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: IDENTIFICATION OF A NOVEL FOUR-LNCRNA SIGNATURE AS A PROGNOSTIC INDICATOR IN CIRRHOTIC HEPATOCELLULAR CARCINOMA
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript was mostly clear with professional use of language. I have a few grammatical suggestions:
1.	Line 41: “Liver Cirrhosis is an important prognostic risk factor in patients with liver cancer”.
2.	Line 49: please omit “a total of 204”. It is understood that the 204 cirrhotic HCC samples came from 204 patients, unless otherwise stated.
3.	Line 75: please remove “as a highly fatal disease”. The next sentence reiterates that point.

Sufficient background provided barring a few references:

1.	Line 75: please provide statistics for liver cancer related deaths, with references
2.	Line 76: please provide statistics for increasing mortality and why “new interventions” have become absolutely necessary.
3.	Line 101: Since the study is based on lncRNAs as prognostic indicators in HCC, it would be great if the authors could cite an example of a lncRNA used for patient stratification.

Professional article structure, figures and tables.
Experimental design: The article falls within the scope of the journal. The introduction is well laid out with a description of liver cancers and HCCs. It also sets about addressing the association between liver cirrhosis and the risk of developing HCC. It then goes on to explain why lncRNAs may be important clinical indicators and therapeutic targets for patients with liver cancer. However, the purpose of this study is unclear, thus It would be useful to include a sentence or two about “aims” or “objectives” of the study at the end of the introduction.

It is also unclear how this particular study is addressing a knowledge gap. The authors need to explicitly state what the knowledge gap is i.e. the stratification of HCC is poorly done and therefore the authors wish to add a more robust prognostic strategy or no one has studied cirrhosis as a prognostic marker in HCC and therefore the authors endeavor to establish that association?

Materials and methods are generally well written with enough information to allow replication of study. Here are a few comments:
1.	How was the correlation coefficient cutoff of 0.4 determined? R = 0.4 seems like a low threshold for association.
2.	Some of the material included in results should be moved to Methods. For example, lines 190 – 193 describe patient stratification using a software. This can be moved to methods as the authors are not reporting any findings here. Lines 201 – 207 talk about cinstructing prognostic signatures and calculating risk scores. This can be moved to Methods as the authors are still describing steps that they used to get to their results.
3.	Lines 205-207: what are the numbers/coefficients -0.4818, 0.4404, 1.2845 and -1.5170 in the risk score calculation? How were these numbers derived?
Validity of the findings: Data seems statistically sound and analysis appears to be robust, with the conclusion well stated. The original questions needs to be well defined, together with the aim of the study and then the conclusion needs to be restated in that context, if necessary.
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: IDENTIFICATION OF A NOVEL FOUR-LNCRNA SIGNATURE AS A PROGNOSTIC INDICATOR IN CIRRHOTIC HEPATOCELLULAR CARCINOMA
Review round: 1
Reviewer: 2

Output:
Basic reporting: Summary: The authors identify a novel four-lncRNA signature as a prospective candidate biomarker in cirrhotic hepatocellular carcinoma  through a publicly available dataset that they analysed.   A combination of machine learning heuristics and statistics are supplemented with their results.

Strengths:  The authors identify perhaps a very important dataset and come up with a pipeline that could simply be based on an in silico approach

Weaknesses: The narration could have been nicer.  A few important references could be a good addition  besides having a methodological flowchart.
Experimental design: The rationale of the work appeared to be lost.  On a hindsight, the authors MUST describe why lncRNA signatures, after all.

Moving further to methods,  the dataset the authors considered  could have some textual insights like population, ethnicity etc.,  They could describe in a sentence or two pointing to the erstwhile tables they cited.

To the end, they could also subtly point what if there are RNA-Seq datasets that they could consider.

Although Kaplan-Meier survival curve is drawn through R, a better reference must be pointed out in lines 160-165.

A good flowchart for methods would be very nice addition.

The Pearson coefficient between  0.2 and 0.4 with moderate effect could have been considered.  How good is this justifiable ( >0.4) ?

Lines 189-194  could move to  Materials and methods
Validity of the findings: The lncRNAs were not checked for curation/bona fidelity.  Did the authors verify them?

For example, they could have subtly checked for their role in NONCODE etc.

Although in  lines 226-230,  the authors mention the cut-off values for training and test datasets, arbitrary division of test datasets  is perhaps not a good idea.  It might lead to bias, if they are divided based on high/low risk on the test datasets as well. How good was the precision/recall in this case?  Did the authors check this?

When the SRA dataset is mapped to the genes,  PLPBP is shown to be a candidate protein-coding gene associated with tumorigenesis of HCC.  Did the authors check for lncRNA-mRNA interactions. A gamut of tools for such predictions do exist.  This would have been an awesome to come up with their role for biomarker analyses.

Where are these signatures localised?  To which chromosome? A pictorial representation would be very good.
Additional comments: Line 47: The word  'dataset'   could be one word
Please check typos all through and  a few words are capitalised in between

Line 75: Introduction could have Globocan statistics as a reference with thorough HCC prevalence details.

Lines 90-104:  A good number of works in the recent past have dealt identification of lncRNAs as candidate biomarkers.  For example., Hu et al., 2017; Yuan et al., 2017.   These authors have also discussed on the role of  circulating biomarkers towards prognosis/diagnosis making a good way to consider the role of ncRNAs,viz. SVUGP2/GIHCG etc.

These sections could have a very good rationale to start with.  For example., why lncRNA signatures alone?  The role of miRNAs and their respective interaction studies with mRNA etc. An absolute rationale for this study is missing.

The article must be proofread thoroughly in the next version.

The authors contributions and funding source etc., may be added.


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: IDENTIFICATION OF A NOVEL FOUR-LNCRNA SIGNATURE AS A PROGNOSTIC INDICATOR IN CIRRHOTIC HEPATOCELLULAR CARCINOMA
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have addressed my concerns satisfactorily and I do not have any further comments and inputs to add
Experimental design: The authors have addressed my concerns satisfactorily and I do not have any further comments and inputs to add
Validity of the findings: The authors have addressed my concerns satisfactorily and I do not have any further comments and inputs to add
Additional comments: The authors have addressed my concerns satisfactorily and I do not have any further comments and inputs to add


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: IDENTIFICATION OF A NOVEL FOUR-LNCRNA SIGNATURE AS A PROGNOSTIC INDICATOR IN CIRRHOTIC HEPATOCELLULAR CARCINOMA
Review round: 2
Reviewer: 2

Output:
Basic reporting: I am satisfied with all the comments rendered.Thank you.
Experimental design: I am satisfied with all the comments rendered
Validity of the findings: I am satisfied with all the comments rendered.Thank you.
Additional comments: I am satisfied with all the comments rendered.Thank you.


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: COMPLEXITY OF HUMAN WALKING: THE ATTRACTOR COMPLEXITY INDEX IS SENSITIVE TO GAIT SYNCHRONIZATION WITH VISUAL AND AUDITORY CUES
Review round: 1
Reviewer: 1

Output:
Basic reporting: Review: PeerJ#37165
Title: Complexity of human walking: the attractor complexity index is sensitive to gait synchronization with visual and auditory cues

This manuscript describes a study undertaken to assess a novel measure of complexity of gait, which is compared to DFA.  To this aim, the attractor complexity index is introduced, which is basically the rate of divergence (as used for local dynamic stability calculations, also sometimes referred to as Lyapunove exponents) after different tracking times. It is shown that a state space constructed from AP signals yields ACI which are highly correlated to DFA results, and also show similar changes with cueing. All in all, this is a well written and interesting manuscript. I have only two major comments, which may require re-analysis of the data.
Experimental design: Major comment:

1)	If I understand correctly, for DFA, all steps measures were included in the calculation (at leats, there is no mention of a specific selection of strides), whereas for ACI and LDS calculations, only 300 strides were used. While this is OK, it could well be that if DFA and ACI (and LDS) were calculated over the same strides (which is not the case now, as DFA is calculated over more strides), agreement between the two measures would be even higher. It may thus be good to redo the calculations, such that all measures are calculated from exactly the same data.
Validity of the findings: See 2.
Additional comments: Major comment:

2)	While data are shared, I do not consider these the “raw” data, as they are simply the calculated parameters. There can of course be debate about whether that constitutes “raw” data or not, but I feel it my duty to at least mention this here.

