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: IN SILICO PREDICTIONS OF PROTEIN INTERACTIONS BETWEEN ZIKA VIRUS AND HUMAN HOST
Review round: 1
Reviewer: 2

Output:
Basic reporting: The main idea of this article is to use machine learning algorithms in order to evaluate novel ZIKV-Human protein interactions based on two viral strains, one of them related to neurological development issues.
The manuscript is well balanced, well organized, and the references used are sufficient for this study. Additionally, the study was presented with good scientific rigor, as well as with the quality of its figures, tables, and raw data.
The achieved results are well related to the manuscript hypothesis and give us a novel ZIKV-Human interactions mechanism, as well as confirms other studies cited in the discussion.
On the other hand, it should have a general English language revision in different parts of the text for ensuring the most appropriate formal and scientific language, but nothing that compromises the quality of this work.
The authors even referred “Eid, ElHefnawi & Heath, 2015” as its main reference for the training method. Is there any other study that used the same approach? I think it is important to cite another reference to reinforce the use of this methodology. Additionally, please check the same reference in the Pubmed, but for 2016:

Eid FE, ElHefnawi M, Heath LS. DeNovo: virus-host sequence-based protein-protein interaction prediction. Bioinformatics. 2016 Apr 15;32(8):1144-50. doi: 10.1093/bioinformatics/btv737. Epub 2015 Dec 16. PMID: 26677965.
Experimental design: The study was defined with a rigorous experimental design, using previous studies methods and validated databases and tools, as well as commonly used machine learning algorithms for evaluating protein interactions. On the other hand, it is not clear why the authors choose just that specific strain for comparison and did not include other ZIKV strain data for comparison. In this case, the only way to do this comparison is by using published results. I suggest, if it is possible that the authors include in the training set at least one more ZIKV strain for positive interactions. Additionally, please describe explaining the reasons you choose these specific strains.
Validity of the findings: Even other authors have used similar approaches to find the ZIKV-Human PPI network, this manuscript brings novel information about the possible mechanisms of this viral infection mainly in cases with neurological issues. The authors included additional training and validation steps, in comparison to other studies, and they found possible new mechanisms of interaction. Additionally, the authors confirmed other studies' findings which were cited in the discussion section. The results and discussion of this manuscript are well described and it is related to its main hypothesis, as well as is in the same way as other previous studies described for ZIKV-Human interactions and for other viruses.
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: IN SILICO PREDICTIONS OF PROTEIN INTERACTIONS BETWEEN ZIKA VIRUS AND HUMAN HOST
Review round: 2
Reviewer: 1

Output:
Basic reporting: The edits made in the revision introduced multiple grammatical and typographical errors.
Literature references were enhanced and additional references were included but their critical evaluation can be improved instead of "listing".
The "hairball" figure remains unacceptable in presentation and information content.
Experimental design: Thank you for the clarification on Kegg vs GO enrichment. Regardless of the source of enrichment, my concern remains that generic pathways were identified.
The research question is broad but valid, but the conclusions lack specificity.
Validity of the findings: Conclusions need to be specific to Zika and explained in the context of Zika biology.
Additional comments: "respectful disagreement" is not sufficient to address my concerns


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: IN SILICO PREDICTIONS OF PROTEIN INTERACTIONS BETWEEN ZIKA VIRUS AND HUMAN HOST
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors have fixed all issues and followed all recommendations in this second version of the manuscript. Thus, I have no comments in this section.
Experimental design: The authors have fixed all issues and followed all recommendations in this second version of the manuscript. Thus, I have no comments in this section.
Validity of the findings: The authors have fixed all issues and followed all recommendations in this second version of the manuscript. Thus, I have no comments in this section.
Additional comments: Thank you for accepting my suggestions. I understand that the manuscript is now suitable for publication.


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

Input:
Title: IN SILICO PREDICTIONS OF PROTEIN INTERACTIONS BETWEEN ZIKA VIRUS AND HUMAN HOST
Review round: 2
Reviewer: 3

Output:
Basic reporting: In this work, Pitta and colleagues applied Random Forest and SVM algorithms to predict the interaction between Zika virus and human proteins. The article is of broad interest and can help in finding targets for the development of new drugs. However, there are a few points that should be addressed before its ready for publication. Of note, the presentation of the data could be improved.
Experimental design: no comment
Validity of the findings: no comment.
Additional comments: Minor
The English language should be improved to ensure that an international audience can clearly understand your text. This includes some mistyping errors, such as abstract  "on the other hands"->"on the other hand"; Line 232 Virus Mentha, but LIne 242 -> VirusMentha, what is the right way? Line 335 "non-interaction: ...

Important
The authors should use a figure to show the data in Table 2. There are many types of visualization for enrichment data, such as observed here: (https://bioconductor.statistik.tu-dortmund.de/packages/3.8/bioc/vignettes/enrichplot/inst/doc/enrichplot.html -  Dot Plot).

Check figure quality. Figures 2, 3 and 4 seem below acceptable quality. However, it could be a product of PDF conversion. Consider checking prior publication.

Why the author did not account differentially structural and non-structural proteins, since structural proteins are more likely to be exposed to host proteins due to their role in particle formation?


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

Input:
Title: IN SILICO PREDICTIONS OF PROTEIN INTERACTIONS BETWEEN ZIKA VIRUS AND HUMAN HOST
Review round: 3
Reviewer: 1

Output:
Basic reporting: This reviewer has been invited to comment on the manuscript starting from the rebuttal. Thus, my comments and arguments were built after suggestions from other reviewers. In general lines, the manuscript is sound and fit for publication after minor improvements described below.

Important: Authors should include a supplementary table with access ID for each protein shown in figure 2, divided by ZIKV-protein interactant. It must contain columns listing all important parameters obtained from PPI predictions. This will allow readers to get in touch with the data and further validate findings, thus improving visibility of this work. Moreover, will also allow the investigation of interactions with borderline significance as exemplified in lines 405-413.

I agree with other reviewers that Figure 4 is pointless. No information is added other than a ‘beautiful plot’ with different numbers of circles. The information contained in this figure should be better shown as a simple Venn diagram (or Euler diagram) associated with figure 2, where these numbers stand out without being rationally compared. Then, rephrase lines 345-348 and move where figure 2 is described. Also, a Venn diagram would allow quantification when this data is discussed in lines 420-425.
Experimental design: Experimental design seems suitable for their findings and is well documented.
Validity of the findings: Despite no validations, authors make it clear that their work is based on predictions and should be validated prior any assumptions.
Additional comments: This work is important as it highlights mechanistic differences between African and Asian strains of Zika virus. An example of these differences has been recently published by Aubry et al, 2021 Nat Comm (DOI 10.1038/s41467-021-21199-z), however Aubry et al do not propose any mechanism for their observations. This work by Pitta et al is important as it shed light on the differences between host-pathogen interaction of ZIKV strains. Moreover, their methodology could be easily implemented to investigate PPI between flaviviruses of medical importance and the human host.


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: GENOME-WIDE ANALYSIS OF BPDOF GENES AND THE TOLERANCE TO DROUGHT STRESS IN BIRCH (BETULA PLATYPHYLLA)
Review round: 1
Reviewer: 1

Output:
Basic reporting: A few sentences in this manuscript are ambiguous.
Experimental design: The author needs to describe the methods of gene screening in detail.
Validity of the findings: no comment.
Additional comments: In this study, the authors identified 26 Dof family genes from birch tree. Then the phylogenetic relationship, gene structure, conserved motifs of these BpDofs were analyzed. Expression analysis showed that several BpDofs response to PEG treatment significantly. Three BpDof genes with the most significant expression changes under PEG treatment were selected for subsequent transgene using a transient expression method. Determination of stress-related indexes showed that these genes could enhance the drought tolerance of transgenic plants. In general this manuscript is interesting and provides valuable information. However, the manuscript was poorly written and had many errors. The results section is oversimplified in some places. The discussion section is logically confusing and needs to be rewritten, and some of the sentences in this section are duplicated with the results section. Part of the bioinformatics analysis results and experimental results need to be further confirmed.

Keywords:
“Abiotic stress”, change to “Drought stress”.

Introduction:
The third paragraph (lines 61-85) in this section is too long. I think this paragraph can be divide the into two parts at line 80. In addition, the fourth paragraph (line86-89) should be merged into the preceding paragraph.

Materials & Methods:
Line 106, how were the 26 BpDofs genes screened? The author needs to describe the methods of gene screening in detail.

In the title of Table S1, The letter “BpDof” should not be italicized when described as a transcription factor. Letters are italic only if they are the name of a gene. There are many of the same mistakes in this manuscript.

Line 107-111, this sentence is ambiguous and needs to be rewritten. In addition, if the author want to identifiy the conserved domains contained in these BpDof proteins, they should not directly use the Dof domain file (Pfam PF02701) for identification. They do not know whether these DOF proteins contain other conserved domains.

Line 121-123 (Table S2), 39 AtDof genes are listed in this Table. However, only 26 BpDof genes were obtained in this study. This makes it doubtful whether there are many omissions in the screening results of this study.

