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: KERAPHYTON GEN. NOV., A NEW LATE DEVONIAN FERN-LIKE PLANT FROM AUSTRALIA
Review round: 1
Reviewer: 1

Output:
Basic reporting: All of this is just fine.  I have made a few minor suggestions linked to the text by line number.  See General Comments section below.
Experimental design: Experimental design is sufficient although I would suggest addtion of preservation minerals in the Materials and Methods.  All of this is explained in my review below.
Validity of the findings: I find the paper more-or-less as it stands perfectly acceptable.  See General comments below for explanation
Additional comments: Review of:
Keraphyton gen. nov., a new Late Devonian plant genus of iridopterid affinities from New South Wales, Australia
Antoine Champreux, Brigitte Meyer-Berthaud, Anne-Laure Decombeix

I think this is an interesting report of important Devonian fossil material from Australia – a part of the ancient Earth from which we desperately need more information.  In general, the description and analysis are well done, adequate for the purpose of typifying a new genus, and deserving of publication in PeerJ.  I recommend the work more-or-less as the text already stands.
I list several comments by line number below mostly designed to clarify the presentation somewhat.
I offer to the authors the following comments that might be helpful:
1.	Amplifying the presentation to include the nature of preservation is important, I think.  See comment for text line 77.
2.	I realize that all has been done that probably should be done given the limited nature of the specimen at hand.  I certainly do not dispute the basic findings.  However, Keraphyton represents a significant extension of the concept of Iridopteridales in time, morphology/anatomy, and geographic range that may or may not be useful.  Only time will tell. In my opinion, Chinese, Moroccan and Australian material, from the late Devonian especially, seems to indicate the presence of groups at ordinal or class level that probably need to be established and defined in their own terms rather than appended to Middle Devonian high-level taxa from Europe or North America.  Please note that this is not intended as a criticism.  This is an exciting time in plant paleontology of these generally unknown regions, and I can think of no group in a better position to accomplish this task than the current authors.
Wm Stein
Line 21: I think you simply mean ‘pattern’ instead of ‘division pattern’ since a static pattern of the vascular system is observed and divisions of this system are in fact not observed.
Line 23: By ‘partly specific’ do you mean endemic or something else?
Line 49: The reference for Arachnoxylon perhaps suggests that Stein et al. were responsible for the generic name, when in fact this was Read’s original.  We merely added a new species to an already standing genus in this paper.  The same applies to Iridopteris; this name is Arnold’s.  I think this is merely a matter of citing references (in parenthesis) apart from formal designation of taxonomy, i.e., Arachnoxylon Read or Iridopteris Arnold.
Line77: In this paragraph, no mention is made of the minerals that make up the specimen.  For proper interpretation, I think adding this information would be very helpful.  The specimen looks similar to late Devonian phosphatic permineralizations seen in the midcontinent North America.  However, mention is made below of pyrite in the phloem region, I believe.
Line 78: In the previous paragraph reference is made to transverse and longitudinal ‘sections’.  Were these all ‘thin sections’ as described here?  If not, please specify where thin sections were made and where, perhaps, thick sections as opposed to blocks A-E still remain.
Line 165: I think I see what you refer to as protoxylem in transverse section, although it might be useful to label these cells in a figure as such.  Were you able to verify identification of protoxylem in longitudinal section?  Nothing is said about that in the text.  I must say that this protoxylem, if that’s what it is, seems very different than the conspicuous permanent mesarch strands observed in the Iridopteridales.
Line 166: Here’s the mention of pyrite (=dark filled cells??) mentioned by me above.  Although Fig. 3H shows some degree of compression, and therefore fairly densely packed with dark material, it appears from other views in Fig. 3, that the dark material comprises only one type of cell in the phloem region.  In phosphatic permineralizations, we also observed dark-filled cells but, if memory serves, not pyrite.  Dark-filled cells are also present in the pyrite & limonite permineralizations of Middle Devonian Iridopteridales.  However, the dark stuff is, we guess, organic in origin.
196: I agree with your conjecture here. Thus, this might be reason to have concern about assignment with the Iridopteridales of the Middle Devonian at least.  Might early ferns be an equally valid assignment?
Line 212:  I might suggest saying something like ‘differential hormonal prominence of the shoot apex…’ or being more specific. Our original idea was that the central protoxylem in the Aneurophytales and others likely represented an image of the shoot apical meristem written by hormonal influence on developing provascular tissue much like, by analogy, light exposes film.  The interpretation of Iridopteridales and others, by contrast, seemed to indicate less influence by the shoot apex and perhaps greater influence by the meristems of lateral branches or appendages.  Your sentence as it stands might be interpreted to mean the opposite.
Line 216: I agree with this assessment, although some degree of intermediacy is observed within the Pseudosporochnales.  Pitting in the xylem matches as well, but hypothesized organotaxy not, protoxylem organization not, and general form of the protostele probably not as well.  So, it seems to me, the fossil presents us with a mixed message.  How interesting!
Line 255: How so consistent?  The Middle Devonian Iridopteridales all have whorled organotaxis.  Keraphyton organotaxis, as hypothesized, seems to indicate a difference.
Line 269: The term “temporary” - a feature of time - is used to describe a static structural relationship.  Perhaps it would be better to reword more precisely.
Line 286: I think these comparisons cover the situation in a reasonable way, but see my general comment 2 above.
Line 300: Compare Fig. 4P with 4I (and also seen in the Arachnoxylon specimen of Fig 4K).  Note the difference in that a permanent strand remains in each rib of Asteropteris and Arachnoxylon with paired protoxylem strands ultimately becoming part of departing traces.  A similar pattern is not apparent in Denlongia, although I haven’t studied the latter to know if this difference is important or not.
Line 316:  You might correct me on this, but the Venezuela region might in fact have been part of Laurussia in the Devonian, later separated.


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: KERAPHYTON GEN. NOV., A NEW LATE DEVONIAN FERN-LIKE PLANT FROM AUSTRALIA
Review round: 1
Reviewer: 2

Output:
Basic reporting: This paper is generally very well structured and clearly written (I have noted a few corrections by line number, indicated in Comments to the Authors). The comparison of Keraphyton with other iridopterid taxa is particularly clear. The figures are excellent, and require no revision.

IMPORTANT CORRECTION: The authors make a very clear case for describing a new genus and species for their specimen. They have followed all the technical requirements for describing a new taxon under the ICN. HOWEVER, their species is orthographically incorrect. Since the species is named in honor of a woman, Ruth Mawson, the epithet should be spelled: mawsoniae, in the feminine genitive singular case (See Art 60.8(b) of the ICN (https://www.iapt-taxon.org/nomen/pages/main/art_60.html) Please correct on lines 21: 102: 109: 195: 258: & elsewhere in the paper.

The only portion of the paper that should be strengthened is in the the discussion of the relationships between the ferns, cladoxyls, iridopterids and sphenopsids (lines 31-43).  The discussion as written is rather confusing and light on details. There are several questions at issue here: 1) the relationship between iridopterids and sphenopsids; 2) whether the 'permanent protoxylem group' (Moniloformopses) represents a clade and 3) whether sphenopsids are ferns. The comment "The relevance of the Moniliformopses has been heavily discussed (Pryer et al., 2001; Rothwell & Nixon, 2006)" points to the dispute regarding 2 & 3, but the comment is cryptic, and the references cited are out of date. If the authors want to wade into this debate (although it would add context, it's not strictly necessary for the purposes of this paper), they should cite more up-to-date literature. The attempt of Rothwell & Nixon (2006) to critically evaluate gene trees has been superseded by numerous molecular phylogenies since, virtually all recovering sphenopsids as a basal clade within a broad, monophyletic fern clade (See PPGI: http://dx.doi.org/10.1111/jse.12229 & references within, as well as Qi & al. (2018): https://www.sciencedirect.com/science/article/pii/S1055790318301854 and Shen & al. (2018): https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5795342/).

Perhaps more importantly for the purposes of this paper would be a somewhat expanded discussion of the relationship between iridopterids and sphenopsids. As the authors note, the relationship between the two groups is somewhat tentative, but it remains a viable hypothesis, and it strengthens the significance of iridopterids as not just curious Devonian plants but as progenitors of an important modern group of terrestrial plants. Regardless of whether the 'permanent protoxylem group' is a clade, it clearly unites cladoxylopsids and iridopterids, and a brief discussion of this, especially from the developmental perspective, would also be useful.
Experimental design: This is a descriptive paper of a fossil plant, so experimental design is not applicable. Significance of fossil is clearly defined and described.
Validity of the findings: No comment. The validity and novelty of the new taxon is clearly laid out.
Additional comments: The Middle and Late Devonian was a time of rapid radiation of terrestrial plants, setting the stage for the appearance of modern major groups. In their description of a new genus and species of Iridiopteridales from the Late Devonian of Australia, Champreux et al. make a valuable contribution to our understanding of the early evolution of modern ferns sensu lato, as well as the diversity and paleogeography of Late Devonian megafloras.

The rest of these comments are mostly minor corrections indicated by line number.

22-23: "It also supports the view that this flora was, at least, partly specific to this part of the world at this time." Sentence unclear. Do you mean that Late Devonian Australian floras displayed significant endemism?

41: "The discovery of new Devonian fossils affiliated" WITH

66: Mandowa Mudstone [This is apparently a formal name, so I think that 'Mudstone' should be capitalized.]