Minor comments:
1)	Please avoid the use of abbreviations for the conditions. This makes reading unnecessary hard for readers. PeerJ does not have strict word limits, so no need to make your readers suffer through the abbreviations.
2)	In the abstract, an Area under the curve of 1 is mentioned, but this cannot be found in the results?
3)	Figure 5 is rather unreadable; maybe it can be made somewhat bigger?


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: COMPLEXITY OF HUMAN WALKING: THE ATTRACTOR COMPLEXITY INDEX IS SENSITIVE TO GAIT SYNCHRONIZATION WITH VISUAL AND AUDITORY CUES
Review round: 1
Reviewer: 2

Output:
Basic reporting: Abstract:
-	It would be good if the author provides an explanation on what would be the benefits of using the new measure (ACI) that uses continuous time series over DFA in terms of extra information it can provide.
-	I would suggest using AUC for the abbreviation of the area under the ROC curve as it is more common.
-	If ACI and DFA perform equally, then what is the point of using ACI. Please refer to my first comment.
Introduction:
-	I commend the authors for the comprehensive background on DFA.
-	Line 94-101- how the interpretation of alpha in its classical form is changed according to the study of Dingwell and Cusumano, Gait Posture. 2010 Jul;32(3):348-53. doi: 10.1016/j.gaitpost.2010.06.004. I suggest discussing this paper in this paragraph as well.
-	I still don’t get what extra benefit ACI provides over DFA alpha. I think more rationale is needed to convince the reader.
-	In addition, more theoretical background is needed on the relevance of ACI to DFA from a theoretical point of view as ACI is simply the long-term LDS.
Experimental design: -	Line 183- doesn’t filtering affect the calculation of ACI and DFA? Any reference?
-	Line 189- 192- please cite appropriately for delay embedding and average mutual information method. Also the Rosenstein’s study
-	Line 219-p=0.5 or 0.05?
-	According to Figure 1, it doesn’t seem that AC1-4 (slope over strides 1 to 4) is a good linear fit of the region? What is the rationale for this measure?
Validity of the findings: -	The findings are valid and support the hypothesis. But my concern again is the theoretical connection between ACI and DFA.
-	In a similar manner, is there any ACI value equivalent to alpha=0.5?
-	As also mentioned by the author in the Introduction, previous studies didn’t find different long-term LDS between stability condition. What is the difference between the method of calculating ACI in this study and previous ones that makes it unique to be used as a fractal measure? Considering that it can differentiate between conditions in this study. This becomes more important considering that short-term LDS in AP could efficiently differentiate the conditions in this study with AUC=0.82 implying that short- and long-term LDS are measuring the same thing (which indeed they are)
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: COMPLEXITY OF HUMAN WALKING: THE ATTRACTOR COMPLEXITY INDEX IS SENSITIVE TO GAIT SYNCHRONIZATION WITH VISUAL AND AUDITORY CUES
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript is clear overall. The figures looks clear, although I suggest to shift x and y axis in Figure 2 and 3. In Figure 2 I suggest to use the same scale for both ML and AP LDS, as it is it seems that in No cueing they both present the same range but AP LDS is much wider. While a 'Raw Data' file was provided, it appears it contains only the main outcomes for the analysis, not the actual raw data necessary if one would like to replicate the author's results.
Experimental design: A few more details are necessary in the Methods section: notably, while I agree that a full description of DFA is not necessary, it is crucial to report the parameters selected (e.g., n_min and n_max, evenly spaced algorithm or not?). While I understand that the DFA results can be found in previous publication, I think enough information should be provided in the present article to allow replication.
Validity of the findings: The results may shed new lights on previously published work investigating long-term LDS (or ACI). Overall the conclusions are supported by the results, although access to the raw time series would benefit potential for replication.
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: COMPLEXITY OF HUMAN WALKING: THE ATTRACTOR COMPLEXITY INDEX IS SENSITIVE TO GAIT SYNCHRONIZATION WITH VISUAL AND AUDITORY CUES
Review round: 2
Reviewer: 1

Output:
Basic reporting: the author covered almost all comment. The only remaining comment is:

in response to my previous comment "In addition, more theoretical background is needed on the relevance of ACI to DFA from a theoretical point of view as ACI is simply the long-term LDS." he replied: "The reason of the sensitivity of ACI to long-term gait complexity is still an enigma for me. I cannot provide more information than that I gave in the introduction (L 162-168). I hope that specialists in nonlinear gait analysis can further investigate this point."

he also continues to another comment "...the sensitivity of ACI to noise structure of stride intervals is purely based on observations and has no theoretical justification for now."

These responses confirm that the results of this study are based on observations and future studies are needed to investigate the theoretical relationship between DFA and ACI. this should be added and underlined in the Abstract and Conclusion.
Experimental design: The author covered all comment
Validity of the findings: the author covered all comment
Additional comments: No additional comments


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

Input:
Title: COMPLEXITY OF HUMAN WALKING: THE ATTRACTOR COMPLEXITY INDEX IS SENSITIVE TO GAIT SYNCHRONIZATION WITH VISUAL AND AUDITORY CUES
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors has made substantial revisions to address each reviewer's comments. The figures are clearer, but most important the rationale as to why it is important to compare LDS (ACI) to DFA measures is better introduced and discussed.
I also appreciate that the author does not try to answer some questions with some convoluted explanations, but simply state 'I don't know'.
Experimental design: The revisions in the Methods (comparing two techniques on the 'same' signal) certainly enhance the potential impact. I am personally curious to see if the results holds if ACI is computed from shorter time series; in particular, if the relationship between ACI 2 (and ACI 3) with Alpha-DFA is still present. One of the limitations of DFA (and similar techniques) is that it requires lots of stride to obtain reliable estimates, which can limit its use for pathological populations. But if DFA and ACI are highly correlated even when ACI is computed on shorter time series, this may open the door to analyzing gait dynamics in under-studied populations (granted, a correlation between DFA and ACI does not necessarily mean they provide the same information!).
Note that I do NOT recommend this analysis in the present paper (although I think it may add another layer of information!). I just think it is something the author might consider in the future.
Validity of the findings: The addition of the actual raw data is very much appreciated.
Additional comments: Very good paper in general, I do not have more comments at this time. I would like to apologize to the editor and the author for being a bit late in my review.