Line 143, the method of PEG treatment needs to be described in detail. For example, how much PEG solution is poured in this treatment?

Line 175, the author states that the “pROKII-35S was used as a control”. However, in the results section (line 273; Figure 7), the authors used WT plants as controls

Results:
Line 189-190, the authors should briefly describe how they screened for Dof genes in birch.

Line 196-198, there are no charts or supplementary materials in the paper to show the results described in this sentence.

Line 199, as a genome-wide analysis of Dof family genes, collinearity analysis should be performed in this study.

Line 201, two AtDof genes were randomly selected for sequence alignment, which is not as good as selecting representative genes in this family that have been studied extensively.

Line 206-209 (Figure 2), based on the phylogenetic tree analysis, the authors divided BpDof proteins into five groups, which were named II to VI. However, in Figure3 (line 215), the authors divided these Dof proteins into 6 groups, and the naming method was changed to A-F. The author needs to further verify the grouping results and unify the naming method. In addition, 38 AtDofs protein sequences were used in Figure 2, while 39 Atdofs protein sequences were listed in Table S2. Please check whether the figure legend of Figure 2 is incorrect or indicate which sequence of AtDofs is not used for the construction of the phylogenetic tree.

I suggest combining Fig4 and Fig3 into one figure.

Line 262-263 and line 266, “BpDof417、BpDof424、BpDof411” ? Please check.

Line 263-264, Dof4 and 17 are up-regulated, while Dof24 is down-regulated. The description should be clear. In addition, the expression of Dof14 in leaves also changed greatly (Figure 6), the expression level even higher than that of Dof17, which should not be omitted here.

Line 269, the methods of transgene and PEG treatment should be briefly described here. In particular, the author should make it clear that the transient expression method is used in this study.

Line 286, the authors said that the SOD activity of the transgenic material was significantly higher than that of WT, but there was no difference between the enzyme activity of two transgenic lines (Dof4, Dof17) and WT (Figure 8).

Line 291, please check whether there is a significant difference between Dof17-overexpressing plants and WT in Figure 8D.

Discussion:
Line 302-304, the AtDofs were divided into 7 groups in other studies. Why are there fewer groups in this study when all the sequences of AtDofs are used for phylogenetic tree analysis? Further verification and discussion are needed.

Line 304-305, delete this sentence or put it in another appropriate place.

Line 306, merge this paragraph with the previous one.

Line 314, delete this sentence or move it to the results section.

Line315, this part needs to be further discussed in combination with the similarity of gene structure within the same group and the differences between different groups.

Line335-336, further discussion should be conducted on the differences in expression patterns of the same gene in different tissues (roots, leaves), such as whether the response and regulatory functions of some Dof genes under drought stress are tissue-specific.

Conclusions:
Line 353, “molecular analysis” is ambiguous.

Line 356, change “BpDof genes” to “BpDof proteins”

Line 361, showed “enhanced” ROS scavenging capability


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: GENOME-WIDE ANALYSIS OF BPDOF GENES AND THE TOLERANCE TO DROUGHT STRESS IN BIRCH (BETULA PLATYPHYLLA)
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The manuscript by Sun et al. reports Genome-wide analysis of BpDof genes and the tolerance to drought stress in birch. The author analyzed the characterization and expression of the BpDof genes and the tolerance to drought stress, indicating that some of them can enhance the resistance to drought stress. I think this work is interesting, but, there are several issues need be revised.
1. Please check the full text. The name of gene is in italics and the name of protein is in normal.
2. Please keep the format consistency of all figures.
3. Line 149: What size of the birch seedlings was treated with 20% PEG6000?
4. Line 174: Please add the restriction endonuclease used in the vector construction.
5. Line 184 and 185: Please add the dissolving solutions of DAB and Evans blue.


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: GENOME-WIDE ANALYSIS OF BPDOF GENES AND THE TOLERANCE TO DROUGHT STRESS IN BIRCH (BETULA PLATYPHYLLA)
Review round: 2
Reviewer: 1

Output:
Basic reporting: Although this is the first round for me to assess the academic quality of the manuscript submitted by Sun et. al., I can find the manuscript has been extensively revised according to the tracking changes. The current (revised) version of the manuscript is much improved. Dofs are plant specific TFs that play various roles in developmental processes and environmental stresses. Genome wide identification and analysis of Dof genes is useful to further reveal their molecular functions. The manuscript is overall well designed and organized. However, some minor reversions are needed before publication.
Experimental design: The manuscript is well designed, I have no special comments to this part.
Validity of the findings: The results and findings obtained in this study is meaningful for researchers who are interested in this field.
Additional comments: 1. For the method section, the authors should refer to the literatures of the softwares, such as BLASTX (L111),  MEGA (L127) .
2. L233 Populus trichocarpa, not Populus trichocarp. In addition, please the genome version used in this analysis.
3. For the conclusion section. please do not simply repeat the results 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: GENOME-WIDE ANALYSIS OF BPDOF GENES AND THE TOLERANCE TO DROUGHT STRESS IN BIRCH (BETULA PLATYPHYLLA)
Review round: 3
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Based on my suggestion in the first review, the authors have addressed my concerns appropriately. The authors did a commendable job in putting together all the Betula platyphylla available Dof genes resources and performing in-silico analysis, this work is meaningful. So, the manuscript can be accepted for publication in 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: GENOME-WIDE ANALYSIS OF BPDOF GENES AND THE TOLERANCE TO DROUGHT STRESS IN BIRCH (BETULA PLATYPHYLLA)
Review round: 3
Reviewer: 2

Output:
Basic reporting: The authors have addressed all my concerns and the revised manuscript is much improved now. I have no further comments on the manuscript.
Experimental design: I have no special comments on the experimental design part.
Validity of the findings: No comment.
Additional comments: Thanks for your quick response, I have no other comments now.


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

Input:
Title: MOLECULAR AND MORPHOLOGICAL EVIDENCE FOR A NEW SPECIES OF LEPTOPUS (PHYLLANTHACEAE) FROM SOUTHEAST YUNNAN, CHINA
Review round: 1
Reviewer: 1

Output:
Basic reporting: The English needs some improvement, I indicated a few instances. Overall quite good English (but I am not a native speaker).

Literature references are fine.

Overall the structure is fine, but I miss a section Discussion. The conflict in phylogenetic results between both markers can be discussed and the combined results can be compared with those of Vorontsova et al.'s phylogeny (which are likely the same, as most are their data); here you can also explain the difference in number of species between their and your data. In the discussion, the Indian species, likely not a Leptopus, can be discussed, now at the end of the manuscript.
Experimental design: It is difficult to comment on the molecular work as all used techniques are only represented by references, idem with the phylogenetic analysis. I expected at least a discussion for the Bayesian analysis about sufficient cycles and high enough scores with ESS values (program Tracer). Therefore, I think that adding the programs used and settings of the phylogenetic analyses will be an improvement, not just a simple reference.

For the rest fine..
Validity of the findings: The findings seem fine.
Additional comments: It is a short and good paper, showing that the new species is morphologically and phylogenetically distinct. The key also demonstrates even better than a discussion that the species is different.

Only you put a lot of focus on the different habit. But bear in mind that that is field character, generally not visible on herbarium specimens nor mentioned on the labels. Thus you cannot be certain that all other species have a different habit!


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

Input:
Title: MOLECULAR AND MORPHOLOGICAL EVIDENCE FOR A NEW SPECIES OF LEPTOPUS (PHYLLANTHACEAE) FROM SOUTHEAST YUNNAN, CHINA
Review round: 1
Reviewer: 2

Output:
Basic reporting: The English while mostly clear and unambiguous has many grammar problems, and a few typos. I have flagged and/or suggested edits in this regard as tracked changes in an attached version of the manuscript. The literature cited covers all relevant materials related to the current taxonomy of Leptopus. A mention of the medicinal/biochemical studies in Leptopus could be added to make the reader aware of this aspect to the genus, even if not strictly relevant to this systematics study.