103: and φυτό (phyto), plant.

125: Mandowa Mudstone

211: a hormonal [the 'h' is aspirated ('huh') so is preceded by 'a']

279-280: "On the contrary" -> better to phrase this, "in contrast"

284-286: Do the authors want to comment on the functional significance of the broad metaxylem tracheids?

289: COMPRISING [not 'comprised of' - comprise is an active verb meaning the same thing as 'encompass']

315: only COMPRISES Compsocradus ...

318: Mandowa Mudstone


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: KERAPHYTON GEN. NOV., A NEW LATE DEVONIAN FERN-LIKE PLANT FROM AUSTRALIA
Review round: 1
Reviewer: 3

Output:
Basic reporting: The paper is well written, concise and conforms to the standards of Peer J in terms of clarity, presentation of methods, data and conclusions.  The photos and diagrams are of good quality.  The authors present an interesting interpretation of this fossil.   I think some alternatives should be considered, and at least briefly presented, and that some parts of the interpretation are not as certain as presented.   I strongly recommend that the images be more extensively labelled.  This is elaborated below.

1.	The specimen shows signs of distortion- parts of the xylem ribs are broken, some cells are squashed.  The authors present information, including their view of what the stele would look like if not distorted, well.   The diagrams are helpful.
2.	The division within each rib/arm is unusual and should be described a little more carefully.   I am not sure one should use the term “ultimate” on a product of dichotomy; however, other terms I can think of are equally problematical.  I recommend in line 150 that another sentence or two be inserted to describe the two ways the dichotomies occur in the main ribs, because it jumps too suddenly into how there are two of each type.
3.	 The emission of small branches off the fundamental ribs is a unique feature of this plant.  It really does not occur in plants usually included in iridopterids.
4.	Having a central region is unusual- a limited amount is present in a few iridopterids but not in many.   Two plants that are rather similar- and possibly should also be compared- are Dixopodoxylon (Middle Devonian), whose affinities are uncertain and which may represent a root, and the Early Devonian Gothanophyton, probably a basal euphyllophyte whose affinities are uncertain.  Lateral traces are known in the latter.
5.	This leads me to ask, have the authors considered that the structure might be a root, especially given the presence of an endodermis.   The authors should state why they don’t think it is a root.
6.	I also question if this is an iridopterid- it lacks any evidence of producing laterals and how laterals occur anatomically or morphologically is important in defining iridopterids.  It anatomy is rather different too.  Might it also represent a unique lineage (if it is a stem).
7.	In line 196, the authors suggest emission of laterals is opposite and decussate but there is very little evidence in favor of that.  It depends on which ribs produce traces at which level, and whether one or several traces supplied laterals.  One could argue equally for helical or whorled as well.
8.	One more small question- line 153: cells are xx wide?
9.	Concerning labels:  label all ribs, not just some of them in Fig. 1 and 2.  For example, lateral 1b is not labelled in Fig 1B.  is one of the laterals (1D) printed in reverse?  Add some arrows to point out specific structures.
Experimental design: OK
Validity of the findings: See item one above; back off on interpretation by stating "possible" but include alternatives.
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: KERAPHYTON GEN. NOV., A NEW LATE DEVONIAN FERN-LIKE PLANT FROM AUSTRALIA
Review round: 2
Reviewer: 1

Output:
Basic reporting: This is a second review. The authors have addressed many of the reviewers' concerns.  I still have one statement which they don't have to address and a few questions they could-
statement:  in reference to fern-like, while it depends on what the authors mean, if related to ferns then I think any relationship to ferns is by default and I am in a minority of not thinking all so-called moniloforms are monophyletic and that they relate to ferns.  I think we may find that many represent individual euphyllophyte lineages that began and ended...this plant might be one of them.  It may very well turn out to be a member of an as yet unrecognized lineage.

questions/comments:
line 141is this Qi or QIU???
line 387: are the higher beds in the formation similar in lithology? is deposition continuous (or can that be determined).  For stating late Late Devonian it seems it could be as old as Frasnian and no younger than LL Devonian or Tn1a, but the data are sparse
line 387, 388:  after  suggesting (or so I thought) POSSIBLE iridopteridalean, language here is more "certain iridopterid"... I suggest inserting "possible" before iridopteridalean,  and in line 388 "may indicate...."   because there are some differences.
line 391- again perhaps use "Iridopterid-like plant"?

the opposite, decussate nature is one way the actinostele can be viewed, and this is rather supported by the presence of a central segment- but the array of arms and their divisions also would argue for a more helical departure of laterals.  So I think that aspect should be clearly indicated as the authors' interpretation.

Thus I recommend accept after minor revision.
Experimental design: OK
Validity of the findings: OK with the above caveats regarding interpretation of data, although authors support their interpretations
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: VISUALIZATION AND QUANTIFICATION OF MIMETIC MUSCULATURE VIA DICECT
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript is very well written in excellent English. The introduction is concise but provides sufficient background to the topic with all major previous literature cited. The results section is relevant to the stated aim of the paper.

It isn't stated that the diceCT scans or the 3D reconstructions are going to be shared. Will these be made available via one of the morphological repositories?
Experimental design: This manuscript reports a very interesting study into the facial musculature of a strepsirrhine primate species. It’s really nice to see diceCT being used to look at facial muscles as the focus is so frequently the masticatory musculature and the more superficial muscles tend to get overlooked. The methodology seems sound and well-reported. The subject of the study is well-suited to publication in PeerJ and I am always pleased to see descriptive anatomy papers – this is important information that needs to be made available to other researchers.
Validity of the findings: The findings of this study seem valid and the results have not been over-interpreted. However, I feel that at the moment, this manuscript is somewhat superficial and requires a great deal more detail before it is suitable for publication. I outline what I think is missing in the general comments section.
Additional comments: 1.	The results section needs greatly expanding in order to describe each muscle in detail. I realise that the method used, in which the face is removed from the bone before staining may make this a bit more difficult, but I’m sure there is something that could be said where each muscle runs from and to, the orientation of the fibres, how the muscle is positioned with respect to the other muscles under study, and some indication of its function.

2.	Related to the above point, at the moment there is only one figure in the whole manuscript. I was wondering if there were any other figures that would help reveal the morphology of the muscles under study. For instance, are there any slices from the microCT stack that would show the arrangement of some of the muscles (it’s a little unusual to not have at least one CT slice in a diceCT paper), or that could highlight some of the detailed anatomical points you raise in the discussion? Also, given that you’re dealing with superficial musculature, I was wondering whether it might be possible to produce a figure similar to 1B but with the reconstructed mimetic muscles laid over the pre-dissected head? That last point is just a suggestion – it may not be as useful a figure as I think it is, and you may not have a pre-dissection photo of the specimen in the correct orientation.

3.	You’ve given us the mass of each muscle, but this information is only useful if we know how big the specimen was. Can you please report the body mass and/or cranial length of the specimen.

4.	In the discussion, where differences between the morphology reported here and that described in other strepsirrhines are highlighted, I think it would be good to mention the function of the muscles that are being compared. Ideally, you could then speculate on the impact of the anatomical differences that you are reporting. I suspect that the differences are quite minor so it will be difficult to determine the functional consequences, but by at least reporting the function of the muscles, the reader can begin to assess the importance of these differences.


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: VISUALIZATION AND QUANTIFICATION OF MIMETIC MUSCULATURE VIA DICECT
Review round: 1
Reviewer: 2

Output:
Basic reporting: The language is clear and unambiguous. Professional language is used throughout. I did not have a problem understanding the goals, scope, findings, or conclusions of the study.

I recommend drawing more widely from the literature. For example, the blue-eyed black lemur is referred to as a model taxon, but no references are given to demonstrate this. My primatology colleagues don’t necessarily agree that it is a model, so references would help to bolster this statement. Likewise, sourcing of interpretations for mimetic musculature from the literature should be more fleshed out.

Visual aids and data provided are sufficient and necessary. I could see an argument being made to show some 2D diceCT images for mimetic musculature, but I also recognize that such thin slips of muscle are unlikely to be visually informative to the reader. What the authors have chosen to present is clear and well delinated.

This study does not incorporate a hypothesis because it is an anatomical description and proof of concept.
Experimental design: The experimental design revolves largely around specimen preparation, staining, micro-CT scanning, and digital dissection. All are well articulated, and the study is repeatable.

I recommend elaborating on what standard approach was used to distinguish overlapping musculature (e.g., could fascicle orientation be distinguished clearly at 50-micron scale resolution?). If multiple reference guides were used when interpreting overlapping muscles, this would be helpful to the reader as well.

The study does not appear to be ethically flawed. The sample was sourced opportunistically, and the interpretative work for 3D rendering musculature is based on comparative analyses with closely related taxa.
Validity of the findings: The study is novel. The authors make a good argument for the difficulty in working with mimetic musculature as a result of how thin it is and its placement within the skin. DiceCT and other contrast-enhanced 3D visualization methods are well suited to finely parse thse muscles in 3D space. Utilizing reverse dissection and a “mask” approach for staining the tissues of interest was clever, allowing the authors to minimize iodine concentrations. This should have enabled them to develop nice 2D images on which the 3D reconstructions are based. I suspect others will mirror this approach in the future when undertaking diceCT work on restricted regions of interest.