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: INDIRECT DOPPLER FLOW SYSTOLIC BLOOD PRESSURE MEASUREMENTS TAKEN WITH AND WITHOUT HEADPHONES IN PRIVATELY-OWNED, CONSCIOUS DOGS
Review round: 1
Reviewer: 1

Output:
Basic reporting: This manuscript is well written. The language is professional throughout with minimal if any grammatical errors. The figures are relevant and easily read.  The raw data is supplied and does raise some questions which will be directed in other portions of this review. The references were adequate . The introduction was adequate.
Experimental design: The research is original in nature. The questions are well defined, the main one being if determining hypertension using headphones is a more sensitive manner of blood pressure determination than without headphones. Exclusion of difficult to handle patients might have caused bias, but not clear if could be performed in any other way with out sedation of patients which would have induced additional bias.
Methods were described sufficiently.
The stats as described were a bit confusing for most general readers. These should be reviewed by a statistician and perhaps clarified in the methods section in more easily understood manner by general readers
The study does need to be expanded to increase the number of dogs and then to divide categories of co-morbidities which were not addressed at all. How might this have impacted the blood pressure, a fair number were on medications?
Your most important issue in terms of research question about headphone use is addressed, expanding numbers of cases might also help reinforce the results. However, the effect of age, weight , anxiety should be expanded upon in a more rigorous manner since you elected to include this as part of the study. In addition, you elected to include , " a typical hospital" population with a variety of presenting problems but did not address any impact that these co-morbidities or the medications used to treat these patients may have impacted the study, or induced bias. Were patients with CKD more used to being in the hospital having blood pressure determinations?  If these parameters were looked at statistically and not significant would be helpful for readers to know
Validity of the findings: I thank the authors for providing raw data as well as statistical analysis.
The areas that this reviewer has the most problem is listed above and repeated here.
Your most important issue in terms of research question about headphone use is addressed, expanding numbers of cases might also help reinforce the results. However, the effect of age, weight , anxiety should be expanded upon in a more rigorous manner since you elected to include this as part of the study. In addition, you elected to include , " a typical hospital" population with a variety of presenting problems but did not address any impact that these co-morbidities or the medications used to treat these patients may have impacted the study, or induced bias. Were patients with CKD more used to being in the hospital having blood pressure determinations?  If these parameters were looked at statistically and not significant would be helpful for readers to know
Additional comments: Well written.
Overall a good discussion
A study that truly needed to be performed
Most of areas of weakness or need for further study have been addresses.
No real discussion of impact of co-morbidities or medications patients receiving were discussed.
Your most important issue in terms of research question about headphone use is addressed, expanding numbers of cases might also help reinforce the results. However, the effect of age, weight , anxiety should be expanded upon in a more rigorous manner since you elected to include this as part of the study. In addition, you elected to include , " a typical hospital" population with a variety of presenting problems but did not address any impact that these co-morbidities or the medications used to treat these patients may have impacted the study, or induced bias. Were patients with CKD more used to being in the hospital having blood pressure determinations?  If these parameters were looked at statistically and not significant would be helpful for readers to know


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: INDIRECT DOPPLER FLOW SYSTOLIC BLOOD PRESSURE MEASUREMENTS TAKEN WITH AND WITHOUT HEADPHONES IN PRIVATELY-OWNED, CONSCIOUS DOGS
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Review “Indirect Doppler flow systolic blood pressure measurements
taken with and without headphones in privately-owned,
conscious dogs”  Peer J

General comments:
The study is well designed and carried out. I am glad such a study was done and will be a good addition to the literature on BP measuring.

Minor specific comments:
Lines 101-102: “A soft measuring tape was used to measure the circumference of the mid-antebrachium, and a cuff was selected that was 30-40% of the appendage” – this second part of the sentence is not so clearly written it should be  that the width of the cuff should be 30-40 % of the limb (extremity) at the site of cuff placement. It  would be more understandable to the reader.

Line 149 why authors repeat “at the start of measurements” twice, is this necessary?
Lines 178-182- the results of median measurements with or without headphones dffer from those in the table 1. Please refer to the comment bellow.
Table 1: BP should have its unit written
It is not clear: the results section BP results are written as median and range that is ok, but in the Table one it is confusing what is BP 1 – a mean +/-SD of the group using headphones and than no headphones and vice versa in the second column? And in the next row  mean of BP 2-6 – the same comment.


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

Input:
Title: INDIRECT DOPPLER FLOW SYSTOLIC BLOOD PRESSURE MEASUREMENTS TAKEN WITH AND WITHOUT HEADPHONES IN PRIVATELY-OWNED, CONSCIOUS DOGS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have addressed the reveiwers comments. Well written. Complete literature review.
Experimental design: No Comment
Validity of the findings: No comment
Additional comments: Well written.  Thank you for this study. The response to reviewer comments were thoughtful and well articulated


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: INDIRECT DOPPLER FLOW SYSTOLIC BLOOD PRESSURE MEASUREMENTS TAKEN WITH AND WITHOUT HEADPHONES IN PRIVATELY-OWNED, CONSCIOUS DOGS
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Reviewer 2.
I am pleased with the authors' corrections and I have no further comments.
Congratulations.


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: TAXONOMIC, BIOLOGICAL AND GEOGRAPHICAL TRAITS OF SPECIES IN A COASTAL DUNE FLORA IN THE SOUTHEASTERN CAPE FLORISTIC REGION: REGIONAL AND GLOBAL COMPARISONS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript is written in very good English with a few typographical, grammatical and general errors which I have indicated below.
Literature references and sufficient field background and context are provided.
The article conforms to the structure required by PeerJ.
Relevant results are presented on a documented flora. As this is descriptive research, this research is not strictly hypothesis driven.
Experimental design: The experimental design of this study is simple although labor intensive. It involves the careful documentation and identification of the flora of the area and analysis according to geographical traits, biological traits and species conservation status.
Validity of the findings: In this study, the coastal dune flora of the Cape St Frances area has been assembled over a number of years by experts in the field, and identifications were made by them and by using iNaturalist. This data is summarized in appropriate bar graphs and tables as well as a species listing of the flora in a supplementary file. Consequently, the study can be viewed to be original, rigorous, and the findings valid. Some clarification is required for the presentation of some of the data as I have indicated in the comments to the author.
Additional comments: I wish to bring the following points to the attention of the authors:
1.	The reference, Le Roux, (2000), as referred to in Line 130 is not in the reference list.
2.	Line 92 and 93: At this stage, some 13 900 km2 of the PAP is comprised of calcareous dunes, a 24-fold increase relative to the present time. This last phrase does not make sense in relation to the previous sentence and needs rephrasing. Should this not be a decrease?
3.	Line 130: The sentence “Contrary to Le Roux (2000), no Cenozoic aeolianites (Nanaga Formation) occur in the region.” needs clarification. This is too brief to be understandable as it gives the reader no indication of the area to which it is compared to, and needs a phrase such as “Contrary to Le Roux (2000), who investigated the …… of ….., no Cenozoic aeolianites (Nanaga Formation) occur in the region.”
4.	The authors make use of the area definitions (defined in lines 226 to 230) of the Greater Cape Floristic Region (GCFR) as defined by Born et al. (2007), the Cape Floristic Region (CFR) as defined by Manning and Goldblatt (2012) and the southeastern centre of the CFR (CFRse). A reference for the definition of the CFRe should be given. Species are then classified according to these criteria, and the results of these analyses are shown in Figure 3 C. According to the classification, the CFRe forms a smaller part of the area of the CFR, whilst the CFR in turn forms part of the even larger GCFR. Thus, in my opinion, if a species is scored as present in the CFRe, it can also be scored as present in the CFR and in turn in the GCFR. If one inspects the bar graph in Figure 3 C, this is not apparent, because then the GCFR bar should be the tallest representing the cumulative number of these species. This needs clarification please. I assume that species that have been scored for presence in the CFRe, have not been scored as present in the rest of the CFR and the rest of the GCFR, and the species occurring in the CFR have not been scored in the GCFR. This needs clarification in the methods or else this is not understandable, and the statistics do not make sense.
5.	It s not clear from the presentation of the data, who generated the floras of Walker Bay and Koeberg. References to the authors of these floras should be specifically indicated in the text in lines 286-288 and again in Table 1, as this data was not generated as part of this study.
Typographical and grammatical corrections are required as follows, and are indicated:
Line 66: “  coastal dunes, especially on their seawards margin, are subject to frequent, strong, salt-laden winds…”
Lines 133 to 135: due to the long sentence construction the end of the sentence is not correctly structured (plural required of "systems", as indicated):  “Geomorphologically, the study area comprises vegetated parabolic dunes and several more recent plumes of mobile sand forming and impressive (maximum 14 km-long) bypass dune systems (Tinley, 1985; Illenberger and Burkinshaw, 2008).
Line 135: Add comma for clarity: On the inland margin, dunes reach a maximum altitude of 62 m.
Line 166: spelling Salvia africana-lutea
Line 183: spelling Senecio halimifolius,
Line 216:  spelling Pterocelastrus tricuspidatus
Line 360: There “are” none on California (not “is”)..
Line 388: Fig. 6 Some of the wide-spread species (not “wides”)
Line 419: are MCE endemics and none have a tropical distribution….
Line 460: “Trees, however, trees are absent and lower……” delete “Trees”and start sentence with “However”.
Line 517: “Fababceae” should be spelt “Fabaceae”
Line 520 and 521: “…..no non-sprouting shrubs are unlikely to be found on the coastal dunes of Chile.” The double negative needs rephrasing.
Line 589: Two errors: “The growth form mix of CFR dunes floras shows strongest convergence with Australian dunes in that in both, evergreen hemicryptophytes and shrubs share dominance, and annuals are floristically and subordinate.” The first "dunes" should not be plural and delete the last “and”.