The full alignments as used for the phylogenetic analyses need to be made available, either as supplements here or in public databases such as Dryad. The analyses can’t be replicated without the alignments and complex alignments (likely the case for ITS) are difficult to replicate without knowing minute starting details. The fasta files made available at review only include the sequences of the new taxon and no context of the alignment. The disposition of the raw data for the sequence assemblies is unclear and preferably it would be deposited in an archive such as the SRA. This is especially relevant if long multi-locus assemblies are presented (see below) or if the remainder of the genome skim data is being ‘thrown away’ when it could be mined for other purposes by future investigators. The authors should be consistent on how they phrase the DNA they use for phylogenetic analyses; they use the term “fragment” or “DNA region”, but genes, genetic markers, or loci would be more appropriate. The figures are relevant, clear, and ample, and the 2 plates of live-plant photographs are equivalent to a botanical illustration in showing all the diagnostic parts. The support values on the phylogeny graphics should be carefully positioned so as not to touch or overlap the branch lines; for those on short nodes the numbers could be moved to underneath node. The multiple accessions for some species need to have identifiers (e.g., Leptopus australis 1, Leptopus australis 2) to tie positions in trees to accessions in Table 1. The legend for Fig.4K should specify units for scale (presumably each tic is a mm). I do not understand why Fig.4J is moldy – is this fruit taken in the wild and typical, or is it a poorly preserved specimen. Dot leaders in keys should be uniform to allow species to line up. Legend for Fig.5: “red circular” should be ‘red circle’.
Experimental design: The manuscript presents original primary research on a newly discovered species that is within the scope of the journal. The Methods associated with the DNA sequence generation and phylogenetic analyses are incompletely presented. In particular, how the data was generated is not detailed in this text (i.e., how were libraries prepared, what sequencing platform, read length, how many reads, how were the reads filtered, what was the coverage of the assemblies). It is not sufficient to simply cite this all under “Zeng et al. (2018)” which likely has many details different and slight technology changes from what the authors used here. It is also not sufficient to cite away most details of phylogenetic analyses under Yao et al. (2020); minimally the run parameters need to be specified here and, burn-in for BI. Were the data sets partitioned in any way? The Methods discuss using genome skimming to yield a full cp genome, and it is a lost opportunity not to present that genome with this manuscript, rather than just extract a single gene. Similarly, the reads should allow the assembly of the full rDNA repeat (ETS-26s) while only ITS was extracted here. Presenting those full sequences with some minimal descriptive aspects (length of cp genome, number of genes, etc.) would be a valuable addition to this manuscript and it remains odd to describe assembling and annotating a plastid genome but show nothing of it. If a full cp genome is to be presented elsewhere (such as a data release note in Mitochondrial DNA: Resources), then those intentions should be so indicated. “nrITS” can just be referred to as ITS.
Validity of the findings: The new species is validly presented (i.e., type designated, diagnosis) according to nomenclature rules and the plant itself is distinctive (both morphologically and in sequence divergence) enough within Leptopus to be considered a new species by a specialist taxonomist such as this reviewer. The phylogenetic evidence is sound; those analyses appear sufficient, but as I noted before the details on their quality rely on citation rather than explicit statements. The authors make some comparisons (morphology and close phylogenetic relationships) between the new species and L. fangdingianus, but they should do more to address biogeographic and ecological similarities. It’s striking that both these species are very rare, live relatively near each other, and live in similar habitats. Generally, the biogeography of Leptopus with its wide range across Eurasia (in both temperate and tropical regions), and mix of narrow endemics and broadly distributed species is interesting. It is unfortunate that not all species have been sampled for a phylogeny to enable greater evolutionary and biogeographic inferences.

As a plant with a narrow distribution and given that the authors have visited the type locality, it would be valuable to provide a conservation assessment (following IUCN criteria and categories). Was the new species brought into cultivation? The specimen localities are vague and could use coordinates; if conservation status is a concern in disclosing locality details, then the accuracy of those coordinates could be limited for publication. The reader should be able take those coordinates and use GoogleEarth to look at imagery of the type locality or plot its distribution without having to extrapolate from the described locality. The plant description could use more details and in particular is incomplete on measurements of described structures (i.e., shrub height, length and width of stipules, sepals, petals, disc-segments, stamens/filaments/anthers, styles); it should more closely match those details in Vorontsova & Hoffmann (2009). What is leaf venation type? Explicitly state 2 ovules and/or seeds per locule. What floral parts are persistent in fruit (i.e., sepals, styles)? Fruit type should be specified (presumably an explosive schizocarp splitting into 3 equal 2-valved mericarps). Describe columella and fruit valves after dehiscence. Having technical details that are parallel to the recent monograph on Leptopus is important for this new taxon as few specimens are available in 3 Chinese institutions.
Additional comments: This is a distinctive new species, for which the authors provide all the ingredients for a thoughtful taxonomic paper (description, revised key to species, phylogeny, illustrations, map). The “Custom Checks” for DNA, field study and new species have been followed as much as the state of the manuscript allows; the 2 new sequences are not publicly released yet (but have GenBank numbers and those data are also as supplements), and the LSID number must await ms. acceptance. It remains surprising that the authors don’t make broader use of their ample molecular data. Even though they need only a small amount of orthologous data to insert their new species in prior phylogenetic frameworks to determine relationships, describing more of their genomic sequencing efforts would make the manuscript more modern in approach and allow the authors’ results to be incorporated into broader future studies.


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

Input:
Title: MOLECULAR AND MORPHOLOGICAL EVIDENCE FOR A NEW SPECIES OF LEPTOPUS (PHYLLANTHACEAE) FROM SOUTHEAST YUNNAN, CHINA
Review round: 1
Reviewer: 3

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The manuscript has interesting results and appropriately conducted. However, there are some information that need to be reconsidered. The major comments are as following and please see some minor comment and suggestion in the annotated MS.
1. In the Introduction.
Introduction should include all important information for the studied genera, thus information of Leptopus nepalensis should be mentioned in the Introduction as well.
2. The diagnostic character of the new species.
The new species has several different characters compared to other Leptopus species, however the leaf size is not clearly distinct as present in the description that the leaf size is 2 – 15 cm x 1.2 – 5 cm. So it does not distinct from the other species that is less than 10 cm. So this character need to be reconsidered.
3. The key to species:
In the couplet 7 and 8, the leaf size was used. It very difficult to separate the species based on leaf size as it was not clearly distinct. So this characters need to be reconsidered.


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

Input:
Title: MOLECULAR AND MORPHOLOGICAL EVIDENCE FOR A NEW SPECIES OF LEPTOPUS (PHYLLANTHACEAE) FROM SOUTHEAST YUNNAN, CHINA
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors implement most of the original Review comments and the manuscript is greatly improved editorially. The technical plant description has fleshed out details, but the molecular/phylogenetic methods are still sparse in that regard. A few new editorial comments:

Line numbers here relate to the pdf version.
Line29: “A key to all the Leptopus species in the world is provided.”> ‘A key to all 10 accepted Leptopus species is provided.’ Note: Extraneous to add “in the world”; ‘worldwide’ could be acceptable.
L50: “The species is much different...”> ‘The species is very different...’
L90-91: “To study the phylogenetic position of the new species, a phylogenetic study of the genus Leptopus was performed,”> ‘To resolve the relationships of the new species, a phylogenetic study of the genus Leptopus was performed,’
L154-155: “and mature seeds with copious endosperm (Fig. 4I)”> This detail about copious endosperm also should be added to the seed part (L220) of the plant description, given that it’s stated prior as a generic diagnostic character.
L164-165: “longitudinally strongly ribbed stems), some of its leaves laminas could up to 15 cm long and 7 cm wide (vs. leaf laminas all less than 10 cm long and 3 cm wide), leaves”> ‘longitudinally strongly ribbed stems), laminas up to 15 cm long and 7 cm wide (vs. laminas all less than 10 cm long and 3 cm wide), leaves...”
L172-173: “from the rest four congeneric species”> ’from the remaining four congeneric species’
L217: “2-valed”> ‘2-valved’
L281-282: “Petioles of mature leaves far less than 1/4 of leaf lamina length; leaf apex not rounded (except several specimens of L. chinensis)” > Would it be accurate and cleaner to qualify as ‘Petioles of mature leaves far less than 1/4 of leaf lamina length; leaf apex not rounded (rarely rounded in L. chinensis)’
L242-243: “and L. fangdingianus seems to be reasonable because”> ’and L. fangdingianus is expected because’
L246: “by karst environment”> ‘by a karst environment’
L321: “Leptopus lolonum”> Italics
L327: “Leptopus cordifolius”> Italics

Figure legends need italics for scientific names and plastid genes (presently inconsistent in that regard).
Experimental design: I disagree with the author rebuttal: “The technique of genome skimming is mature enough to date and most of relevant phylogenetic studies did not describe the detailed information about procedures of DNA sequence generation and also some other information, such as the read length, the number of reads, the coverage of the assemblies. So, the method associated with the DNA sequence generation in the present taxonomic study was not described in detailed, but a reference (Zeng et al. 2018) was cited in the manuscript.”    I do not accept that no sequencing methods are needed, and most papers do provide the basic information I originally suggested (i.e., how were libraries prepared,
what sequencing platform, read length, how many reads, how were the reads filtered, what was the coverage of the assemblies). While genome skimming may be conceptually well established (or “mature”), how it is executed is very variable today among published studies and improves with time. Skimming with 50 bp reads 5+ years ago is very different from Illumina today or using Pac-Bio, and how Zeng et al. did it 3+ years ago likely differs in details that affect data quality and coverage from what the present authors undertook. Zeng et al. doesn’t even provide basic sequencing details to allow quality to be assessed to begin with, so its citation is not especially informative. While the authors have indicated they plan to do more with the skimming data and may present more details there, the manuscript currently in hand cannot cite that future effort.