The authors do not appear to provide the underlying data. These could be in the from of the CT-generated images stack and metadata, and/or in the form of 3D .STL/.PLY/.OBJ files for the skull and mimetic musculature, and/or in the form of fly-through videos that provide 2D anatomical references to the 3D models. PeerJ typically requires data sharing with publications, and I would encourage the authors to make their datasets available as one or more supplemental files.

Conclusions are well stated and speculation is constrained to the topic of variation and comparisons with other approaches for studying memetic musculature. It is a sober and useful examination of the method and the opportunities
Additional comments: Please find below a few line-by-line suggestions and comments that I anticipate will further strengthen the manuscript:

Line 27–28 : the authors should in the neck in their introduction about memetic musculature. They note the platysma later in the study (and, reasonably, why the muscle volume is not quantified), but at this stage it would be more complete to include the neck in addition to regions of the face as locations of mimetic musculature.

Line 37: I recommend removing the “/” between  “directionality/presence/absence” and instead structuring the content as “presence, absence, and orientation”, which is easier to read and does not equate the value of presence/absence with directionality as is implied by the current construction

Line 39: change “them” to “mimetic musculature” for clarity

Line 44: capitalize the “X” in “x-ray”

Line 57: delete the first “the” and delete the only comma

Line 58: delete the hypthen before “the blue-eyed” and replace it with a comma

Line 59: change “within” to “from”

Line 61: the blue-eyed black lemur is not considered a model taxon by all practitioners—I recommend supporting this statement with references or, failing that, deleting the word “model”

Line 71: Please clarify if any muscle tissue was left behind on the head during reverse dissection.

Line 91: What standard approach as used to distinguish overlapping musculature. Was one or more reference guides used?

Line 98: delete the duplicate “as the”

Line 116: Add “We observed that” to the beginning of this sentence.

Line 118: change the comma to a period and replace “who note” with “The latter noted” (this serves to shorten an overly long sentence

Line 118: hybrid with what other species?

Line 120: I recall that prior work was only done on adults, but it would be useful to reiterate here that you are comparing your work with muscles described in adults. As you discuss variation, the potential difference in ontogenetic staging would seem like a relevant point.

Line 136: Formal writing in American English recommends that a period follows the abbreviation “vs.”

Line 148: ~ 30–35-micron resolution is often considered the threshold beyond which individual fascicles cannot be discerned reliably.  I agree with the point made in line 148, but
it would also be helpful if the authors clarified whether they were segmenting by identifying fascicles themselves or bundles of fascicles, including what was interdigitating
between mimetic muscles. At 50-micron resolution, I suspect the authors are working with bundles of fascicles. This point may be important others deciding how scanning
resolutions for their diceCT projects.

Line 160: change the first comma to a period. Chance “and” to “This would in turn”

Line 161: I am not confident that it can state accurately that no one has visualized mimetic muscles from their origins to their insertions. Even gross dissection will allow for this. I suspect the authors mean in 3D, or digitally, or with a certain level of accuracy/precision. They should adjust this statement so that says when they specifically mean it to say.

Line 163: Change “3D” to “three-dimensional” — although the term three-dimensional has been used several times already in the manuscript, it has not been abbreviated previously.

Line 165: add a “for” in between “potential” and “contrast-enhacned”

Figure 1 Caption: Abbreviations appear to inconsistently use capital and lower cases and parentheticals. If the abbreviations used here are considered standards in the field, providing a reference would be helpful. If not, I would suggest standardizing the abbreviation scheme. This
will make it easier for the reader to go back and forth between the abbreviations in the figure and their definitions in the legend.

Figure 1: Please insert a scale bar


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: VISUALIZATION AND QUANTIFICATION OF MIMETIC MUSCULATURE VIA DICECT
Review round: 1
Reviewer: 3

Output:
Basic reporting: This paper is written clearly and is well structured overall. Please correct minor spelling/formatting issues commented in the attached PDF.
While the brevity of the paper is good, this should not come at the expense of reporting key aspects of the method to allow reproducibility, or further exploration of results – there are several important aspects of the paper that require elaboration.
I feel that in its current form this work does not meet the criteria for a ‘minimum unit of publication’, and requires additional data from the literature to give meaning and context to the results reported here (please see results and discussion comments).
Experimental design: The scope of literature covered establishes the knowledge gap to be filled, however some additional implications and justification should be provided (please see general comments).
Validity of the findings: Some additional underlying data should be provided (please see general comments regarding Figure 1).
Additional comments: General Comments:
1.	Understanding of 3D facial muscle anatomy in lemurs is a worthy and very interesting goal, but application of these staining and visualisation methods to an intact cadaveric head, where the facial muscles lie in situ, would be much more appropriate and informative (stain times for the very superficial facial muscles would not increase much from those reported here). It is understandable that the data here may represent a convenient/opportunistic sample acquired through past research, but the authors should discuss this more plainly in the paper, as their apparent choice not to use an intact head seems an important and obvious oversight for a study of this kind. The aims of the study (identification of anatomical differences between taxa and quantification of muscle volumes) cannot be adequately met using only a single facial ‘mask’ specimen that has been excised from the skull and dried (!) before staining. Via the method used here, muscle volumes will be modified (especially in muscles with high relative surface area) and meaningful data on muscular attachments to bone or associated tissue are lost (please see comments on method). Because of these limitations, the authors need to use more circumspect language in their stated aims and discussion of their findings.

2.	A key justification for this study is that facial muscles have never before been visualised in 3D using DiceCT – but this can be taken further. In the introduction, the authors should expand their rationale for and implications of understanding facial musculature configuration in lemurs – i.e. why look at facial muscles in lemurs in particular? Can this tell us anything e.g. about the evolution of facial communication in primates (obviously beyond the scope of the present study, but worth noting as a future avenue of inquiry)? This would help to better establish the relevance of the current work.

3.	Figures: The superimposition of the muscles on the skull in the lower Figure 1 panel is a good inclusion and provides important context for the segmented volumes above. However, the stained CT image stack data with which such images were produced is necessary to demonstrate to the reader the efficacy of your stain protocol and ease with which the muscle could be discerned from the surrounding tissue. In the absence of an included complete image stack as supplementary material, please add at least a panel to this figure (or an additional figure) showing select representative slices from which your volumes were segmented. You may also consider including slices through the rostral region to demonstrate how the integration of tissues here made segmentation of muscles in this area impossible.

Results & Discussion:
1.	Shrinkage. The authors state in the methods that the dissected mask is deliberately desiccated prior to fixation and staining (line 70) – for how long, at what temperature? These need to be reported. Drying the tissues out will reduce the muscle volumes and affect the results reported. While I understand how this drying would be necessary for manual (traditional) wet dissection to better distinguish muscle from connective tissues, it should not be necessary in this case as the iodine stain more readily binds to muscle than connective tissue. In the discussion the authors say the deformation and shrinkage of muscles ‘appeared relatively minimal’ (line 157), but as no baseline or progress scans/photos are provided there is no basis on which to make this assessment. I believe the extent of this limitation on the study is strongly understated in the present manuscript, and the nod to this is the discussion (lines 153-161) is insufficient. The stain concentration was indeed quite low (though not as low as reported, see my comments on the method) and the stain duration was short, which both act to reduce the effects of shrinkage. However, multiple authors have shown that even at low (~2%) I2KI concentrations, muscle volume shrinkage can exceed 20% (Bribiesca-Contreras, F., & Sellers, W. I. (2017). Three-dimensional visualisation of the internal anatomy of the sparrowhawk (Accipiter nisus) forelimb using contrast-enhanced micro-computed tomography. PeerJ, 5, e3039; Vickerton, P., Jarvis, J. and Jeffery, N. (2013), Concentration‐dependent specimen shrinkage in iodine‐enhanced microCT. J. Anat., 223: 185-193. doi:10.1111/joa.12068). Please address in more detail the possible effects of shrinkage on the outcome of this study.
2.	Comparative context. Because muscle volumes form the primary quantitative component of this work, lack of context is a critical issue with the paper. Simply stating the volumes in isolation (especially considering shrinkage and ex situ issues identified above) has minimal scientific value and these data need to be presented with some sort of comparative perspective. My suggestion to rectify this would be to express these volumes as proportions of overall total mimetic muscle volume (though this assumes that all tissues have shrunk equally relative to one another and this should be discussed). Then, these proportions can be compared to any existing data on mimetic muscle volume in other primates (or indeed mammals) – please seek this out and incorporate these comparative data into the paper. The authors are probably aware of this recent paper providing image stacks of several DiceCT stained lemur heads on MorphoSource.org (Yapuncich GS, Kemp AD, Griffith DM, Gladman JT, Ehmke E, et al. (2019) A digital collection of rare and endangered lemurs and other primates from the Duke Lemur Center. PLOS ONE 14(11): e0219411. https://doi.org/10.1371/journal.pone.0219411), which can offer raw comparative data to this end (these would also provide additional qualitative comparisons which would strengthen the anatomical portion of the paper). If the authors are able to find older myological studies that may instead report mimetic muscle masses, these would also be appropriate and could be converted to volumes using standard mammalian muscle density values.

