 475ff.: combination of data from different operators
-- Of course, combining data from different sources (i.e. different observers/operators) needs some internal checking for compatibility. Nevertheless, results should have the aim to be reproducible that by itself means data collection by a different operator. If the variation of interest is that small that it is overwritten by the interobserver error choosing an entirely different approach or method for addressing these research questions should be considered instead of obscuring the issue by collecting data by single operator.

4) Speculation is welcome, but should be identified as such:
- fulfilled
Additional comments: Dear authors,

I am happy that I was asked to review your manuscript. In the majority of papers intra- and inter-observer as well as inter-method error tests are just carried out as small part hidden in the supplements or reported in a few sentences without presenting the actual data. Therefore, I think it is important to systematically evaluate potential sources of error.

Although it is not within the scope of this paper, but out of curiosity, I wondered if there has been a test of between-device error between the SeeMaLab and other surface scanners?

I hope that I could express my review in a constructive way that allows you to directly identify the critical points and address them.