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: TAXONOMIC, BIOLOGICAL AND GEOGRAPHICAL TRAITS OF SPECIES IN A COASTAL DUNE FLORA IN THE SOUTHEASTERN CAPE FLORISTIC REGION: REGIONAL AND GLOBAL COMPARISONS
Review round: 1
Reviewer: 2

Output:
Basic reporting: I think the paper is too long with too much of the data presented in the text, and I suggest using tables or figures for much of this data.

I also think the paper would benefit from some restructuring and a tighter focus - see attached review.
Experimental design: No comment.
Validity of the findings: The findings are valid and interesting.
Additional comments: Please see attached review file.


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

Input:
Title: DISCOVERY OF NOVEL ASTROVIRUS GENOTYPE SPECIES IN SMALL RUMINANTS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Line 41 as well as in the figure legend of fig. 3: “five newly discovered full-length genome sequences” indicates that the authors determined the complete genomic sequences of the five sheep and goat astroviruses, although there are otherwise contradictory information found in the manuscript about completeness or even the exact lengths of sequences of the identified novel sheep and goat astroviruses. e.g. lines 252 – 253: “these five scaffolds represent complete or almost complete viral genomes of novel MAstV strains” or in lines 346-347: “Five novel, almost full-length astrovirus sequences were discovered…”

Lines 82-84: Additional animal species other than humans and ruminants from which neurotropic astroviruses were also detected (e.g. minks and swine) should be mentioned at least in the introduction because the neurotropic astroviruses identified from those animals are highlighted in figure 2.

Lines 84-87: The source materials (e.g. brain or CNS samples etc…) of these neurotropic astroviruses should be included here.

Lines 223-226: Please consider that this section should be moved to the Detection of various astroviruses using Pan-astrovirus RT-PCR (line 208) section.

Lines 267 – 271: Please consider that this section should be moved to the Discussion section

Figure 2: Please consider including “clades” to the phylogenetic tree according to ICTV taxonomy guidelines (https://talk.ictvonline.org/ictv-reports/ictv_9th_report/positive-sense-rna-viruses-2011/w/posrna_viruses/248/astroviridae-figures) Please consider to use different colors of existing and newly discovered ovine strains (OvAstV). Same color of previously known and novel ovine astrovirus strains is confusing.

genbank submissions: Why tissue_type="brain" could be found in all submission files? According to the manuscript these sequences were identified from fecal samples.
Experimental design: Lines 126-128; lines 132-133 and supplementary table 1: The positions of the primers and probes should be described in more detail, e.g. nt positions on a reference genome or designations of the targeting genomic regions (e.g. RdRp, protease, capsid-conserved/hypervariable part…) should be specified.

Lines 226 – 230: Please consider that the results most likely originated from contamination should be omitted because it is not relevant to the original scope of the manuscript

Lines 285 – 286 as well as in the figure legend of fig. 3: Please consider to include the nt/aa identities between the CapAstV-G2.1 and BoAstV/GX27 as well as between CapAstV-G2.1 and BoAstV/JPN/HK. It is most likely that the exact parental sequences are not BoAstV/GX27 and BoAstV/JPN/HK but other astroviruses related to these viruses. Therefore this section should be rephrased like “Another putative recombination event (2) was identified between BoAstV-GX27/CHN/2014 and BoAstV/JPN/Hokkaido11-55/2009 or closely related sequences as potential parental sequences”
Validity of the findings: Line 290 – 291: Although some of the identified ovine and goat astroviruses are fulfilled the criterion of novel species but these viruses are still candidates therefore this sentence should be rephrased accordingly.
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: DISCOVERY OF NOVEL ASTROVIRUS GENOTYPE SPECIES IN SMALL RUMINANTS
Review round: 1
Reviewer: 2

Output:
Basic reporting: In this study, Kauer and colleagues screened a panel of 164 fecal samples from small ruminants in Switzerland for known encephalitis-associated virus astrovirus strains in sheep and cattle. Furthermore, they tested the samples by a published heminested panastrovirus RT-PCR for other astroviruses, sequenced positive samples and performed recombination analysis.
The manuscript is well written and the conclusions stated by the authors are comprehensible. As different astroviruses in encephalitic animals were described independently several times in the last years and nothing is known about the excretion or pathogenesis, the topic of this manuscript is relevant for a better understanding of these viruses.
Experimental design: The original primary research presented in this work is within the scope of the journal. The research question is well defined, relevant & meaningful.
Nonetheless, the single sequences obtained by NGS should be verified by RT-(q)PCRs and a test for the frequency of the detected new genotypes would strengthen the work.
Validity of the findings: The authors could not detect any so far known neurotropic astroviruses in the sample panel but were able to discover new astrovirus genotypes in small ruminants which further broaden the genetic diversity of this virus family.
Unfortunately, the number of investigated samples is low and no information about the health status of the animals is available.
The analysis of recombination events indicates possible interspecies transmission, highlights the possible zoonotic potential and thus once again shows that the former opinion of species-specific astroviruses appears to be wrong.
Additional comments: Line 17: Space character missing.
Line 43: Explain why unexpected? Divergent Astroviruses in feces of animals have been shown before.
Line 48: Space character missing.
Line 135: Were internal controls used to check extraction efficacy and quality of sample materials?
Line 157: Revise word order.
Line 183: What percentage of the scaffold consists of sequence information? Where are the gaps located? A figure in the supplements representing the genome coverage would be helpful (at least for the five large scaffolds).
Line 224: Could it be that the benzonase treatment affects not only the amount of free nucleic acids but also reduces to some extent the overall concentration of astroviral RNA within the capsids? What sequences were generated from the RT-PCR amplicon of the other ten samples before benzonase treatment which scored negative after the treatment?
Line 299: RT-qPCR. Please check the spelling throughout the manuscript.
Line 309: Were the other sequences found by NGS confirmed with specific RT-(q)PCRs? This should be done.
Line 346: Further information about the frequency of the five newly detected astrovirus genotypes would improve the manuscript. Would it be possible to screen the panel of feces samples with RT-PCR systems for theses variants? The panastrovirus heminested PCR is maybe not that sensitive and you may have missed positive samples.
Figure 2: Please include all encephalitis-associated strains described in animals.