3.	Intraspecific variation. The anatomical discrepancies found by the authors between theirs and existing work on closely related lemurs are quite minor. Facial communication in lemurs has not been demonstrated (yet!), so it is likely that facial muscles are under low selective pressure and therefore may vary intraspecifically to a large degree. Even in humans, where facial communication is extremely important, facial muscle presence/absence, arrangement and characteristics vary considerably (e.g. see Watanabe, K. (2016). Facial Muscles and Muscles of Mastication. In Bergman's Comprehensive Encyclopedia of Human Anatomic Variation. doi:10.1002/9781118430309.ch24). Whether the anatomical differences noted here are typical or not for Eulemur flavifrons cannot be stated based on a single specimen. Please elaborate on this in the discussion using more cautious language, addressing the ‘n=1’ sample size and strong possibility of wide intraspecific variation in these traits.

Method:
1.	Missing details. More detail is needed in the methods to make them reproducible and align with emerging conventions in DiceCT literature:
•	How many slices were in the final CT image stack?
•	Did the authors do any resampling etc when dealing with a large (?) image stack in Amira? If so, please report this.
•	How were the boundaries of muscle tissues discerned from surrounding soft tissues? (by eye is fine of course, but the authors should provide examples of their image slices so the reader can see the efficacy of the stain, and thereby the reliability of visual distinction between muscle and non-muscle)
•	Details of software tools used, and any automated software processes. Was every slice manually segmented or was there inter-slice interpolation?
•	Did the segmented muscles undergo any smoothing when generating the 3D meshes? This may affect the volumes calculated and should be reported if used.

2.	Lugol’s concentration reporting issue. I believe the concentration of Lugol’s solution has been misreported and is slightly higher than stated (this is minor). The authors cite a paper (Burrows, A. M., Omstead, K. M., Deutsch, A. R., Gladman, J. T., & Hartstone-Rose, A. (2019). Reverse Dissection and DiceCT Reveal Otherwise Hidden Data in the Evolution of the Primate Face. Journal of Visualized Experiments, (143). doi:10.3791/58394) for the reverse dissection protocol, which I followed up as the stain concentration was not one I had come across before. The stain concentration reported in Burrows et al. 2019 may be incorrect (1.75g iodine plus 3.5g potassium iodide in 200mL water is 2.625% w/v I2KI, not 1.75% as reported). If this is the method used to mix the Lugol’s iodine for this study, please check this and report the correct concentration in the methods here.


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

Input:
Title: VISUALIZATION AND QUANTIFICATION OF MIMETIC MUSCULATURE VIA DICECT
Review round: 2
Reviewer: 1

Output:
Basic reporting: The language used throughout is good. Sufficient background has been provided and the study is self-contained with results relevant to hypotheses. I'm very pleased to see the new figure of a diceCT slice - I think it makes it very clear how difficult the reconstruction would have been in some cases. The new table is excellent - a very useful summary of the anatomy.

I think there is still a problem regarding the data sharing - I followed the link to Morphosource and I can find the author's project, but the diceCT stack doesn't seem to be there. It says 'This specimen has no media'.
Experimental design: I am satisfied that the experiment is well-designed and described in sufficient detail.
Validity of the findings: The results are well-described, particularly now that Table 2 has been included. The conclusions are justified.
Additional comments: I am happy that the authors have addressed all my comments and, as far as I can see, the comments of the other reviewers.


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: VISUALIZATION AND QUANTIFICATION OF MIMETIC MUSCULATURE VIA DICECT
Review round: 2
Reviewer: 2

Output:
Basic reporting: Thank you for providing the complete image stack data on Morphosource.
The addition of the colour coded 2D slice in Figure 1 is very helpful.
To assist the reader in interpreting this figure, please add some orientation labels to the figure itself or to the caption (e.g. superior at top, superficial at right etc.), and please add in the caption which plane the section is in (coronal, sagittal etc.).
Experimental design: The methodological clarifications and inclusion of some more cautious language have addressed all the concerns I had.
Validity of the findings: No comment
Additional comments: The additional work the authors have put into contextualising their observations and measurements, drawing together data from the literature in a systematic way, has really paid off. I believe this paper will be a valuable (and very citable) resource for future workers seeking to understand the configuration of the muscles of the mammalian face.


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: HIDING IN PLAIN SIGHT: DNA BARCODING SUGGESTS CRYPTIC SPECIES IN ALL ‘WELL-KNOWN’ AUSTRALIAN FLOWER BEETLES (SCARABAEIDAE: CETONIINAE)
Review round: 1
Reviewer: 1

Output:
Basic reporting: The background information is mostly clear although could be improved by directly stating the number of diversity of Australian genera in the first paragraph of the introduction, or quantified  following statement on 65% of genera on line 56.

The second paragraph is largely a non-sequitur and while the biology is important, this paragraph seems to largely be irrelevant as there are no-discussions or conclusions relating to biology. If the authors decide to include this information, it would be better suited towards the end of the Introduction, and prehaps relating to the utility of generating this barcode library.

Goals are stated clearly but there is room for a hypothesis that barcodes are likely to identify numerous undescribed species. This could be supported by the limited taxonomic attention in the last 65 years,  high levels of endemism, cryptic species complexes already identified and estimates of undescribed proportion of Australian fauna.

English is clear. Citations are sufficient to provide context.
Experimental design: This study provides the first insights in to the hidden diversity of Australian cetoniines and fills an important gap in our knowledge of scarabs as well as the Australian fauna.

The design and investigation is rigorous and methods are described in detail.  The authors should be commended that this first investigation provides barcodes for almost 50% of species and all common genera. It provides a clear framework for further investigation and the utility of barcodes for identification.
Validity of the findings: I'd like to see a short discussion of the utility of this barcode library beyond taxonomy, e.g. the potential agricultural importance mentioned in the introduction, or ability to match life-cycles.

I was intrigued when I read in the introduction that both larvae and adults were sampled and that one of the goals of this paper were to facilitate life-cycle matching but these larvae didn't get mentioned in the discussion or conclusion. Were they from easily identifiable species or were they matched using these barcodes?

The taxonomic interpretations were robust. I think its great that when possible, multiple samples were included per species. It would be useful to include locality information either on the trees or in a table so readers don't need to refer to BOLD to decipher locality  information from this data set.

Overall, this is an important contribution to the field.
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: HIDING IN PLAIN SIGHT: DNA BARCODING SUGGESTS CRYPTIC SPECIES IN ALL ‘WELL-KNOWN’ AUSTRALIAN FLOWER BEETLES (SCARABAEIDAE: CETONIINAE)
Review round: 1
Reviewer: 2

Output:
Basic reporting: In this study Mitchell and co-authors Liu et al. analyzed about 280 specimens of Australian cetoniine flower beetles using the DNA barcode fragment. During the last years, DNA barcoding has become an important and popular tool in modern biodiversity studies as well as taxonomic research. Based on the given data and results, I think that the analysis and results are interesting and likely helpful for other researchers in this research field. In my eyes, a clear, unambiguous and professional English language has been used. The article structure and figures look professional. The literature provided is also context-related, but some important references are missing (see sticky notes in the uploaded pdf). The figures and associated legends are understandable without going back to the main text.

I feel, however, that a discussion of possible pitfalls of using mtDNA in terms of species delineation and specimen identification, e.g., incomplete lineage sorting, the presence of Wolbachia, NUMTS etc. as part of the introduction or discussion will improve the quality of the manuscript. Such effects can cause unexpected mitochondrial diversity as well.

Furthermore, it is obvious that DNA barcoding will play an essential role in modern molecular biodiversity research, e.g., the effective use of high-throughput sequencing technologies (meta-barcoding) highly relies on detailed barcode libraries. These aspects should be also discussed.
Experimental design: The research question is well defined, relevant and meaningful. The authors state how their research fills a serious knowledge gap in the taxonomy of Australian flower beetles. Methods are described in sufficient detail to replicate. However, the authors may consider using additional species delimitation approaches (e.g., GMYC, bPP). Do you will get similar results in comparison to the BIN/RESL approach? This would be interesting to know. For some species cluster, e.g., Chondropyga dorsalis, you may also think about using statistical parsimony networks as implemented in PopART (http://popart.otago.ac.nz/index.shtml) to visualize observed molecular patterns.
Validity of the findings: I wonder if the results would be different if more range wide samples of different species were included. In some cases the samples sizes were quite small (i.e. one individual). However, the conclusions are well stated, linked to original research question and limited to supporting results. Overall, data and analyses are robust.
Additional comments: Please check the uploaded pdf for more specific comments.