L115-117: “All the three datasets were analyzed using two approaches: Bayesian Inference (BI) (Ronquist & Huelsenbeck, 2003) and Maximum Likelihood (ML) (Stamatakis, 2006).” >The references cited are for specific programs and this sentence needs to be rephrased around those programs (i.e., MrBayes v???, RAxML v???) and not merely indicate the BI and ML optimality criteria. Just as in the sequencing methods, attempts here to cite away critical details (in that case to “Yao et al. 2020) fall flat and at least ML BS replicate numbers should be specified here along as noted above, with clarifying the programs used.
Validity of the findings: From the author rebuttal comments: “Actually, we have the GPS coordinates of the two wild populations of the new species, but detailed information about the coordinates are not provided in the manuscript, because of the two populations are not located in nature reserves and some of the new species reported recently in China may be digged after their publication, especially those have good values in medicine or horticulture.”    This conservation concern reason regarding not specifying coordinates should be reiterated in the manuscript rather than letting their absence seem like a major lapse. Perhaps along the lines of inserting a parenthetical statement in the type locality, or inserting a statement under ‘Distribution and habitat’ or ‘Ethics statements’. See inserted qualification in this regard in Ethics section: ‘The collection location of the new species reported in this study is outside any natural conservation area and no specific permissions were required for the location. Since this species is currently undescribed, it is not currently included in the China Species Red List (Wang & Xie, 2004). <<However, due to conservation concerns and lack of habitat protection, exact locality coordinates have been withheld from our published specimen records.>> Our field studies did not involve any endangered or protected species. No specific permits were required for the present study.’ (Line 57-61)
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: THE MODULATORY PROPERTIES OF ASTRAGALUS MEMBRANACEUS TREATMENT ON ENDOMETRIAL CANCER: AN INTEGRATED PHARMACOLOGICAL METHOD
Review round: 1
Reviewer: 1

Output:
Basic reporting: ABSTRACT:
1.	Wrong spelling of Formononetin in line 42.
2.	ER-beta in line 45 should be written in full name

INTRODUCTION:
1.	There is lack of coherence between paragraphs.
2.	Introduction did not address what other people have done to solve the same problem, to reason why this study was conducted.
3.     The introduction should include sufficient backgrounds

FIGURES
1. Overall, captions are too short and do explain the significant findings in the figures.

TABLES
1. Please be specific in the caption and avoid using word such as "some" (eg Table 1)
Experimental design: 1.	In many sections, please avoid defining the database/ software (eg: Cytoscape, TCMSOP, GO, KEGG etc) but straight away tell how database/ software was used in the study. For example, what materials were used to construct the network using Cytoscape.
2.	What is the basis for selecting OB >30% and DL > 0.18?
3.	The top 40 DEGs were mentioned once and in line 212 and did not see if these identified genes were used anywhere in the subsequent methods.
4.     Methods should be described in detail so that it is reproducible by others.
Validity of the findings: RESULTS:
1.	Table 1 only shows 18 examples (some are repetitive compounds). Please be specific for showing only these 18 examples.
2.	Line 222-225 belongs to Materials and Methods.
3.	In line 222, what is EC regulated genes and how many? How many Astralagus targets?
4.	Line 234-235 belongs to Materials and Methods. Please clarify what does it by “the original PPI network was screened twice”?
5.	DC & BC in line 229 are not defined.
6.	How is the network validated? Any comparison to EC networks that have been published?

DISCUSSION:
1.	ER-beta was not mentioned anywhere in the Materials & Methods and Discussion, why it was selected for experimental validation?
2.	Discussion looks very brief and selective. For example: Why the discussion only focuses on KEGG when GO was also used.

CONCLUSION:
1.	The conclusion is far-fetched which the compounds in the plants show potential for EC treatment not a promising one.
2.	The aims mentioned in the Introduction were fully answered (ie mechanism of action)
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: THE MODULATORY PROPERTIES OF ASTRAGALUS MEMBRANACEUS TREATMENT ON ENDOMETRIAL CANCER: AN INTEGRATED PHARMACOLOGICAL METHOD
Review round: 1
Reviewer: 2

Output:
Basic reporting: The title, abstract, introduction, methods, results and discussion are appropriate for the content of the text. Furthermore, the article is well constructed, the bioinformatic analysis is well performed. The figures are relevant, high quality, well labelled and described.
Experimental design: The method design is original and novel. However, the research is within the scope of this journal. Research question is well defined, relevant and meaningful. The methods are highly technical, ethical and logistical. Statistical methods are chosen correctly.
Validity of the findings: All underlying data have been provided in detail. The findings are meaningful. The conclusions are well stated and relevant to the research questions. One of the disadvantages is the lack of in vitro or in vivo validation experiments.
Additional comments: This paper investigates the effect of Astragalus membranaceus on endometrial cancer. The authors identified a series of active ingredients of Astragalus membranaceus using GEO and TCMSP datasets. To explore further about the potential targets and related pathways, the authors demonstrated that Astragalus membranaceus may play a role in the treatment of endometrial cancer through p53 signalling pathway, transcriptional dysregulation in cancer, and endometrial cancer signalling pathway. Furthermore, the authors verified one of the identified components - forononetin to be able to inhibit the proliferation of endometrial cancer cells and exert anti-cancer effects by promoting the expression of ERβ. In short, this study identified the active components and targets of  Astragalus membranaceus, and also suggested that Astragalus membranaceus could be considered as a potential therapy for endometrial carcinoma patients.

Editorial Criteria
BASIC REPORTING
The title, abstract, introduction, methods, results and discussion are appropriate for the content of the text. Furthermore, the article is well constructed, the bioinformatic analysis is well performed. The figures are relevant, high quality, well labelled and described.
EXPERIMENTAL DESIGN
The method design is original and novel. However, the research is within the scope of this journal. Research question is well defined, relevant and meaningful. The methods are highly technical, ethical and logistical. Statistical methods are chosen correctly.
VALIDITY OF THE FINDINGS
All underlying data have been provided in detail. The findings are meaningful. The conclusions are well stated and relevant to the research questions.
Overall, I think this paper is well written and will be of interest to the community of endometrial carcinoma pharmacogenetics and Astragalus membranaceus research in cancer. The pharmacological method and statistical analysis is valid and makes sense. The authors make it comprehensive by integrating analysis of multiple sources including GEO and TCMSP. The main strengths of this paper is that it addresses an interesting and unexplored question about the effect of Astragalus membranaceus in endometrial cancer, finds a novel discovery based on a carefully selected set of pharmacological procedures. What makes it better is the in vitro validation experiments. As such this article represents an excellent and elegant pharmacogenetics study which will almost certainly influence our thinking about the function of Astragalus membranaceus in endometrial cancer. In general, the work is convincing except some major and minor comments below:

Major Comments:

I’m wondering if there are any ongoing clinical trials focusing on any of the active ingredients identified in this study to treat endometrial  carcinoma or even ovarian cancer? It will be very strong evidence for the significance of the current study if so. Please carefully search through https://clinicaltrials.gov

Considering the sample size of the GEO dataset(GSE63678) is relatively small. I recommend using the data from ICGC(International Cancer Genome Consortium) and TCGA(The Cancer Genome Atlas Program), which are national or international consortiums with much more samples. Please check the NCI Genomic Data Commons (https://portal.gdc.cancer.gov) to find more information about TCGA data and ICGC Data Portal(https://dcc.icgc.org) to find more information about ICGC data. At least, TCGA should contain Endometrial Carcinoma samples: https://www.cancer.gov/about-nci/organization/ccg/research/structural-genomics/tcga/studied-cancers/uterine

For the validation of the findings, I wonder if the CCLE dataset (Cancer Cell Line Encyclopedia https://portals.broadinstitute.org/ccle) and GDSC (Genomics of Drug Sensitivity in Cancer https://www.cancerrxgene.org/) could be used. Both CCLE and GDSC collect cell lines expression data with drug response information (eg., IC50). Please check out if any of the identified active compounds were included in CCLE or GDSC.

Minor Comments:
I would recommend adding a section for Abbreviations to include the full names for TCMSP, GO, GEO, KEGG, etc.

Please add “Cell lines were routinely checked for mycoplasma contamination.” in the Reagents and Cells section.