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: DISCOVERY OF NOVEL ASTROVIRUS GENOTYPE SPECIES IN SMALL RUMINANTS
Review round: 1
Reviewer: 3

Output:
Basic reporting: No comment.
Experimental design: I think further effort could have been made to extend the sequence information of some of the viruses and that partially sequenced viruses could have also been included in the phylogenetic analysis (see below for details).
Validity of the findings: Recombination analyses need some clarification (see below for details).
Additional comments: Kauer et al. report the identification of several potentially novel astroviruses of ruminants, although they only obtained extended sequence information for 5 of them. These novel viruses belonged to 3 species and affected goat and sheep. There was evidence for cross-species transmission and viral recombination. The paper is well written and properly structured, the objective is well-defined and proper methods were used, analyses are thorough, and results properly discussed. However, there are some points that, in my opinion, deserve a more in-depth analysis/explanation.

1. First of all, obtaining as much sequence information as possible is crucial for this kind of studies. The authors have chosen to consider for an in-depth investigation only viruses for which the complete coding sequence was determined. However, I believe that more effort could have been made to try and extends obtained scaffold sequences (unless it was attempted but not described). For example, extended sequences could be obtained by designing primes and connecting fragments, and through genome-walking or RACE methods. I realize that such laboratory investigations are not always possible as they are laborious and costly, but bioinformatics methods can be used for this purpose too. For example, although blast and diamond settings were not provided, did the author try to use a less strict search to identify viral fragments with a lower identity to reference strains? Also lose-setting mapping and cross-assembly (between different samples) could help in identifying viral fragments and their relationships (obviously such assemblies would have to be PCR-confirmed). For example, from a quick analysis, it looks like samples G8 and S5 contain the same virus and I could successfully assemble fragments G8-3/4/5 and S5-2 into a ~2.7Kb contig. This study would benefit a lot from obtaining further complete genomes.

2. Although for some viruses only partial sequence was obtained, for some of them a sizable portion of the genome was obtained that allows to perform meaningful phylogenetic analysis. I think partially sequenced strains should not be dropped and, although definitive classification cannot be provided, an additional phylogenetic analysis should be performed to describe partially sequenced strains. These strains, may not have the requirements to be classified as novel species, but they are still interesting (I blasted some of them and many may be additional new viruses) and their phylogenetic relationship to other astroviruses should be investigated. Such analysis would also help to prove the point you make at line 335 about co-infections: you do have overlap between different fragments from the same sample but you never show how similar they are to each other (for example, you clearly have three different viruses in sample G1).

3.  Recombination analysis. Aren’t the setting used a bit strict? Normally you would choose events detected by at least 2-3 methods, not by all. Can you justify your choice? Furthermore, multiple contigs were found in samples involved in recombination. Did you make sure that only one virus was present in these samples and that recombination patterns were not due to artificial assembly of reads? Were recombination events confirmed by building trees with portions of the alignments included between breakpoints? If not, this should be done.

Minor.
- Line 26. It should be “aims” and “were”.
- The introduction is very well written and complete. However, I think that a clearer classification of bovine/ovine astroviruses at a species level could be provided to help the reader distinguishing between strains (e.g. which strains are included in species MAstV-13 and which other likely belong to different species). More information can be found in https://doi.org/10.3390/v9050102.
- Figure 1. There are some weird arrows in the middle panel coming down from the first box.
- Supplementary Table 1. I would add references to papers including primers previously published.
- Lines 150 and 153. Are you sure primers were used at a final concentration of 10uM? Normally protocols use a final primer concentration of 0.05-1uM. Please, verify.
- Line 157. This sentence is a bit weird and English should be checked.
- Lines 176 and 216. I think the correct form is BLASTn (n in lower case).
- Line 163. I suggest using a more elegant title without abbreviations.
- Line 164. To facilitate reading, I think a comma after “(NGS)” should be introduced.
- Line 189. How was this model chosen, based on a modeltest analysis or on literature?
- The first three paragraphs of the results can be combined into one unique paragraph which summarizes the information contained in these three paragraphs, which also contain irrelevant information (e.g. the first paragraph could be summarized into “None of the samples were positive for BoAstV-CH13 and OvAstV-CH16 ”. Also, lines 209-211 can be removed).
- Lines 215-220. First of all, how did you get a >600nt sequence when the amplicon was 450nt? Were some bands bigger or were you sequencing something else? Can it be that this number was reported wrong (according to supplementary table 2, no sequence was longer than ~300nt)? Secondly, blasting a 6nt seems a bit absurd! These results clearly indicate that, in many cases, there were some issues with sequencing and the question “why these amplicons were not cloned before sequencing” comes into mind. I strongly suggest removing this analysis and simply stating that 19 samples were astrovirus-positive and those were used for NGS (results of this sequencing are ignored anyway as all samples were used for NGS). (The same is true for the results of the PCR performed after pre-treatment).
 - Line 223. “de novo” should be italicized.
- Line 243. I assume your selection criteria was that sequences should cover the full-length of the three ORF. You should specify if this is the case because it is not entirely clear.
- Line 253. I would say these represent novel species as species demarcation criteria for astroviruses (if I am not wrong) is 75% aa identity in the capsid.
- Line 257. The word “isolate” is referred to viruses that have been isolated on cell-culture. I would use the word “strain” instead.
- Supplementary table 4. I would remove the last column as it is not informative. Also, the sentence below the table is a bit weird.
- Figure 2. I would mark more clearly the strains identified in this study and indicate clades on the tree (e.g. accepted species and/or HMO clade).
- Figure 3. Which of the methods used in RDP was used to generate these plots? This must be indicated.
- Line 335. You can only talk about co-infections if you compared the different contigs identified in the same sample and show they are different…
- Line378. There is a typo (astroviruses/in).
- Did I understand correctly that raw sequences were submitted to GeneBank? If so accession numbers should be provided in the text.


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: DISCOVERY OF NOVEL ASTROVIRUS GENOTYPE SPECIES IN SMALL RUMINANTS
Review round: 2
Reviewer: 1

Output:
Basic reporting: Lines 83 - 85 of the revised manuscript: As far as the reviewer knows the first a neurovirulent porcine astroviruse type 3 was discovered in 2017 by two independent research teams in the USA and Hungary (Arruda et al., 2017: Emerging infectious diseases, 23(12), 2097 and Boros et al., 2017: Emerging infectious diseases, 23(12), 1982). The two references given by the authors (Laurin et al. 2011; Reuter et al. 2012) described only enteric porcine astroviruses unrelated to CNS infections.
Experimental design: Adequate
Validity of the findings: All underlying data have been provided and the conclusions are well stated.
Additional comments: None. Nice work!


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

Input:
Title: DISCOVERY OF NOVEL ASTROVIRUS GENOTYPE SPECIES IN SMALL RUMINANTS
Review round: 2
Reviewer: 2

Output:
Basic reporting: All necessary changes were done. No further comment.
Experimental design: All necessary changes were done. No further comment.
Validity of the findings: All necessary changes were done. No further comment.
Additional comments: All necessary changes were done.


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: DISCOVERY OF NOVEL ASTROVIRUS GENOTYPE SPECIES IN SMALL RUMINANTS
Review round: 2
Reviewer: 3

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: I am satisfied with the revisions. I found 2 typos and I have one remark:
- line 84: typo: astroviruse.
- line 424: typo. There is a wild "of" at the end of the line.
- lines 428-433. Given the fact that these viruses seem to be very species-unspecific and the fact that some are pretty divergent, I don't think you can't conclusively determine from which hosts parental viruses came from and also, likely, parental strains are not exactly those used for the RDP analyses, but relatives to those strains. I suggest rephrasing this paragraph.