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

Input:
Title: HIDING IN PLAIN SIGHT: DNA BARCODING SUGGESTS CRYPTIC SPECIES IN ALL ‘WELL-KNOWN’ AUSTRALIAN FLOWER BEETLES (SCARABAEIDAE: CETONIINAE)
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript is generally well written and conforms to the PeerJ standards on structure. There are only a handful of relatively small issues which should be easy to address. The introduction provides a good overview of Australian Cetoniinae and previous taxonomic work on the group. Sequences are made available through both BOLD and GenBank, and detailed specimen metadata and images are provided through BOLD. The figures are of good quality. In particular, the illustrated and color-coded supplementary trees are visually appealing, and I wonder if at least one of them (or parts of one) could be transformed into a proper figure in the manuscript instead of being “hidden” in the supplements. Some parts of the manuscript should be moved elsewhere to improve the flow of the text and avoid confusing the reader - see detailed comments below under General comments.
Experimental design: The need and motivation for the present study and its goals are clearly stated. The methods are sufficient to achieve the stated goal, i.e. providing an initial DNA barcode library for the focal taxon to serve as a starting point for more thorough taxonomic studies. Some basic details of PCR and sequencing protocols (primers, sequencing platform, etc) should be added in Materials and Methods instead of simply referring to the previous publication. Rooting of the trees is not justified in any way in Material and Methods, but the justification is briefly mentioned in Discussion (lines 297-299). This note should be moved to the last section of M&M where the tree inference methods are described. Apart from these minor issues, the methods are well reported, and I see no ethical or technical problems.
Validity of the findings: All primary data are made available through both BOLD and GenBank. Data and image quality on BOLD are mostly exemplary, although collection data beyond the country are not reported for many specimens. I assume that this is due to deficient label data on the sampled specimens rather than an omission on the authors’ part. Conclusions are not overarching and are supported by the results. A few more specific comments are listed below under General comments.
Additional comments: Line 71: 4000 is a gross underestimate. A quick search on Web of Science with the term “DNA barcod*” as topic resulted in 7262 articles and 1000 or so hits for other document types. The 2003 paper by Hebert et al. (https://doi.org/10.1098/rspb.2002.2218) has been cited more than 10000 times according to Google Scholar.
Line 76: “…aid taxonomic research in many families” - Consider citing a few studies as examples
Line 185-187: A public project on BOLD works fine for data sharing in this case, but in future work, I suggest using the dataset feature on BOLD instead. Specimen records from multiple projects can be combined into one dataset, and the same records can be added into multiple datasets, without any need to move records between projects. A dataset can also be assigned a DOI. I believe the dataset feature will be useful in your future work on Australian Cetoniinae when more sequence data are added to many of the taxa covered here.
Line 214: As mentioned above, please move the note on lines 297-299 here to justify your approach to rooting the trees.
Line 218: How were the larvae identified – by associating them with adults based on barcodes or morphologically? Were some taxa represented by larvae only? Consider adding a note on sampling larvae as well as adults in Material & Methods. If larvae were sampled in order to test identification of larvae based on barcodes, add text appropriately in M&M, Results, and Discussion.
Line 239-246: Mainly a matter of personal preference, but I think the information in this paragraph would be better presented in a table including both monophyletic and non-monophyletic genera.
Line 251-252: Representatives of two genera appearing in the same BIN is highly unusual and is almost always due to operational errors. Is this possibly a case of poorly resolved genus-level taxonomy? Can you exclude the possibility of cross-contamination or specimen mix-up?
Line 291-293: This seems to belong to the Results section rather than here, especially since there is no mention of these results in Results in the current version of the manuscript.
Line 306-313: Move this paragraph up before the previous one to make the text more coherent
Line 314-320: Join to a single paragraph
Line 339: probabilities -> probability
Fig. S1 legend: Baysian -> Bayesian


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 RETROSPECTIVE STUDY ON THE PROGNOSTIC VALUE OF PREOPERATIVE C-REACTIVE PROTEIN TO ALBUMIN RATIO IN PATIENTS WITH ORAL CAVITY SQUAMOUS CELL CARCINOMA
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comment.
Experimental design: Please have a look at Major concerns: 1 to 2.
Validity of the findings: No comment.
Additional comments: Oral cancers are the sixth most frequent cancer with a high mortality rate. Oral squamous cell carcinoma (OSCC) accounts for more than 90% of all oral cancers. Increasing incidence of OSCC of the oral cavity and oropharynx is reported in young adults who habit of alcohol, cigarette and betel. People’s cigarette smoking, betel quid chewing, and alcohol consumption not only cause field cancerization but are also correlated significantly with systemic inflammation. C-reactive protein (CRP) is a commonly used marker of systemic inflammation, routinely measured in serum blood samples and easy to detect. It is a good idea to investigate the correlation of CRP with OSCC prognosis, and it might provide new ideas for the diagnosis and treatment of OSCC. In this manuscript, Dr. Fang and colleagues looked at the role of the C-reactive protein-to-albumin ratio (CAR) in diagnosis and prognosis of OSCC. The study provides the conclusion, which showed that preoperative CAR could constitute an independent prognostic indicator for OS and DFS prediction in OSCC patients treated with curative surgery. Although, the current study is interesting and well but there are some major concerns that need to be addressed.

Major concerns:
1. Although, there were some reports [1-3] have pointed out the prognostic value of C-reactive protein/Albumin Ratio (CAR) in oral squamous cell carcinoma (OSCC), this study used more complete and larger sample size clinical data. However, the factors affecting the survival of patients are complex, and the patient's physical state and treatment strategy are also different in different clinical stages. Therefore, I suggest that the authors can further analyze the diagnostic efficiency separately in early (might Stage I-II) and late stage (might Stage III-IV) of OSCC. In this study, there were 135 patients in the early stage (Stage I-II) and 191 patients in the late stage (Stage III-IV). I believe there should be enough data for relevant analysis, and it will help to increase the application value of this research.
[1] Wang Q, Song X, Zhao Y, et al. Preoperative high c-reactive protein/albumin ratio is a poor prognostic factor of oral squamous cell carcinoma. Future Oncol. 2019;15(19):2277–2286. doi:10.2217/fon-2019-0063
[2] Park HC, Kim MY, Kim CH. C-reactive protein/albumin ratio as prognostic score in oral squamous cell carcinoma. J Korean Assoc Oral Maxillofac Surg. 2016;42(5):243–250. doi:10.5125/jkaoms.2016.42.5.243
[3] Hasegawa T, Saito I, Takeda D, et al. Risk factors associated with postoperative delirium after surgery for oral cancer. J Craniomaxillofac Surg. 2015;43(7):1094–1098. doi:10.1016/j.jcms.2015.06.011
2. C-reactive protein (CRP) is a commonly used marker of systemic inflammation, and there are some studies have showing that CRP level can be used as a prognostic and diagnostic indicator for esophageal squamous cell carcinoma and tongue squamous cell carcinoma [4-5]. Thus, I suggest that the authors should compare the efficacy of CRP level and CAR in predicting the prognosis of OSCC. I believe that it will enhance the significance of this study.
[4] Katsurahara K, Shiozaki A, Fujiwara H, et al. Relationship Between Postoperative CRP and Prognosis in Thoracic Esophageal Squamous Cell Carcinoma. Anticancer Res. 2018;38(11):6513–6518. doi:10.21873/anticanres.13016
[5] Du J, Hu W, Yang C, Wang Y, Wang X, Yang P. C-reactive protein is associated with the development of tongue squamous cell carcinoma. Acta Biochim Biophys Sin (Shanghai). 2018;50(3):238–245. doi:10.1093/abbs/gmy004

Minor comments:
1. “RESULTS – Baseline characteristics” Line 171-172. “Nearly half of the patients were diagnosed as having stage IV disease…” Please add the number and percentage of patients with stage IV in manuscript as same as other characteristics.
2. Figure 1-3. The text in the picture is too small. Please increase the font size.
3. “RESULTS – Association of CAR with survival outcomes” Line 196-215, Figure 3A-B. Please present the median survival time, hazard ratio (HR) and 95% CI of ratio at survival curve.


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 RETROSPECTIVE STUDY ON THE PROGNOSTIC VALUE OF PREOPERATIVE C-REACTIVE PROTEIN TO ALBUMIN RATIO IN PATIENTS WITH ORAL CAVITY SQUAMOUS CELL CARCINOMA
Review round: 1
Reviewer: 2

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: The paper is relevant for the current literature, minor revisions are needed. It is a really good study. The english is correctly written, clear and unambiguous.

Introduction:
It is rigth

Material and methods:
- Study patients
You should add the literatura about the guidlines followed by your department to the complementary treatment after radical surgery.
Do you request any radiological explorations for the follow up?
- Inflammation – based prognostic scores
It is rigth
- Statistical analysis
It is rigth

Results:
- Baseline characteristics
It is rigth
- Inflammation -  based prognostic score cutoff and ROC curves
It is rigth
- Association of CAR with clinicopathological charasteristcs
It is rigth
- Association of CAR with survival outcome
It is rigth
- Nomogram models
It is rigth

Discussion:

In the third paragraph  you should cited a current study which AUCs based on inflammation are inferior to CAR in OSCC.
For Example:
Jariod-Ferrer ÚM, Arbones-Mainar JM, Gavin-Clavero MA, Simón-Sanz MV, Moral-Saez I, Cisneros-Gimeno AI, Martinez-Trufero J. Are Comorbidities Associated With Overall Survival in Patients With Oral Squamous Cell Carcinoma? J Oral Maxillofac Surg. 2019 Sep;77(9):1906-1914.