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: THE MODULATORY PROPERTIES OF ASTRAGALUS MEMBRANACEUS TREATMENT ON ENDOMETRIAL CANCER: AN INTEGRATED PHARMACOLOGICAL METHOD
Review round: 1
Reviewer: 3

Output:
Basic reporting: This study explored the modulatory properties of Astragalus membranaceus treatment on endometrial cancer by using network pharmacology analysis coupled with experimental validation. The studies about the progression and treatment of endometrial cancer are important work. This study provides a scientific basis for clarifying the effects and mechanism of Astragalus against endometrial cancer. However, this article was not well organized; some of the methods and results were not shown clearly; moreover, the English writing and grammar must be extensively improved. Some major and minor concerns need to be solved before acceptance for publication, these points as discussed below:
Experimental design: no comment
Validity of the findings: no comment
Additional comments: 1, The authors are encouraged to supplement in vivo experiments.
2, High quality TCM Database should be retrieved and collected in Database Construction, such as ETCM, TCMSP and TCMID database, etc., and should not be limited to the TCMSP database.
3, Please consider whether it is reasonable to use OB ≥ 30% and DL ≥ 0.18 as the judgment index.
4. The clinical application of Astragalus membranaceus against endometrial cancer should be supplemented.
5, The background of this study should be descripted in more detail in the introduction section.
6, Many active compounds were screened in the Astragalus membranaceus, why only selected formononetin to conduct the experimental validation? The activities of other active compounds need to be evaluated.
7, Why did the authors only focus on ERβ rather than other important targets?
8, Please increase the pixels of the figures, especially the figure 4.
9, Only detection of ERβ expression could not confirm the mechanism of Astragalus against endometrial cancer. More experimental validation is encouraged.
10, The role of ERβ in endometrial cancer needs to be discussed in more detail in the discussion.
11, The English writing and grammar must be extensively improved. For example, in the ABSTRACT section, the wrong spelling “forononetin” and “formonitine” should be changed to “formononetin”. The authors need to check the manuscript again to avoid some spelling mistakes.


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: THE MODULATORY PROPERTIES OF ASTRAGALUS MEMBRANACEUS TREATMENT ON ENDOMETRIAL CANCER: AN INTEGRATED PHARMACOLOGICAL METHOD
Review round: 2
Reviewer: 1

Output:
Basic reporting: Overall the manuscript has improved by addressing the comments from reviewers
Experimental design: No comment
Validity of the findings: 1. Figure 5: The process for constructing the core PPI network of the 13 targets

Why 13 targets when in the results say: The core PPI network consisted of 48 nodes and 528 edges.
Brief explanation for clarity will be good.
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: THE MODULATORY PROPERTIES OF ASTRAGALUS MEMBRANACEUS TREATMENT ON ENDOMETRIAL CANCER: AN INTEGRATED PHARMACOLOGICAL METHOD
Review round: 2
Reviewer: 2

Output:
Basic reporting: The title, abstract, introduction, methods, results and discussion are appropriate for the content of the text. Furthermore, the article is well constructed, the bioinformatic analysis is well performed. The figures are relevant, high quality, well labelled and described.
Experimental design: The method design is original and novel. However, the research is within the scope of this journal. Research question is well defined, relevant and meaningful. The methods are highly technical, ethical and logistical. Statistical methods are chosen correctly.
Validity of the findings: All underlying data have been provided in detail. The findings are meaningful. The conclusions are well stated and relevant to the research questions.
Additional comments: Major Comments:

I’m wondering if there are any ongoing clinical trials focusing on any of the active ingredients identified in this study to treat endometrial  carcinoma or even ovarian cancer? It will be very strong evidence for the significance of the current study if so. Please carefully search through https://clinicaltrials.gov

RESPONSE: Thanks for this comment. We searched Astragalus and the selected compounds in the database. Unfortunately, we did not find any relevant clinical trials.
However, we searched the PUBMED database and found that the compound Chinese medicine of Astragalus membranaceus, Angelica gigas, and Trichosanthes kirilowii was in phase I clinical trials for the safety of solid tumour patients. And there is also some clinical trial data in China. We add these data in the Discussion section.

COMMENTS: The Discussion part looks great. Thanks!

Considering the sample size of the GEO dataset(GSE63678) is relatively small. I recommend using the data from ICGC(International Cancer Genome Consortium) and TCGA(The Cancer Genome Atlas Program), which are national or international consortiums with many more samples. Please check the NCI Genomic Data Commons (https://portal.gdc.cancer.gov) to find more information about TCGA data and ICGC Data Portal(https://dcc.icgc.org) to find more information about ICGC data. At least, TCGA should contain Endometrial Carcinoma samples: https://www.cancer.gov/about-nci/organization/ccg/research/structural-genomics/tcga/studied-cancers/uterine
RESPONSE: Thanks for this suggestion, and we concede the previous version is weak. We added the UCEC RNA-Seq data in the TCGA database and found 587 cases, including 35 normal samples and 552 tumour samples.

COMMENTS: The revised version of “Data mining based on the GEO and TCGA database” part in Methods looks good to me.

For the validation of the findings, I wonder if the CCLE dataset (Cancer Cell Line Encyclopedia https://portals.broadinstitute.org/ccle) and GDSC (Genomics of Drug Sensitivity in Cancer https://www.cancerrxgene.org/) could be used. Both CCLE and GDSC collect cell lines expression data with drug response information (eg., IC50). Please check out if any of the identified active compounds were included in CCLE or GDSC.

RESPONSE: Thanks for this comment. However, it is a pity that we searched the selected compounds in the two databases CCLE and GDSC, and found no helpful information. We guessed that these might be due to traditional Chinese medicine complexes. And we are honoured that you have provided us with two important databases to provide more guidance for our future research on drug information.

COMMENTS: It makes sense. Thanks for double checking the databases.

Minor Comments:
I would recommend adding a section for Abbreviations to include the full names for TCMSP, GO, GEO, KEGG, etc.
COMMENTS: The Abbreviations section looks good.
Please add “Cell lines were routinely checked for mycoplasma contamination.” in the Reagents and Cells section.

COMMENTS: I see the recommended sentence added. Thanks!


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: THE MODULATORY PROPERTIES OF ASTRAGALUS MEMBRANACEUS TREATMENT ON ENDOMETRIAL CANCER: AN INTEGRATED PHARMACOLOGICAL METHOD
Review round: 2
Reviewer: 3

Output:
Basic reporting: This article predicts the active ingredients and potential targets of Astragalus membranaceus for application to endometrial cancer, which may help to illustrate mechanism of action on a comprehensive level. This manuscript has been intensively revised and improved, and could be accepted for publication after some minor revisions, for example, the words in the figure 6 are too vague to read.
Experimental design: The experimental procedures and results were clearly described.
Validity of the findings: The findings were validation.
Additional comments: This manuscript has been intensively revised and improved, and could be accepted for publication after some minor revisions, for example, the words in the figure 6 are too vague to read.


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: PROTEIN FUNCTION PREDICTION WITH GENE ONTOLOGY: FROM TRADITIONAL TO DEEP LEARNING MODELS
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: 1 For the survey method part, I don’t think it is a fair comparison of the mentioned tools, since the benchmark set CAFA3 was published in 2019, the DeepGOplus was trained using more recent data released in 2020, and other tools, such as PFP and PANNZER2 were trained using different datasets (training data was not mentioned in the manuscript). The results of DEEPred were extracted from the DEEPred paper, the training data was still not clear. Because deep-learning models are easy to get the over-fitting problem, the sequence similarity between the training data and the benchmark set will strongly affect the performance.  It is not clear whether the sequences in CAFA3 have been used in training those deep-learning methods, the performance may be overestimated for DeepGoplus and DEEPred. Since DeepGoplus and DEEPred both provide source code, they should be re-trained on the same dataset. Because of such a problem, the conclusions are not well supported. I suggest the authors do more strict experiments or discuss more on the performance evaluation.
2 For the machine-learning-based method, there lacks a clear problem formulation. For example, is it a multi-class classification problem for all the GO terms? Or is it a binary-class classification problem for each GO term? This is also related to the performance evaluation. The metrics used to evaluate the prediction performance, such as precision and recall, are designed for binary-class predictions, how to assign a single score per method?
Validity of the findings: 1 Why only sequence-based methods were selected and compared in this paper? The readers expect to see the comparisons of deep-learning methods as much as possible.
2 As pointed out by the authors, one challenge of protein function prediction is the imbalanced GO classes and the multi-label problem, but what is the current state of the field regarding to these challenges?  Do the authors have any insights in coping with these challenges?
Additional comments: In this review paper, the authors briefly reviewed the conventional approaches and focused on the review of recent deep-learning-based methods for protein function prediction. They presented an overview of current automated protein function prediction methods and conducted a mini comparison of several available tools. And they finally highlighted the challenges of the field. I have two other major concerns that are listed below:
1 Except for the deep-learning methods reviewed in this paper, several more advanced and recently published methods should be reviewed. Such as one in https://academic.oup.com/bioinformatics/advance-article/doi/10.1093/bioinformatics/btab198/6182677 that applied transformer and one in https://arxiv.org/abs/2007.12804 that applied GNN.
2 The data used as benchmark is not clearly explained in the Data section. I still don’t understand what does the NK (no-knowledge) and LK (limited-knowledge) mean. Does it mean the sequence has no or limited public annotations but was fully labeled first in the benchmark dataset? What do the partial mode and fully mode mean? The authors should explicit these two terms. I cannot understand the explanation from the current version from this sentence: “partial mode, for a set of proteins with at least one prediction, and full mode, computed for all benchmark proteins.”