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: TLR2 POLYMORPHISM (RS650082970) IS ASSOCIATED WITH SOMATIC CELL COUNT IN GOAT MILK
Review round: 1
Reviewer: 1

Output:
Basic reporting: The paper deals with genetic polymorphism and association to goat milk quality, namely SCC. It is professionally made. The approach of authors is usual in last years and does not bring any novelty.
Experimental design: The design follows usual standards for such analyses. The number of genotyped goats is relatively low, please emphasize it in the Conclusions section. What is the source of primers? Give the reference, or write that you have designed them, give GenBank number, program for design etc.
Validity of the findings: The validity is in relation to the size of the group in analysis. But the research was made on the good professional level, brings reliable results and could be the basis for next studies. Which parity was the best? The A allele was found to be minor, but it is associated with lower SCC. Try to write some short reflection of the fact.
Additional comments: Please revise the paper as suggested above.
Some formal notices:
Abbreviations of genes, genotypes and alleles must be written in italic, revise it throughout the text.
Part C of the Fig. 1 is not necessary, but this notice is not crucial.
R. 102, T allele?


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: TLR2 POLYMORPHISM (RS650082970) IS ASSOCIATED WITH SOMATIC CELL COUNT IN GOAT MILK
Review round: 1
Reviewer: 2

Output:
Basic reporting: Well written, only minor changes suggested below to improve writing.
Experimental design: Fine; no issues with any of the criteria listed as standards. A couple of minor concerns regarding figure 1, as indicated in comments to the authors
Validity of the findings: Strong impact since it is only the second study to find the association reported, which may be of benefit for improvement of dairy production.
Additional comments: Methods: clarify whether SCC data was first averaged per goat, and then averaged per genotype; or alternatively whether the SCC dataset was not averaged per goat, and only separated by genotype regardless of number of recordings per goat.

Do the authors want to test association by genotype for maximum SCC values?

Line 149: N=2 is too low a number to support or not support any hypothesis, so this statement needs to be greatly qualified, e.g., "There were not enough A/A goats to examine the possibility of heterozygote advantage, although it is notable the average SCC value for the two A/A goats was lower than the average for G/A heterozygotes or G/G homozygotes."

Figure 1A: If GG has one band, and AA has two bands, then why are there not 3 bands for GA?

Figure 1C: There is great overlap in the 95% confidence intervals for the two genotypes. How is this consistent with a significant difference? Is there a better way to plot this to indicate the difference is significant?

Minor comments, by line
5-Indicate correspondent with an asterisk
18-"pathogens invading THE mammary gland"
Throughout: "Slovenian Alpine goats"  might be best written as "Alpine breed Slovenian goats"
25-"in the milk recording" should be "in the dataset"
Throughout: Gram positive should be capitalized
37-"one of the major issues" should be "is of major concern"
41"pathogens in" should be "pathogens by the"
45 "precisely" should be "well"
47 "represent" should be "are"
48 "and is" should be ". TLR2 is"
60 "known for cow" should be "established for cow's"
67 remove "the"
70 "milk recording" should be "the dataset"
92-93 Are the primer pair original or previously published? Please specify.
127, if not significant p should be greater than, not less than 0.05
142 expectedly should be "as expected"
149"the lowest SCC in the milk of goats with A/A genotypes" should be "the SCC in the milk of goats with A/A genotypes was lower than that of the other genotypes"
149 N=2 is simply too low a number to support or not
172 "Dr." should be capitalized
References: all taxonomic names (Capra hircus, E. coli) should be italicized
Table 1 header: according to the genotype" should be "by the genotype of TLR2 (rs65...


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: TLR2 POLYMORPHISM (RS650082970) IS ASSOCIATED WITH SOMATIC CELL COUNT IN GOAT MILK
Review round: 2
Reviewer: 1

Output:
Basic reporting: The paper is made on good professional level.
Experimental design: Good, usual in the research of this kind.
Validity of the findings: The paper brings new knowledge in the field.
Additional comments: The authors revised the manuscript, and I agree with publishing.
R. 105, G allele must be written in italic.


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: TLR2 POLYMORPHISM (RS650082970) IS ASSOCIATED WITH SOMATIC CELL COUNT IN GOAT MILK
Review round: 2
Reviewer: 2

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


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

Input:
Title: FUNCTIONAL AND EVOLUTIONARY PERSPECTIVES ON GILL STRUCTURES OF AN OBLIGATE AIR-BREATHING, AQUATIC SNAIL
Review round: 1
Reviewer: 1

Output:
Basic reporting: This is an exceptional study on the anatomy and fine structure of the gill of the invasive apple snail, Pomacea canaliculata. The authors bring together 3D reconstruction with histology, SEM and TEM to bring valuable new insight into the structure and function of the gill in this family, and the implications for the evolution of amphibiousness. The manuscript is clearly and carefully written, with professional English used throughout. The anatomical descriptions are straightforward and easy to understand. They have taken great care to ensure that the language is clear and precise. The literature cited is comprehensive. The figures are of outstanding quality and clarity. Goals are clearly defined, and the authors have thoughtfully placed their study into context and discussed the implications of their findings.

My main criticism is in their occasional introduction of terminology that differs from established terms in caenogastropod anatomy, and especially compared to those used by Andrews in her exemplary study on the functional anatomy of the mantle cavity, kidney and blood system of ampullariids. This creates confusion, makes it difficult to compare the results of different studies, and obscures homology with other caenogastropods (e.g., ureter, efferent ureteral vessel). In some cases, the terms they have introduced are not adequately descriptive of the feature they describe (e.g., mantle fold; see below). I would also like to see a discussion of any differences in interpretation regarding circulation in the mantle roof between this study and that of Andrews (1965), highlighting new results and insights of this study.
Experimental design: no comment.
Validity of the findings: see above.
Additional comments: Comments and suggestions:
In several instances, the authors use colloquial language to describe the shape of structures (e.g. comparisons to a shark’s fin or to cauliflower) which I would remove.

Lines 73-74: there have been TEM studies of vent caenogastropod gills (ie Provannidae) that have symbionts.

Lines 76-77: “because we were stricken by the morphological parallelisms found between the gill of P. canaliculata and those of somewhat phylogenetically distant taxa.”: Delete

Line 91: dilute Bouin’s fluid

Line 108: “material was fixed and embedded as described above”; two methods were described above. Presumably you are referring to embedding in histoplast here.

Line 119: dilute Bouin’s fluid

Line 135: Referring to the anterior chamber of the kidney as the ureter is not standard terminology in the anatomical literature of ampullariids, and confuses the homology of this structure. For example, the ureter of viviparids is a de novo extension of the kidney into the mantle roof alongside the intestine. What you have termed ureter, is an anterior kidney chamber. Many caenogastropods are known to have differentiated regions of the true kidney that are specialized for different functions. The term ureter has been used uncritically in studies of caenogastropods, and I would advise against perpetuating this practice.

Line 143: among “siphonate” gastropods, there are those with a true siphon (=siphonate clade), and those with simply an extended mantle edge (Ponder et al 2008). In both cases, in contrast to ampullariids, the siphon is derived from the mantle roof.

Line 144: why is pallial fold in quotation marks? This is certainly more accurate than “mantle fold”, as it is termed in Figure 1, because it is not a fold in the mantle, it is a fold on the floor of the mantle cavity. I also don’t understand the need to introduce different terminology, when the existing terminology is perfectly adequate.