Conclusions:
It is rigth


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 RETROSPECTIVE STUDY ON THE PROGNOSTIC VALUE OF PREOPERATIVE C-REACTIVE PROTEIN TO ALBUMIN RATIO IN PATIENTS WITH ORAL CAVITY SQUAMOUS CELL CARCINOMA
Review round: 1
Reviewer: 3

Output:
Basic reporting: the english language is clear.
the author should revise the use of comma. ( before AND comma is not necessary)
Experimental design: The reasearch is in line with the aims of the journal
the identification  of patients afftected by OSCC that need to more aggressive adjuvant strategy is an open issue.
a retrospective study has a lot of bias
It's not clear if the patients gave the IC.
Validity of the findings: In the multivariate analysis  the 5yrs OS is atatically associted with  pT, pN , G,rading, CAR and NLR.
 it's not clear the reason why tha author used in the nomogram characteristics that weren't associated ( at MVA)  with OS such as sex, age and stage.
the patients were enrolled from 2008 to 2017, it's not clear why the authors considered 5years OS.
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 RETROSPECTIVE STUDY ON THE PROGNOSTIC VALUE OF PREOPERATIVE C-REACTIVE PROTEIN TO ALBUMIN RATIO IN PATIENTS WITH ORAL CAVITY SQUAMOUS CELL CARCINOMA
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: Oral cancers are the sixth most frequent cancer with a high mortality rate. Oral squamous cell carcinoma (OSCC) accounts for more than 90% of all oral cancers. Increasing incidence of OSCC of the oral cavity and oropharynx is reported in young adults who habit of alcohol, cigarette and betel. People’s cigarette smoking, betel quid chewing, and alcohol consumption not only cause field cancerization but are also correlated significantly with systemic inflammation. C-reactive protein (CRP) is a commonly used marker of systemic inflammation, routinely measured in serum blood samples and easy to detect. It is a good idea to investigate the correlation of CRP with OSCC prognosis, and it might provide new ideas for the diagnosis and treatment of OSCC. In this manuscript, Dr. Fang and colleagues looked at the role of the C-reactive protein-to-albumin ratio (CAR) in diagnosis and prognosis of OSCC. The study provides the conclusion, which showed that preoperative CAR could constitute an independent prognostic indicator for OS and DFS prediction in OSCC patients treated with curative surgery. The paper is significantly improved and all concerned raised by the reviewer have been addressed. I think it is suitable for publication at this point for this version of revised manuscript.


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

Input:
Title: A RETROSPECTIVE STUDY ON THE PROGNOSTIC VALUE OF PREOPERATIVE C-REACTIVE PROTEIN TO ALBUMIN RATIO IN PATIENTS WITH ORAL CAVITY SQUAMOUS CELL CARCINOMA
Review round: 2
Reviewer: 2

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: The minnor reviews have been improved. The structure and the design are adecuate. The conclusions are clear and the results are important for the literature.


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: DIVERSITY AND SPOILAGE POTENTIAL OF MICROBIAL COMMUNITIES ASSOCIATED WITH GRAPE SOUR ROT IN EASTERN COASTAL AREAS OF CHINA
Review round: 1
Reviewer: 1

Output:
Basic reporting: 1 BASIC REPORTING
Clear, unambiguous, professional English language used throughout.
The English language used throughout the manuscript is clear and unambiguous. Some words used in the title and throughout the manuscript are not technically correct. For example, “pathogenicity” and “pathogenic” should be replaced by “spoilage potential” and “spoilage”, respectively. Moreover, the terms “morbidity” and “disease index” should be replaced by “percentage of incidence” and “McKinney index”. Finally, the terms “cultivable” and “cultivated bacteria and fungi” could be replaced by “culturable” and “culturable microbial community”.
Intro & background to show context. Literature well referenced & relevant.
The introduction needs revision. The section at lines 86-92 could be shortened. I suggest to remove lines 93-101 (in my opinion the literature cited is not relevant for this work) and to replace this part with metagenomics studies or studies using culture-independent approaches related to grape rots (in particular sour rot) and wine and table grape microbiota.
Structure conforms to PeerJ Standards, discipline norm, or improved for clarity.
The structure conforms to PeerJ Standards.
Figures are relevant, high quality, well labelled & described.
Figures are relevant, of good quality, well labelled and described as shown by figure captions and in the main text.
Raw data supplied.
Raw data are supplied in the supplementary files.
Experimental design: 2 EXPERIMENTAL DESIGN
Original primary research within Scope of the journal.
Research is in accordance with Scope of the journal.
Research question well defined, relevant & meaningful. It is stated how the research fills and identified knowledge gap.
Research objectives are well defined, relevant, and meaningful:
The objective of this study was to determine: (1) the diversity and abundance of bacteria and fungi in sour rot-infected table grapes collected from Yantai city based on traditional culture methods; (2) the pathogenicity of bacteria and fungi associated with grape sour rot; and (3) the diversity and abundance of bacteria and fungi in sour rot-infected table grapes based on 16S rRNA and ITS high-throughput sequencing analysis.
In my opinion the workflow of the work could be revised. In particular, the spoilage potential of bacteria and fungi associated with grape sour rot could be presented as the third step of the work, while the high-throughput sequencing analysis could follow the characterization of culturable microorganisms. This order should be followed throughout the manuscript.
As regards statement related to the fill of research gap, this part is reported at lines 81-85. I suggest to reinforce this part and to report the statement just before the objectives at the end of Introduction section.
Rigorous investigation performed to a high technical & ethical standard.
Research activities are performed following high technical and ethical standard.
Methods described with sufficient detail & information to replicate.
The methods are described with sufficient information regarding procedures and materials used.
Validity of the findings: 3 VALIDITY OF THE FINDINGS
Impact and novelty not assessed. Negative/inconclusive results accepted. Meaningful replication encouraged where rationale & benefit to literature is clearly stated.
Decisions are made based on impact and novelty of interest to a wide audience. Nowadays, the microbiota of rotten grape berries is limited characterized, so studies related to this field are appreciated and encouraged by the scientific community.
As regards replication, the authors analysed the data from three technical replicates for different analyses. In this kind of study biological replicates should be considered. Grapes of the same variety from different vineyards or vines, and collected in different vintages should be analysed. However, I understand that this kind of sampling is difficult and time-consuming. I think that the goal of this work is the microbial characterization of the table grape sour rot microbiota and mycobiota using two different approaches and not the study of these microorganisms from different cultivation sites and vintages.
All underlying data have been provided; they are robust, statistically sound & controlled.
Data have been provided and they are relevant, statistically analysed and checked.
Conclusions are well stated, linked to original research question & limited to supporting results.
Conclusions are well stated and linked to original research questions. However, I suggest to introduce other findings such the isolation from rotten berries of bacteria such as Cronobacter sakazakii, an emergent pathogen, and Klebsiella pneumoniae, a source and shuttle for antibiotic resistance.
The conclusions as well as the entire manuscript should report only supported results. Sour rot symptoms are well defined: skin browning, ethyl acetate smell, disaggregation of the internal tissues, berry emptying. Sour rot symptoms can be reproduced at lab level using Drosophila flies (doi.org/10.1007/s00248-012-0041-y), or by selected NSY-AAB associations (doi.org/10.1016/j.fm.2017.05.001) that are naturally carried out on grapes by the insects.
The symptoms in figure 3 are not related to sour rot but are examples of spoilage caused by sour rot bacteria and fungi on berries (cracking, colonization of grape tissue by bacteria, or mycelium development on the grape berry surface). Acetobacter sp. did not develop sour rot symptoms on berries probably because the sour development needs the presence of Drosophila flies to prevent wound healing or the co-occurrence of selected yeasts. The etiology of sour rot is still determined by acetic acid bacteria and non-Saccharomyces yeasts, or by Aspergillus spp. that the authors detected by high-throughput sequencing.
However, the authors demonstrated that different bacteria and fungi isolated from sour rotten berries could be involved in grape spoilage. It is an important finding that could be reported in the conclusions.
I suggest to revise the Conclusions section.
Speculation is welcome, but should be identified as such.
See reviewer comments reported above.
Additional comments: 4 GENERAL COMMENTS
The work entitled “Diversity and pathogenicity of microbial communities causing grape sour rot in eastern coastal areas of China” reports the genetic and physiological characterization of culturable fungi and bacteria from sour rotten berries, the description of microbial communities of rotten berries by high-throughput sequencing, and the spoilage potential of isolated fungi and bacteria on grape berries. The study is relevant for a broad scientific community since it describe for the first time the isolation of certain bacteria and fungi from grape sour rot. In addition, the spoilage potential of the isolated microorganisms is presented. However, the weakness of the work is the casual relationship between the isolation of certain microorganisms and the development of the disease. Moreover, a revision of the introduction and discussion section is recommended. English language is of sufficient quality but a revision of technical words is suggested.