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: PROTEIN FUNCTION PREDICTION WITH GENE ONTOLOGY: FROM TRADITIONAL TO DEEP LEARNING MODELS
Review round: 1
Reviewer: 2

Output:
Basic reporting: 1) Professional English used throughout
2) Background context and literature reference have been provided
Experimental design: Investigation into methods have been performed and results presented.
However, more structured details are required in the explanation of methods. While reading, details seemed confusing.
Validity of the findings: Conclusion have been supported by the results and overall review.
Additional comments: The review is well written giving an overview of conventional and new methods for protein function prediction.
Recommendation: Structured details are required in the explanation of methods.


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: PROTEIN FUNCTION PREDICTION WITH GENE ONTOLOGY: FROM TRADITIONAL TO DEEP LEARNING MODELS
Review round: 2
Reviewer: 1

Output:
Basic reporting: Background context and literature reference have been provided.
Experimental design: Including more details and flowchart presented helps in better understanding
Validity of the findings: The conclusion have been supported by the results
Additional comments: The manuscript is more structured and issues have been addressed. However few sections in the manuscript lacks correct sentence formation and is sometimes confusing. Working on improving those is recommended.


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: PROTEIN FUNCTION PREDICTION WITH GENE ONTOLOGY: FROM TRADITIONAL TO DEEP LEARNING MODELS
Review round: 3
Reviewer: 1

Output:
Basic reporting: Background provided, literature referenced, clear writing
Experimental design: Recommendations were worked on and methods described in details
Validity of the findings: The conclusion have been supported by the results
Additional comments: Recommendations have been worked on.


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

Input:
Title: GENETIC DIVERSITY AND POPULATION STRUCTURE OF EUSCAPHIS JAPONICA, A MONOTYPIC SPECIES
Review round: 1
Reviewer: 1

Output:
Basic reporting: The study focuses on analysis of genetic diversity and structure of Euscaphis japonica collected from different regions of China using ISSR markers. The data might be used for establishment of conservation program of this species.
The limitations are by use dominant markers as ISSR markers for this study and the use of too limited statistical methods based on similar studies.
The English language is sufficient quality.
Experimental design: Material &Methods section:
-The Methods used are too limited according to the huge works on genetic diversity and structure in woody species.
Validity of the findings: Results section should be revised according with these comments:
-Description of Fst values and similarity coefficient could be moved to “Genetic differentiation” part.
-The UPGMA and PCA analyses should be described in a part entitled “Genetic structure” along with the results of STRUCTURE analysis.
-Results of STRUCTURE analysis should be more described and the genetic diversity parameters of the identified clusters estimated.
Discussion section:
Discussion was too descriptive and should be rewritten. Its no necessary to describe the obtained results in the discussion section but to compare them with results obtained in previous works based on this topic.
I suggest that this study needs more improvement to considered it for publication in this journal.
Additional comments: The study focuses on analysis of genetic diversity and structure of Euscaphisjaponica collected from different regions of China using ISSR markers. The data might be used for establishment of conservation program of this species.
The limitations are by use dominant markers as ISSR markers for this studyand the use of too limitedstatistical methods based on similar studies.
The English language is sufficient quality.
I suggest that this study needs more improvement to considered it for publication in this journal.

Material &Methods section:
-The Methods used are too limited according to the huge works on genetic diversity and structure in woody species.

Results section should be revised according with these comments:
-Description of Fst values and similarity coefficient could be moved to “Genetic differentiation” part.
-The UPGMA and PCA analyses should be described in a part entitled “Genetic structure” along with the results of STRUCTURE analysis.
-Results of STRUCTURE analysis should be more described and the genetic diversity parameters of the identified clusters estimated.

Discussion section:
Discussion was too descriptive and should be rewritten. Its no necessary to describe the obtained results in the discussion section but to compare them with results obtained in previous works based on this topic.


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

Input:
Title: GENETIC DIVERSITY AND POPULATION STRUCTURE OF EUSCAPHIS JAPONICA, A MONOTYPIC SPECIES
Review round: 1
Reviewer: 2

Output:
Basic reporting: No comment
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: The manuscript focused on a woody species Euscaphis japonica and revealed its genetic diversity and population genetic structure using ISSR markers. The authors found that E. japonica had an overall high level of genetic diversity and the results of clustering analysis showed limited gene flow between some populations especially between the deciduous and the evergreen populations. Generally, while this manuscript provided some valuable data, the significance of studying the genetic variation of E. japonica and the potential applications of the results are still unclear. In addition, the authors misunderstood the meaning of genetic differentiation and genetic clusters, leading to very confusing results. I therefore think that a major revision is necessary before the acceptance of this manuscript.

Major concerns
1.	In the introduction, I suggest the authors to focus on the importance of genetic variation to species persistence especially for endangered species and species with small population size, rather than comparing the genetic diversity of woody species. Moreover, I can’t see why E. japonica must be studied, and the authors should address the reasons why this plant is so important (e.g., endangered species and/or with great potential to be applied in agriculture).
2.	In the methods and results, the authors should re-organize their statements in genetic clustering and genetic differentiation. Fst and AMOVA showed the pattern of genetic differentiation among populations, while other analysis like STRUCTURE, UPGMA and PCA allocated individuals into different clusters. I therefore suggest to put the results to the right places.
3.	I feel very confused when reading the results of clustering analysis, because it is difficult to conclude whether the clustering patterns obtained from different analyses were converged. For example, whether all individuals assigned into Cluster I in STRUCTURE were allocated into a particular group in UPGMA or PCA. The authors should revise the relevant contents to clearly illustrate their results.
4.	The discussion is too redundant. The ISSR is not a prevalent or advanced technique in population genetics, so this part is not necessary. The following two parts are hard to follow without clear topics. I suggest the authors to compare the overall genetic diversity with other related species and then clarify the geographic distribution of genetic diversity with speculations of the underlying mechanisms. As to the genetic differentiation and clustering pattern, the authors may conclude the differentiation pattern at the beginning and discuss the underlying mechanisms from both life history of the plant and the geographic characters. Moreover, in the conservation implications, the authors can discuss which populations ought to have high priority for conservation and provide the global conservation strategy for the studied species.

Minor comment
1.	I suggest the authors to provide a sampling map to show the distribution of sampled populations.


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

Input:
Title: GENETIC DIVERSITY AND POPULATION STRUCTURE OF EUSCAPHIS JAPONICA, A MONOTYPIC SPECIES
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 addressed all my major concerns, and the manuscript has been improved greatly. Nevertheless, I still have some suggestions: (1) the writing English should be polished by a native speaker, and (2) the authors must plot the sampling map by themselves rather than directly copying the map from Google Earth.


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

Input:
Title: GENETIC DIVERSITY AND POPULATION STRUCTURE OF EUSCAPHIS JAPONICA, A MONOTYPIC SPECIES
Review round: 3
Reviewer: 1

Output:
Basic reporting: No comments.
Experimental design: No comments.
Validity of the findings: No comments.
Additional comments: The authors have addressed all my concerns.


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

Input:
Title: GENETIC DIVERSITY AND POPULATION STRUCTURE OF EUSCAPHIS JAPONICA, A MONOTYPIC SPECIES
Review round: 4
Reviewer: 1

Output:
Basic reporting: I found the paper interesting, especially that you can show genetic differentiation supporting your previous phenotypic findings. My comments below are mostly about how you present your results and I hope these could improve the clarity of your manuscript and help your potential readers better understand your results.
Experimental design: I did not see any mention of herbarium voucher specimens in the paper. If you did prepare them, even for a previous paper, they should be mentioned/ cited here as well.
Validity of the findings: no comment
Additional comments: Line 31: I am not sure ‘multiple analyses’ is the best choice here, as the evidence for deciduous and evergreen populations originate in your 2019 paper as far as I can tell. Unless you mean that populations are exclusively either deciduous or evergreen, though I think this would still count as observation, very useful and interesting one, rather than analysis.

Line 32: I don’t think the UPGMA tree can show whether evergreen or deciduous E. japonica originated first. It is interesting that across your analyses these appear as distinct groups, however as the UPGMA analysis cannot be rooted there is no directionality to the tree to assume that deciduous gave rise to evergreen. This could be an interesting further study.

Line 80: I think this should be ‘to date’

Line 80-81: This sentence is unclear

Line 105: I would recommend citing the 2019 paper here, as I assume the field observations refer to that.

Line 199-200: See my comment above about UPGMA

Line 203-204: It would be useful to add that the DYC and WYC populations are evergreen (providing I am correctly interpreting Table 1)

Line 210-211: I am not sure what you mean strong overlap between deciduous and evergreen in the STRUCTURE analysis, especially since in line 215 you mention little genetic differentiation between evergreen and deciduous. It would be useful to add lines to mark populations and evergreen/deciduous in Figure 2e to help the readers understand the results better.

Line 221: What do you mean ‘main populations’?

Lines 228: I think it should be ‘indicates’ instead of ‘indicated’.

Lines 234-235: I find this sentence out of place here. I think it would be better in the discussion to mention that the phenotypic variation established in Sun et al. 2019 is supported by the analyses here and these could suggest that E. japonica is two species rather than one.