Lines 148-149: rather than groove, I would suggest channel or something similar. “Groove” suggests to me a much more narrow structural definition (ie. an indentation or furrow).

Line 156: The 3D rendering shows (verb agreement)

Line 157: efferent ureteral vessel. Again, this terminology relating to the “ureter” is obscuring homology of these structures with other caenogastropods. This term has never been used in descriptions of gastropod vasculature, and obscures comparisons with homologous structures in other caenogastropods.

Line 174: supraintestinal. More typically referred to as supraesophageal in the recent literature.

Line 175 accessory supraintestinal ganglion. Given its position, it seems more appropriate to term this structure an accessory visceral ganglion. The presence of accessory ganglia on the visceral loop in caenogastropods is well known.

Line 176: viscero-supraintestinal connective. This is technically incorrect, as the connective joins the supraintestinal and “accessory supraintestinal” ganglia. This is the supraesophageal portion of the visceral loop.

Line 194: what kind of shark? Preferable to use standard geometric terms to describe the shape.

Lines 222-223: “which somehow remind cauliflowers under the scanning electron microscope”: Delete.

Line 242: in region I

Line 266: are also found in the

Line 275: these discontinuities connect

Lines 307-309: “Indeed, in the extreme case of an aquatic animal being brought to air, the gill leaflets tend to stick together thus decreasing the surface area for the gas exchange, and hence, leading the animal to asphyxia (Maina 2002b).”. But the presence of rods in other gastropods means that their presence is not related to facilitating gas exchange while emergent. How do you explain that?

Line 328: The classification of Bouchet et al (2005) has been superseded. In the most recent version (2017), the Architaenioglossa is a grade.

Line 393: “P .canaliculata”. The space needs to come after the period.

Line 418: dwell in the

Line 440: granulocytes occurring in the

Line 470: “The bases of terrestriality”: it should be “basis” not “bases”, and I would argue they aren’t terrestrial. Replace with “The evolution of amphibiousness” or something similar.

Lines 478-479: has received considerable attention

Lines 503-504: “supports the view of the adult’s gill as the post-torsional left gill”: this is the first mention of this in the manuscript. It shouldn’t appear for the first time in the conclusions, but should be mentioned elsewhere in the main body of the results or discussion.

Figure 1: tes, should be testes, not testicle. Upo, should be nephropore, the standard term for the opening of the kidney to the mantle cavity. Pallial fold, rather than “mantle” fold.

Figure 2: What is “the dissection piece”? “Limiting” what with the lung? The meaning here is obscure. Epb is not in the list of abbreviations. “Rectal sinuses” is not correct. There are not multiple sinuses. There is a single rectal sinus and channels that drain it. 2B. I would rotate this figure 180, so that anterior is below, as the other images in this figure.

Figure 3:
Major ganglia vs central ganglia. “Central” implies something positional.
Penial vs penile.
Vsc. As mentioned above, technically this is the accessory supraintestinal-supraintestinal connective. Typically, and more simply, referred to as the supraesophageal part of the visceral loop.

Figure 4:
Diagram of the four regions of the gill leaflet. Presumably these observations are generally applicable to all gill leaflets, not just this one. Change throughout (see e.g. legends for Figs 6, 7, 9, 10)

Figure 5:
“Piece of dissection”: there has to be a more elegant way of saying this. Apparently similar usage to that in the legend for Figure 2 which I found confusing.
α, alpha-cell; β, beta-cell. On subsequent figures, the labels for these cells are α-c and β-c. This should be standardized.

Figure 6:
ics is not in the list of abbreviations.

Figure 7:
Myeloid bodies, and fibrogranular material, not “figures”

Figure 8:
Generally, the black labels are very difficult to make out on the dark background; suggest to use white.
8C. …connect the intercellular spaces….

Figure 10:
It seems far more common to use “vacuoles” rather than “vacuolae”, which I had never seen used before.
Again, some of the black labels are very difficult to make out on the dark background; suggest to use white.

Fig. 11:
11C: “mit” is very difficult to see.

Fig. 12:
“cav, caveolae”: this term appears nowhere else in the manuscript.

Fig. 13:
Myeloid bodies, not figures.

Table 1: Cell types and other features of the gill leaflet regions


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: FUNCTIONAL AND EVOLUTIONARY PERSPECTIVES ON GILL STRUCTURES OF AN OBLIGATE AIR-BREATHING, AQUATIC SNAIL
Review round: 1
Reviewer: 2

Output:
Basic reporting: This is an ambitious study on the (fine-) structure of the gill of an apple snail. It has been carefully carried out with an array of sophisticated methods including LM sectioning plus computerized 3D treatment, SEM and TEM. The results bear the potential for an important publication.
However, at the present stage the MS cannot be recommended for publication because of a number of shortcomings.
Experimental design: This morphological investigation does not contain experiments. The examinations have been carried out carefully with sophisticated methology.
Validity of the findings: The findings are a valuable contribution to the knowledge of gill structure of ampullariids and the extremely large group of caenogastrops in general, which surprisingly have never been investigated in this respect with fine structural techniques before.
Additional comments: I list the most important flaws in the following in form of recommendations for improvement for a future submission:

* Improve the structure descriptions in the results:
The images are of really sound quality but they should be referenced much more intensely in the results text. For example, when a skeletal rod is first mentioned (lines 180-181) there is referred only to Fig. 3, where it is hardly discernible, but the structure is much better visible in Fig. 13.

* Disentangle Results and discussion:
For example, do not give comparisons with literature references in the results, like in lines 260/261. Vice versa, the skeletal rod description in the discussion (lines 328-330) provides details not anywhere found in the results. So, these are details that must be placed in the results.

* Get back to literature
This is the most important problem of the MS; quite some important papers were overlooked/ignored.

These studies
Demian, E. S. (1965). The respiratory system and the mechanisms of respiration in Marisa cornuarietis (L.). Arkiv Für Zoologi, 17(8), 539–560.
Lutfy, R. G., & Demian, E. S. (1965). The histology of the respiratory organs of Marisa cornuarietis (L.). Arkiv För Zoologi, 18(5), 51–71.
on a closely related species must be consulted and quoted as (basis and) previous treatment of the topic.

For the intro (function, organization, homology) on Gastropod “lungs” these papers should be consulted:
Ruthensteiner, B. (1997). Homology of the pallial and pulmonary cavity of gastropods. In Journal of Molluscan Studies (Vol. 63, pp. 353–367).
Lindberg, D. R., & Ponder, W. F. (2001). The influence of classification on the evolutionary interpretation of structure - A re-evaluation of the evolution of the palliai cavity of gastropod molluscs. Organisms Diversity and Evolution, 1(4), 273–299.
These would, for example, show that there are (1) more cases of „lungs“ in gastropods (e.g. all terrestrial prosobranchs) and that these structures are not necessarily homologous (e.g. pumonate lung vs. ampullariid lung).

Also, the original literature source for the finding of “skeletal rods” in ampullariids should be searched for. In the MS there is only secondary literature quoted (e.g. Haszprunar, 1988 [source missing in there too]). This is important for the interpretation/(yet missing) discussion on the “skeletal rods”, which in fact – as clearly evident from the findings in the MS – represent muscles, which ARE NOT skeletal elements (see also Lutfy & Demian, 1965).

Here a hint on the discussion on the gill function: The authors acknowledge the idea (Andrews, 1965) of active water circulation be the gill ciliation but are puzzled by the absence of a respiratory epithelium in the gills. Why not consider a main function of water circulation by the gill and oxygen uptake elsewhere (wall of pallial cavity) as suggested by e.g. Haszprunar 1991 for many molluscs.
Haszprunar, G. (1992). The first molluscs - small animals. Bolletino Di Zoologia, 59(1), 1–16.