Specific comments
Title: Diversity and pathogenicity of microbial communities causing grape sour rot in eastern coastal areas of China
The title did not reflect the results. Microbial communities characterized from sour rotten berries could not be involved in the development of sour rot. I suggest to change the title as follow:
Diversity and spoilage potential of microbial communities of grape sour rot in eastern coastal areas of China
Abstract
L28 “Planting location” and “identified methods” should be replaced, here and throughout the manuscript, by cultivation site and microbial identification methods.
Please, as suggested in the basic reporting, not use the terms pathogens, pathogenicity, pathogenic. It is better spoilage microorganisms, spoilage potential.
L34-L38 Here and throughout the manuscript please replace culture-methods with culture dependent methods.
L48 Incertae sedis sp. is an unknown or undefined taxonomic classification. Thus, it could be removed from the list of fungi and yeasts identified in rotten berries.
Introduction
Introduction section should be revised as requested in the basic reporting part.
L68, L342, L444 Please correct the surname Guerzpni. The correct surname is Guerzoni.
L95 18S rRNA? I did not find 18S rRNA sequencing in Materials and Methods. Are you sure?
Materials and Methods
L113-119 Which grape variety was used?
Which is the total weight of grape tissue collected? 1g or 100 g from 100 rotten grapes? How did you perform the collection?
L121 The samples … Which is the weight of each replicate? Which is the dilution ratio?
L123-124 Did the authors use antibiotics in the medium to prevent the cross-contamination (bacteria on PDA and fungi on nutrient agar)?
L126 ….using the primary medium
L133 …DNA from single colony of each bacterium..
L137 Is the term “quantification” correct?
L153-156 The authors cloned the PCR products. Did the authors find multiple bands on gel? Was this approach used to be sure to work with a single copy of target gene?
L169 …106 conidia/mL or cfu/mL in the case of bacteria.
L202 Remove “that were” and add “and”.
Results
L249-250 It’s better “Sequences of these 15 bacterial species and 10 fungal species showed a 97-100% of similarity with those deposited in NCBI”.
L258 Please replace diseased with spoiled
L260-272 Please use the terms percentage of incidence and McKinney index as suggested in Basic Reporting.
L288-289 I see the Table 3. The microbial diversity (Shannon index and Simpson index) is higher for bacteria than fungi. The richness (Chao1 index and ACE index) is higher for fungi than bacteria. Is it correct? Please verify.
.L298-301 I suggest to describe the dominant species and then that 32.40% of the phyla were not taxonomically classified (Incertae sedis sp.).
L302-313 Which is the prediction accuracy based on yours OTU number/sample of PICRUSt predictions? Which is the accuracy for each functional category in COG and KEGG classification?
Discussion
The Discussion section needs integration. In particular three parts should be added:
1) metagenomic studies on sour rot bacterial and fungal biota.
2) description of new species isolated from rotten berries. For example Cronobacter species, Klebsiella pneumoniae, and Saprochaete gigas. Cronobacter species are emergent pathogens; K. pneumoniae is a multidrug resistant pathogen; S. gigas is of the same genera of  Saprochaete capitata, an emerging fungal pathogen with low susceptibility to echinocandins. Therefore, sour rotten berries could be a reservouir of human pathogens and antibiotic resistance potentially transferable to other bacteria.
3) a discussion on functional classification based on COG and KEGG analysis. In particular, the transport of metabolites, coenzymes, the membrane transport, carbohydrate and amino acid metabolisms are the principal functions. Indeed, these functions are typical of AAB, the main bacterial group found by high-throughput sequencing. The authors could briefly discuss the AAB metabolisms in sour rotten berries (oxidative fermentation, production of acetic acid, amino acid metabolism and so 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: DIVERSITY AND SPOILAGE POTENTIAL OF MICROBIAL COMMUNITIES ASSOCIATED WITH GRAPE SOUR ROT IN EASTERN COASTAL AREAS OF CHINA
Review round: 1
Reviewer: 2

Output:
Basic reporting: This manuscript reports on the characterisation of the microbiota related to the polymicrobial disease grape sour rot.
To identify the causative agents of the disease the authors present a set of microbiological tool and genetic tools. They have studied the non-cultivable microbiome applying next generation sequencing through Illumina MiSeq. In addition they have isolated the bacterial and fungal cultivable microbiome and identified the causative agents by two independent methods, characterising the bacterial phenotype and the taxonomical features of the fungi, and ultimately bacteria and fungi isolated were also genetically characterised by sequencing of plasmids by Sanger sequencing of 16S rRNA gene and internal transcribed spacer (ITS) respectively.
Experimental design: The relevance to disease is well known therefore the research poses interest for the scientific community, however there is a number of issues that make me recommend the rejection of this paper.

1.	It is too risky to identify to the level of species bacteria or fungi through metagenomics. Even Sanger sequencing is not an accurate method when just based in the sequencing of one gene. For fungi for instance the authors should consider to include additional genomic markers such us RNA polymerase subunit II (RPB2) (Thongklang et al., 2014) or the partial large sub- unit (LSU) of ribosomal DNA (Geml et al., 2004), ITS is a highly variable region.
2.	The number of causative agents presented is significantly high and not effectively discussed to consider that pathogenicity tests are efficient to consider all the parasites reported as causative agent of grape sour rot or, in the other hand, some other disease. For instance, the authors cite Fusarium proliferatum as parasite, however, they never discuss that this same species has been reported as an effective biocontrol agent to fight grape downy mildew, Plasmopara viticola (Falk et al., 1996; Baksi et al., 2001), which is remarkable to me. The trial desing of pathogenic tests as reflected in Figure 3 can induce cross contamination due to the proximity of the fruits.
3.	The methodology employed for the construction of the libraries sequenced in MiSeq is non decribed. How many replicates do they sequence? What kind of sample are they sequencing, how did they extract DNA, PCR amplification…? Which is the final concentration of the pool? What kind of control are they introducing in the pool? What is the kit used? What kind of indexing did they performed? How did they check the quality of the library before running?All these technical issues required to reproduced the trial are missing.
4.	Results of NGS sequencing. Non data concerning quality of the sequencing is presented. Quality achieved is missing, number of reads per sample is missing. The mean size of the 16S rRNA (412 bp, is too low, should be around 550 bp before indexing, 620 bp afterwards) fragments and ITS (450 bp, is too low, should be around 450 bp before indexing, 560 bp afterwards) are not accurate to me.
5.	Discussion is poor and difficult to follow. While presenting a good amount of work, they just attached 33 references, what makes me think that a poor approach to the state-of-the-art was performed before and after the trials. For instance I found a lot of literature related to grape sour rot and however the authors sometimes reflect other crops, such as in lines 352-356 when they talk about parasites related to fruits, corn, rice or lily, this is out of scope and not helpful for their work.
6.	Figures and tables. There are so many figures and table, sometimes less is more, I have to ask the authors if all these 9 figures they present are mandatory to expose their results. I think they need to think about this issue. Maybe supplementary material is a good place to fit some of them.
7.	References. As cited above, literature cited is poor, besides 12 out of 33 references are very old, over 10 years, I think we need to make an effort to update the science continuously, and try to be focused within the last ten years (no more than 10% of cites older than that).
Validity of the findings: Due to the number of issues cited and some others I recommend the editor to reject this paper and encourage the authors to fully refurbish their work and try to submit it to the suitable journal.
Additional comments: This manuscript reports on the characterisation of the microbiota related to the polymicrobial disease grape sour rot.
To identify the causative agents of the disease the authors present a set of microbiological tool and genetic tools. They have studied the non-cultivable microbiome applying next generation sequencing through Illumina MiSeq. In addition they have isolated the bacterial and fungal cultivable microbiome and identified the causative agents by two independent methods, characterising the bacterial phenotype and the taxonomical features of the fungi, and ultimately bacteria and fungi isolated were also genetically characterised by sequencing of plasmids by Sanger sequencing of 16S rRNA gene and internal transcribed spacer (ITS) respectively.
The relevance to disease is well known therefore the research poses interest for the scientific community, however there is a number of issues that make me recommend the rejection of this paper.

1.	It is too risky to identify to the level of species bacteria or fungi through metagenomics. Even Sanger sequencing is not an accurate method when just based in the sequencing of one gene. For fungi for instance the authors should consider to include additional genomic markers such us RNA polymerase subunit II (RPB2) (Thongklang et al., 2014) or the partial large sub- unit (LSU) of ribosomal DNA (Geml et al., 2004), ITS is a highly variable region.
2.	The number of causative agents presented is significantly high and not effectively discussed to consider that pathogenicity tests are efficient to consider all the parasites reported as causative agent of grape sour rot or, in the other hand, some other disease. For instance, the authors cite Fusarium proliferatum as parasite, however, they never discuss that this same species has been reported as an effective biocontrol agent to fight grape downy mildew, Plasmopara viticola (Falk et al., 1996; Baksi et al., 2001), which is remarkable to me. The trial desing of pathogenic tests as reflected in Figure 3 can induce cross contamination due to the proximity of the fruits.
3.	The methodology employed for the construction of the libraries sequenced in MiSeq is non decribed. How many replicates do they sequence? What kind of sample are they sequencing, how did they extract DNA, PCR amplification…? Which is the final concentration of the pool? What kind of control are they introducing in the pool? What is the kit used? What kind of indexing did they performed? How did they check the quality of the library before running?All these technical issues required to reproduced the trial are missing.
4.	Results of NGS sequencing. Non data concerning quality of the sequencing is presented. Quality achieved is missing, number of reads per sample is missing. The mean size of the 16S rRNA (412 bp, is too low, should be around 550 bp before indexing, 620 bp afterwards) fragments and ITS (450 bp, is too low, should be around 450 bp before indexing, 560 bp afterwards) are not accurate to me.
5.	Discussion is poor and difficult to follow. While presenting a good amount of work, they just attached 33 references, what makes me think that a poor approach to the state-of-the-art was performed before and after the trials. For instance I found a lot of literature related to grape sour rot and however the authors sometimes reflect other crops, such as in lines 352-356 when they talk about parasites related to fruits, corn, rice or lily, this is out of scope and not helpful for their work.
6.	Figures and tables. There are so many figures and table, sometimes less is more, I have to ask the authors if all these 9 figures they present are mandatory to expose their results. I think they need to think about this issue. Maybe supplementary material is a good place to fit some of them.
7.	References. As cited above, literature cited is poor, besides 12 out of 33 references are very old, over 10 years, I think we need to make an effort to update the science continuously, and try to be focused within the last ten years (no more than 10% of cites older than that).