Line 258-259: The chance of interpopulation bee pollination would depend on distance between populations. It could be useful to add here how far populations are apart in light of bee foraging distance.

Line 274: I believe this should be ‘little’

Line 294: What do you mean ‘inappropriate habitat’, disturbed by human activity?

Line 306: I think this should be ‘storing seeds’

Line 316: See my comments above about the UPGMA analysis.

Line 320-321: I am not sure what analysis you refer to with Structural analysis. The correlation between genetic and geographic distance (usually a Mantel-test) is only mentioned in relation to population ZYL as far as I can tell. Even then this is more derived from understanding where each population is and to which cluster in the analyses they fall into. It could be useful to include a Mantel-test to show correlation, or lack of, or incorporate geography in the figures if possible.

Line 325: I am not sure, but I think there is no need for ‘in’ in front of ex-situ here.

Table 1. I think it would be useful to add deciduous/ evergreen to the ‘Leaf characters’ column or as an extra column on its own to help the reader understand which population is which.

Looking at the previous comments I see your point about choosing 4 clusters in the STRUCTURE analysis to present comparable results between the different analysis. However, I think in the discussion you should mention how the different number of clusters in the PCA analysis relate to STRUCTURE and UPGMA. Furthermore, you could include the STRUCTURE plot for 6-9 clusters as supplementary information.


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: DISCRIMINATING THREE MOTOR IMAGERY STATES OF THE SAME JOINT FOR BRAIN-COMPUTER INTERFACE
Review round: 1
Reviewer: 1

Output:
Basic reporting: The English used in the article needs to be improved as there are a lot of issues with grammar and sentence structure. The subject information is missing from Table 2.
Experimental design: The experimental design looks good, with an identified gap as low recognition rate for MI of same joint. However, this needs more insight and reference. I believe that authors should evaluate other recent works using their dataset and compare them with their work.
Validity of the findings: More analysis needs to be performed to show that the proposed method is superior to existing methods of EEG signal classification. This can be done by comparing the proposed method with other recent works also evaluated using publicly available datasets.
Additional comments: The authors mention that classification of motor imagery (MI) signals relating to same joint are more difficult compared to that of MI signals belonging to different joints. LMD is proposed for signal decomposition and CSP for feature extraction, while twin support vector machine (TWSVM) is used for classification whose parameters are tuned using multi-objective grey wolf optimizer (MOGWO). An average accuracy of 89.84% is obtained using the data collected, which comprises of 7 subjects.
1.	A major concern is that the paper only looks at the classification of MI signals of the same joint using a very small dataset. However, the authors should also evaluate their method on other publicly available datasets (such as BCI Competition IV dataset, EEG GigaDB dataset, etc) and compare with other related works such as those listed below, which have been published in recent years. This is imperative to show that the proposed approach not only works well on the given dataset but also performs well on other datasets for different MI tasks.
•	S. Kumar, R. Sharma, A. Sharma, “OPTICAL+: a frequency-based deep learning scheme for recognizing brain wave signals,” PeerJ Computer Science, 7:e375, 2021
•	M. Miao, A. Wang, and F. Liu, "A spatial-frequency-temporal optimized feature sparse representation-based classification method for motor imagery EEG pattern recognition," Medical & Biological Engineering & Computing, vol. 55, pp. 1589-1603, February 2017.
•	S. Kumar and A. Sharma, "A new parameter tuning approach for enhanced motor imagery EEG signal classification," Medical & Biological Engineering & Computing, April 2018.
2.	It is mentioned that the entropy (En) and super entropy (He) of the cloud model are employed to select effective PF components. However, only PF1 obtained from each channel is used for further processing. This is a one-time evaluation and not an adaptive selection method. The authors should clearly state this in the paper.
3.	No source code is provided. It is highly recommended that the source code for the proposed method be made available as it will enable future research to easily test and compare the proposed method on other datasets as well.
4.	Details of twin SVM should be discussed in detail.
5.	Authors should also discuss other recent works published in the introduction such as those mentioned above and a few others for the benefit of the readers.
6.	The exact number of spatial filters selected should be clearly stated.
7.	There are a number of issues with the English used, a few are highlighted in the annotated document. A few other comments can also be found in the annotated document.


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: DISCRIMINATING THREE MOTOR IMAGERY STATES OF THE SAME JOINT FOR BRAIN-COMPUTER INTERFACE
Review round: 1
Reviewer: 2

Output:
Basic reporting: - Overall, the abstract focuses too much on methods and not enough on the main finding and the significance thereof. The general framework for the proposed decoding technique is properly presented, however, detailed steps such as those involving entropy are not necessary. Furthermore, the authors should briefly mention the significance of the main result (accuracy) relative to other methods and concisely describe its potential influence in the field.

- Line 32: BCI systems "extract" sensorimotor rhythms, etc., rather than "include"

- Line 36: EEG is not random, but rather "noisy"

- Line 39 - 52: The references used to support the different feature extraction and classification methods are a bit excessive. I would consider consolidating these large lists using review articles that go in depth into these topics.

- Line 71: The  LDM-CSP and MOGWO-TWSVM acronyms needs to be written in long form before being used in short form.

- Line 78: It is customary to have a "participants" section or something similar describing the human subjects in detail. At minimum, the authors should describe the sex, age and handedness of these participants. It should also be indicated if informed consent was provided and if this study was approved by an ethics board.

- Figure 1: It would be beneficial to include a visual depiction of the tasks described by the different cues.

- Figure 3-4: I am unsure of how Figures 3 and 4 contribute to the narrative of the current work. These figures provide intermediate and detailed information regarding the methods proposed, but in my opinion, do not add much to the overall results. I would be fine if these were removed or moved to supplementary information.

- Overall, the figures are aesthetically pleasing, are well organized and convey clear messages.

- In general, the English of this article is good but not great. I feel that this work could benefit from a professional proofreading service to correct various language and grammar errors.
Experimental design: - Lines 67-69: The motivation for the current work is unclear in the Introduction. They appear to motivate the current work in lines 67-69, however, as written this language is not strong enough. Why are the authors proposing this new framework? Do current techniques not produce high enough accuracies? Also, what imagery tasks are included in this work and why were they chosen?

- Line 87: please indicate if the 60 trials were the total number or per motor imagery task (i.e. 180 total).

- Line 97: The authors mention no preprocessing steps involved in EEG artifact rejection. This is particularly important as the frontal (C/FC) electrodes used in the current work are specifically prone to eye/muscle artifacts. The authors should describe if and which artifact removal techniques were employed.

- Line 147: The authors should provide justification for the use of CSP with only four EEG channels. CSP is most commonly utilized with large/full EEG montages where spatial dependencies of motor imagery tasks can be leveraged and may be less valid with so few channels. Can the authors please justify this aspect of the work with theory and or previously published works?

- Line 235: The rigor of comparing the proposed technique to numerous traditional techniques in the field is appreciated. Furthermore, these results do well to support the quality of the current work, however, I think this validation should be highlighted much earlier in the manuscript. It would be beneficial and welcomed if the authors were to state in the abstract and introduction that they validated the proposed work against various other approaches and found their technique to outperform them. Along these lines, the validation described here and presented in Figure 8 and Figure 9 appears to be well done and clearly demonstrates the improved performance of the authors' work.

- Related to my previous comment: It is very good that the authors provide statistical evidence in Table 4 to support the performance increase of their proposed approach compared to other techniques (Figure 9). However, the statistical analyses are not described until the last paragraph of the results, rather than in the methods. The authors should correct this and add a brief "Statistical Analysis" section at the end of the methods to describe how these results were obtained. Additionally, it sounds like the authors applied a one-way ANOVA two each pairwise combination of their proposed method and one other traditional method to evaluated significance. If this is the case, I feel that this is an inappropriate statistical test. The authors should in fact apply a one-way ANOVA to all six methods and then post-hoc pair-wise t-tests/Tukey HSD tests. In this case, the authors only need to report the results of the pair-wise tests involving their proposed method. While I dont expect the overall results to change much at all, I feel that this would be a more appropriate methodology.

Furthermore, is there a reason why the authors do not provide the same statistical results for the the values presented in Figure 8? Also, is it necessary to include a table(s) with statistical results when they can also be added to the group-level bar graphs in Figures 8 and 9?

- The main results in the current work involve classification accuracies or similar/related metrics. Would it be possible for the authors to include and physiological results such as the CSP maps that indicate the weighted importance of the different electrodes used?
Validity of the findings: - In the Discussion, the authors need to describe how this work compares to similar work published in the field. Obviously, the authors will highlight the within data set validation that they performed themselves, but will also need to describe the classification performance of similar tasks investigated by other groups/studies. For example, how do these results and tasks used compare to the rest of the field? If there are large discrepancies, potential reasons should be provided.