Due to the number of issues cited and some others I recommend the editor to reject this paper and encourage the authors to fully refurbish their work and try to submit it to the suitable journal.


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

Input:
Title: DIVERSITY AND SPOILAGE POTENTIAL OF MICROBIAL COMMUNITIES ASSOCIATED WITH GRAPE SOUR ROT IN EASTERN COASTAL AREAS OF CHINA
Review round: 1
Reviewer: 3

Output:
Basic reporting: Research into sour rot-associated microorganisms is very relevant, but this article is not updated with the current literature. Four papers in the past year have been published specifically into sour rot causal organisms and associated microorganisms, none of which are mentioned in this paper, and which would significantly impact the interpretation of the results.
Experimental design: The assessment of sour rot infection on inoculated berries is questionable, because the authors seem only be assessing the berries based on area of the berry that is infected. This is not an appropriate assessment for sour rot because it does not take into account the loss of berry integrity that is a pivotal sign of the disease. Then, to assess causal organisms based on an incomplete rating system does not actually determine causality, but instead, association. The authors of determined associated microorganisms, not casual microorganisms. Because it is mentioned that the culture methods are less successful at identifying microorganisms than high-throughput sequencing methods, why is it necessary to include the culture methods?
Validity of the findings: The validity of the findings is questionable because the authors describe these organisms as causal, not as associated. There is little evidence that they are causal organisms of this disease when the method of assessing disease symptoms is incomplete. It would be beneficial to rephrase the purpose of the article as looking at associated microorganisms instead of causal microorganisms.
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: DIVERSITY AND SPOILAGE POTENTIAL OF MICROBIAL COMMUNITIES ASSOCIATED WITH GRAPE SOUR ROT IN EASTERN COASTAL AREAS OF CHINA
Review round: 2
Reviewer: 1

Output:
Basic reporting: 1 BASIC REPORTING
Clear, unambiguous, professional English language used throughout.
The English language used throughout the manuscript is clear and unambiguous.
Intro & background to show context. Literature well referenced & relevant.
The introduction section has been improved. In the references Pinto et al. (2019) is lacking. Please verify
Structure conforms to PeerJ Standards, discipline norm, or improved for clarity.
The structure conforms to PeerJ Standards.
Figures are relevant, high quality, well labelled & described.
Figures are relevant, of good quality, well labelled and described as shown by figure captions and in the main text.
Raw data supplied.
Raw data are supplied in the supplementary files.
Experimental design: Original primary research within Scope of the journal.
Research is in accordance with Scope of the journal.
Research question well defined, relevant & meaningful. It is stated how the research fills and identified knowledge gap.
Research objectives are well defined, relevant, and meaningful:
The workflow has been revised.
Rigorous investigation performed to a high technical & ethical standard.
Research activities are performed following high technical and ethical standard.
Methods described with sufficient detail & information to replicate.
The methods are described with sufficient information regarding procedures and materials used. However, I suggest to add more information on functional analysis.
Validity of the findings: Impact and novelty not assessed. Negative/inconclusive results accepted. Meaningful replication encouraged where rationale & benefit to literature is clearly stated.
All underlying data have been provided; they are robust, statistically sound & controlled.
Data have been provided and they are relevant, statistically analysed and checked.
Conclusions are well stated, linked to original research question & limited to supporting results.
Conclusions have been revised.
Speculation is welcome, but should be identified as such.
No comment
Additional comments: The manuscript has been improved as suggested by the reviewer. It is acceptable for publication in “Peer J” after minor revisions.
Specific comments
1
Please, throughout the manuscript, check spaces, italic typesetting, capital and lowercase letters, removed equations, font style and so on: L73-74, L84, L83-85, L98, L111, L144-L145, L208, L230-1, L234, L248-9, L255-60, L288, L292, L299, L311-12, L330, L353, L360, L396-99, L408-9, Table 3, Table 4. Please add Pinto et al. (2019) to References.
2
The symptoms in figure 3 are not related to sour rot but are examples of spoilage caused by sour rot bacteria and fungi on berries (cracking, colonization of grape tissue by bacteria, or mycelium development on the grape berry surface). Acetobacter sp. did not develop sour rot symptoms on berries probably because the sour development needs the presence of Drosophila flies to prevent wound healing or the co-occurrence of selected yeasts. The etiology of sour rot is still determined by acetic acid bacteria and non-Saccharomyces yeasts, or by Aspergillus spp. that the authors detected by high-throughput sequencing.
Respond: Thanks for your comments, in this experiment, we simulated cracked fruit and feeding of fruit flies and made the wound using needle. This proves that Acetobacter sp. can lead to the symptoms of sour rotted grape in the presence of wounds.

Reviewer comment:

Dear authors,
as I can see in the figures 5 A-16 and 5 B-16 Acetobacter sp. did not develop sour rot symptoms on table grape berries. The figures show spoilage symptoms caused by bacterial colonization but they are far from the typical sour rot. I understand that the reproduction of sour rot symptoms under laboratory conditions is difficult, especially for red skin table grapes. I suggest (for future studies) to better characterize Acetobacter sp. strain through ITS sequencing (in this work the taxonomic position is questionable), sour rot development on grape berries (I suggest to inoculate the strain -10 µL at 108 cfu/mL- in the pedicel cavity after pedicel remove instead in wounds made with needle) and biochemical characteristics.
3
L302-313 Which is the prediction accuracy based on yours OTU number/sample of PICRUSt predictions? Which is the accuracy for each functional category in COG and KEGG classification?

Respond: In our study, three samples were analyzed and 16S rRNA in the samples was annotated according to the COG and KEGG data base. However, the figure and result was the merged result of three samples (replicates). Therefore, no variation analysis was showed in the manuscript. Could you please tell me what is the prediction accuracy? Which result needs to be supplemented, thank you very much.

Reviewer comment:

Dear authors,
16S rRNA in the samples was annotated according to the COG and KEGG data base, using the software PICRUSt. This software works with predictions that can be not accurate. I strongly suggest reading the following document:
https://picrust.github.io/picrust/tutorials/quality_control.html
For these reasons, authors should provide more information related to functional analysis in Materials and Methods and Results. In particular:

- For the standard metagenome prediction workflow, PICRUSt requires that input 16S rRNA data be in the form of a BIOM format table that was picked by mapping your reads to references in the greengenes tree. The most straightforward way to do this is using QIIME’s reference-based OTU picking pipeline as described in the OTU Picking Tutorial. Therefore, OTU IDs must be based on greengenes and sequences that fail to map to references will not be predicted. If the authors used this workflow please add in Materials and Methods.

- Are reference genomes available for the more abundant bacterial species? Please add this information in Results

- Calculate reference genome coverage for your samples using NSTI scores. Please add in Results

- Calculate metagenomics confidence intervals. Please add in Results

- Some details of the reference-based OTU-picking step may be useful to report in order to ensure reproducibility of the results. These include: the version of the Greengenes reference used, the proportion of reads that mapped to reference during OTU picking, the similarity threshold used during OTU-picking. Please include these details in Materials and 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: DIVERSITY AND SPOILAGE POTENTIAL OF MICROBIAL COMMUNITIES ASSOCIATED WITH GRAPE SOUR ROT IN EASTERN COASTAL AREAS OF CHINA
Review round: 2
Reviewer: 2

Output:
Basic reporting: No comment.
Experimental design: No comment
Validity of the findings: No comment
Additional comments: Lines 60-63: Provide citation.
Lines 63-64: Provide citation or remove.
All references to sour rot-infected should be changed to sour rot-affected. Sour rot is not a pathogen, it is a disease, and can therefore not infect a grape, but it can affect one.


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: DIVERSITY AND SPOILAGE POTENTIAL OF MICROBIAL COMMUNITIES ASSOCIATED WITH GRAPE SOUR ROT IN EASTERN COASTAL AREAS OF CHINA
Review round: 4
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: Dear authors,
the manuscript is acceptable for publication in Peer J.


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: DIVERSITY AND SPOILAGE POTENTIAL OF MICROBIAL COMMUNITIES ASSOCIATED WITH GRAPE SOUR ROT IN EASTERN COASTAL AREAS OF CHINA
Review round: 6
Reviewer: 1

Output:
Basic reporting: Line 29: "Microorganisms causing sour rot": this should be changed to associated with sour rot

Line 49-51: As per Hall et al. 2018, sour rot also requires the presence of Drosophila fruit flies, and is also characterized by the smell of acetic acid.

Line 255: "Potential to spoil grapes" is different than causing sour rot symptoms. Sour rot symptoms are characterized by
Experimental design: No comment
Validity of the findings: Line 281: Grape sour rot has been shown to require the presence of fruit flies, in addition to yeast and acetic acid bacteria. Without fruit flies, necrosis occurs but the symptoms do not resemble sour rot in the field (Hall et al. 2018). The organisms associated with sour rot in your study are causing necrosis, but necrosis alone does not define sour rot. Please elaborate in your discussion.

Line 341 is the only mention that Drosophila are required for the disease symptoms to develop but even then, there is no discussion that Drosophila were not used in your assays. Please elaborate.
Additional comments: As the role of Drosophila fruit flies has been documented as a causal organism in sour rot development, I believe it needs to be mentioned in this paper. Also, I believe the lack of inclusion of fruit flies in the pathogenicity assays needs to be addressed.