As is it currently written, the Discussion appears to be repetitive with the methods section and dedicates too much content to the technical details. The Discussion should be of high-level than this and focus on where the results fall within the field at large and how they may impact it in the future. I would therefore suggest that the very large second paragraph of the Discussion to be reworked to accommodate these suggestions.
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: DISCRIMINATING THREE MOTOR IMAGERY STATES OF THE SAME JOINT FOR BRAIN-COMPUTER INTERFACE
Review round: 2
Reviewer: 1

Output:
Basic reporting: Much improved compared to previous submission
Experimental design: Acceptable
Validity of the findings: Performance of the proposed systems as reported looks good.
Additional comments: The comparison made in the revised version (lines 282-294) is not valid. One cannot compare different methods by evaluating them using different datasets. The authors should consider revising the comparisons made.


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: DISCRIMINATING THREE MOTOR IMAGERY STATES OF THE SAME JOINT FOR BRAIN-COMPUTER INTERFACE
Review round: 2
Reviewer: 2

Output:
Basic reporting: Overall, the work has been markedly improved during this recent round of revisions. I believe all of my previous comments from this section have been properly addressed.
Experimental design: - I agree with the editor that the new analysis with the full channel montage is far more valid than the original analysis using only four channels. Furthermore, this step seemed to improve the results overall. Due to the change, however, I feel that the following sentence should be removed from the introduction as channel number is no longer a significant advantage of the current work compared to others.

"However, it requires a larger number of input channels in that study."

- I do not feel that my previous concern regarding the statistical analysis was properly addressed. In the original submission the authors performed pairwise ANOVAs between the classification accuracies of different methods. By contrast, in the current revision, the pair-wise ANOVAs were simply changed to pair-wise t-tests. I believe that this approach could be valid if the authors correct the individual t-test values for multiple comparisons (i.e. Bonferroni or false discovery rate). Alternatively, as I mentioned in the previous round of review, I feel that an ANOVA containing the classification accuracies of all methods is necessary before the pair-wise t-tests. As long as a significant difference exists among any of the included methods, this ANOVA should be significant and would warrant subsequent pairwise t-tests. I recommend that the authors choose to perform one of these options in the following round of review.

- Please add units to the color bar of the new Figure 7. There should also be more details regarding how this group-level topography was created. In the authors' response to reviewer 1 Q6, they state that three spatial filters were obtained for classification (one for each task pair). Ideally, I assume that each one of these filters contains weights to maximize the between-class variance of any two MI tasks. Therefore, it seems more reasonable to show average channel weights for each MI task pair (x 3) to highlight the channels that are maximally discriminable between the different tasks rather than simply their weighted "importance". I think this would provide a deeper level of understanding regarding the influence of different channels on the neural representation of the various MI tasks.

- I feel that further English edit is still necessary throughout the manuscript and that a professional proofreading service would be beneficial.
Validity of the findings: I believe all of my previous comments from this section have been properly addressed.
Additional comments: No additional comments


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

Input:
Title: A NEW THREAT TO LOCAL MARINE BIODIVERSITY: FILAMENTOUS MATS PROLIFERATING AT MESOPHOTIC DEPTHS OFF RAPA NUI
Review round: 1
Reviewer: 1

Output:
Basic reporting: The English was of a quality that allowed this reviewer to understand the system/problem/data.  However, there were some minor grammar issues and the revision would benefit from a review by a native English writer.  PeerJ requirements are met throughout.  My only basic concern is that the Introduction suffers from some misrepresentations of mesophotic reefs: 1) I would not characterize MCEs as “formed mainly by light-dependent corals” [line 42], rather they represent low-light adapted species (= a few coral species, but also sponges & gorgonians, etc.  see for reviews: Lesser et al. [2018] Ann Rev Ecol Evol Syst 49:49-71; Slattery & Lesser [2021] Front Mar Sci 8:654268), 2) while it is true that these are vulnerable systems (Andradi-Brown et al. 2016), it is also worth noting that they may represent a refuge for depth generalists (Bongaerts et al. [2010] Coral Reefs 29:309-327; Slattery et al. [2011] JEMBE 32-41).
Experimental design: The experimental design utilized extensive ROV surveys from around the island to characterize mat and coral cover/health.  Unfortunately, with virtually no data analysis, the paper provides an observational report and speculation regarding the drivers.  I would urge the co-authors to include: 1) percent cover live vs dead corals, 2) numbers of fish (and species) at each site, 3) correlations with mat cover/sites around island.  These quantitative data will be much more useful for the scientific community (ie, MCE vulnerability) and for conservation (ie, return sampling post-COVID), 4) can you include any nutrient data (collections or a meta-analysis of existing datasets).
Validity of the findings: The videos are very impressive and high quality; a little more work (as suggested above) will strengthen this manuscript and the conclusions.  Statistic are necessary in these systems (note the response to cited: Rocha et al. [2018] Science361:281-284, Roberts et al. [2018] “The challenge of the deep should not preclude robust ecological analysis”.  Science. sciencemag.org/content/361/6399/281/tab-e-letters).
Additional comments: This paper has the potential to be a significant contribution to the field.


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: 2

Output:
Basic reporting: The paper by Sellanes et al. is overall a well-written and insightful piece of work that uses a good amount of appropriate references in their background information and interpretation. The manuscript structure and figures are clear and the raw data is shared in the appendix.

I would recommend the raw data to also include the information on coral cover that was collected, and to show the deviation/error of the depth, or perhaps minimum/maximum, as well as the mean. In terms of structure, my only comment is that the conclusion is currently very long, and I suggest the part of future research and its importance should be moved to the end of the discussion.
Experimental design: The research question addressed by Sellanes et al. is important and timely. There has recently been an increase in attention on benthic cyanobacteria and very little is known about their occurrence on mesophotic reef systems (due to the difficulties highlighted by the authors in studying this system). As such, the authors identify a clear research gap and define three clear aims (not research questions) linked to this topic. I do however have some reservations with the experimental design that I hope the authors can address in a revision.

In terms of the methods, I am struggling to understand the survey design in terms of depth at each site which has made it difficult to interpret the findings. The raw data, including coordinates and mean depth of each site, is provided in the appendix which is very helpful, but I suggest it should be clearer in the manuscript itself. The raw data suggests that there were in fact only 24 individual stations (as opposed to 56 as described in the methods) which were measured 1-3 times over 2018 and 2019. While it seems the coordinates of the stations remain similar, the depths change dramatically over the years (i.e. station 2 in 2018 was 200m, but in 2019 was 89.5m). If the stations are monitored more than once, the results need to change to account for this - on the other hand if these should all be considered separate stations then the authors should change the 'Station' numbers in the raw data table.

Depth would be expected to have an impact on benthic community structure (and the authors show this in Figure 1B), yet it remains unclear how the depths of the ROV surveys were incorporated into the study design in a standardised way. For example, are there any deep sites (>150m - where seemingly cyanobacteria are absent as per Figure 1B) on the western side where the authors postulate pollution is driving mat proliferation? This is also important to consider when comparing to the 1999 ROV survey (lines 216-218) to show that coral cover has declined. I would suggest the authors need to spend some time clarifying this throughout the respective sections (perhaps including a map that shows this).

Lastly, I struggle a bit to understand why the authors are basing their semi-quantitative interpretations on only 5 non-overlapping frames when they have 10-20 minutes of video data. How much area does each 'frame' cover? The selected approach provides only semi-quantitative information from a very small area, while there are ways to extract benthic community information from video transects that could provide much more detailed and accurate information. I think it is important the authors justify/explain this approach better in the text and that this needs to be more convincing, or otherwise more data added, prior to publication.
Validity of the findings: Overall the results are reported well, though in places there is certainly room for expansion. The validity of the findings and the authors' interpretations can be strengthened by incorporating more information on site depth (as suggested in an earlier point).

In terms of the taxonomic information, I recommend the authors to present their morphological ID of cyanobacterial taxa with caution. Perhaps the selected approach is much more powerful that bright field microscopy, but with bright field microscopy Lyngbya, Okeania, or Moorea cannot be distinguished from each other (see Duperron et al. 2020) and should thus be referred to as Lyngbya s.l. ([sensu lato], in a broad sense, as opposed to Lyngbya s.s. [sensu stricto], in a narrow sense) (as in Ford et al. 2021). I understand these to only be able to be differentiated by sequencing. Both mentioned references are included in my comments on the PDF, as well as others I recommend the authors to look into.

In terms of coral decline, the authors draw some large conclusions of 'catastrophic loss' but the current manuscript does not have the data to support this. In the absence of previous data, this could be done potentially by showing the amount of dead coral per site - coral loss from 90 to 0% is definitely catastrophic, while from 10 to 0% is less so as the system was never coral-dominated. Among other quantitative/statistical tests the authors could have incorporated to strengthen their interpretations, the authors miss an opportunity to test for correlation between mat abundance and Leptoseris reef health status, though they describe this relationship in the discussion. I recommend the authors to consider including some tests to make their interpretations of their data more robust.
Additional comments: Overall I enjoyed reading this paper and the findings related to cyanobacterial abundance on Easter Island's mesophotic reefs. I have added some specific comments into the PDF document that I hope will help the revision of this manuscript.