In total, the discussion needs to be largely rewritten. In general, I recommend undertaking comparisons with other molluscs (e.g. tons of studies on bivalves) rather than with distantly related taxa (crustaceans, vertebrates) to remain on a solid basis.

Some more remarks can be found in the annotated MS.

With best regards,

Bernhard Ruthensteiner


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: FUNCTIONAL AND EVOLUTIONARY PERSPECTIVES ON GILL STRUCTURES OF AN OBLIGATE AIR-BREATHING, AQUATIC SNAIL
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: I am very satisfied with the revisions to the manuscript. I find the discussion, in particular, to be very well done and much improved. The conclusions are clearly stated and concise, and succinctly summarize the main findings of this study while placing them in a broader context.
I have a few minor editorial suggestions for the authors to take into consideration:
Line 60: extends into the roof of the pallial cavity
Line 134: “Then, the profile of identified structures (objects) were drawn with the mouse using the Trace tool. These traces were used for the 3D visualisation of object”: This process is typically called “segmentation”, in this case manual segmentation. Is the terminology used herein software specific?
Line 148: “in which the main afferent and efferent vessels run”: reference unclear, could be “bases” or “roof”.
Line 150: “Based on their relative position with respect to the blood flow”: This isn’t untrue, but wouldn’t it be more practical (and more conventional) to define the afferent and efferent borders relative to their orientation in the mantle cavity, and hence to water flow?
Line 152: and therefore to the inner mantle? I would take the “outer mantle” to refer to the external body wall of the mantle roof.
Line 154: close to the base of the siphon.
Line 187: presumably the lung roof is the external wall of the mantle?
Line 191: located along the supraoesophageal portion
Line 194: has not been described for P. canaliculata
Line 309: have no abfrontal
Line 313: requires both a large and thin
Line 315: to permit adequate
Line 340: referred to the existence
Line 347: in molluscan gills
Line 373: facilitating O2 extraction from the water
Line 421: “(e.g., it lays gelatinous eggs underwater” – this is repeated twice.
Line 467: immune surveillance: this is a very sophisticated notion for the immune response of gastropods, and one that is distinctly anthropormorphic given the widespread use of this term in human studies.
Line 481: delete “against intruders”
Fig. 1B: “below the outer mantle”: what does this mean?
Supplementary Table 1: I would suggest to restrict the comparisons to those where the interpretation is different, or where confusion may occur due to the use of different terminology. Examples where only the abbreviations differ are not useful.


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: FUNCTIONAL AND EVOLUTIONARY PERSPECTIVES ON GILL STRUCTURES OF AN OBLIGATE AIR-BREATHING, AQUATIC SNAIL
Review round: 2
Reviewer: 2

Output:
Basic reporting: 2nd Review of the Manuscript
Functional and evolutionary perspectives on gill structures of an obligate air-breathing, aquatic snail
by C.Rodriguez, G.I. Prieto, I.A. Vega Corresp and A. Castro-Vazquez

The MS is improved in many ways.
I can recommend publication if the issues annotated in the PDF MS are considered/treated. I must insist in modifying the terminology (“rod”) and discussion of/on the gill muscle.

With best regards,

Bernhard Ruthensteiner
Experimental design: Is o.k.
Validity of the findings: Is o.k.
Additional comments: No additional comments


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

Input:
Title: THE USE OF BIOCHAR IN ANIMAL FEEDING
Review round: 1
Reviewer: 1

Output:
Basic reporting: This is an excellent paper with a lot of data.

It would be much improved if  further summary tables were included.  I would suggest a summary table for each of the animals and highlight whether the  biochars were activated with acids or urea/nitrates/wood vinegar lactic acids added.  Maybe also a separate table that summarises methane production
Experimental design: No Comment
Validity of the findings: No Comment
Additional comments: Congratulations.  This paper has been required for a long time


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

Input:
Title: THE USE OF BIOCHAR IN ANIMAL FEEDING
Review round: 1
Reviewer: 2

Output:
Basic reporting: Well written and comprehensive.  Appreciate authors’ effort to evaluate all trials involving biochar.  I am not aware of another review (at least in English) that covers this subject.
Experimental design: The search seemed fair and complete.  Research results that I am familiar with were all included.  A few suggestions on specific references are given below in the general comments.  Discussion was organized into logical sections which made reading enjoyable.
Validity of the findings: Written with apparent impartiality and an effort to discuss all aspects of biochar relative to animal feeding.  Several places where I intended to ask for further explanation or evidence, but it was then provided in the subsequent paragraph.  Appreciate all of the useful suggestions for further research to improve our understanding and potential uses of biochar
Additional comments: Line 29: it is not clear to me what is meant by ‘blood levels’
L62: large yield increases in what crops?
L79: was this based on anecdotal observations or research data?
L81: please define CEC
L429: I don’t believe the Erickson, 2011 article measured or addressed cow health.  This was a short term study and not intended to quantify health effects.
In the US one of FDA’s concerns is that biochar may mask disease symptoms preventing treatment of the underlying cause of disease.  This is prevalent throughout chapter 3 discussing potential adsorption of toxic substances, when it would be far better to instead limit or eradicate the feeding of these toxic substances.
L609: the link to botulism is suspected, as you write, but is still very much a theory.  It is also very specific to animals that have been fed high levels of glyphosate, which I believe is rare.  Recommend expanding discussion very briefly.
L642: Are these compounds common in animal feed at levels that cause a concern?  Important to also discuss that because these compounds can be bound by biochar, it is critical that we not feed ‘contaminated’ biochar to animals-whether this contamination comes through the production process or by absorption from the environment after production.
L755: I am concerned whether these were otherwise healthy cows or cows with clinical disease(s)?
L756: Leng references---I believe Lines 1778 and 1782 are redundant.  The reference I am most familiar with (Leng et al., 2012) studied biochar inclusion in diets of both male and female cattle, thus the word ‘cows’ is not appropriate. (Also in the table)
L792: would these nutrients have been excreted on the pasture even without the addition of biochar to the diet?  I am not following how biochar changes this process-does it make the nutrients more plant available, make the nutrients more stable once excreted, remove more nutrients from the digestive tract than would normally be excreted (and if so how would that impact animal performance), or some other mechanism?  My simple understanding would be that nutrients are made more stable—less N volatilized and less P leached into groundwater—is this correct?
L803-over what time frame were these measurements made?  Long term or short term study?
L804-809: what type of silage (corn, wheat, grass…)?
L810-814: this paragraph is not overly well developed.  Recommended removing or adding discussion to make it more clear what in vitro measurements were made.
L822: what about feed efficiency (gain:feed)?  With a greater CP intake I assume this is coming from greater overall intake of feed.
L861: recommend stating as ‘without the negative side effects to the environment that antibiotics can have’
L982: reword sentence-not clear as written
L1016-1017: should this be ‘NH3 emissions deceased by 31%’?
L1022: recommend rewording to demonstrate that considerable harm can be done if effluents are not handled correctly.  Nutrient rich effluent can be a resource if used correctly.
L1025: in the aquaculture studies was the biochar fed as part of a complete feed or offered separately from the feed?
L1055: recommend removing first sentence of this paragraph as the issue is much too complex to reduce to 1 sentence.  An entire review could (and has) been written on sources of GHG emissions and interactions with climate change-which seems outside the scope of this review.  The rest of the paragraph sufficiently indicates the importance of reducing CH4 emissions.
L1070: almost entirely through eructation (Murray, Bryant, and Leng, 1976; Br. J. Nutr. 36:1)
L1080: fat has been another feed source studied, biohydrogenation of fatty acids in the rumen utilizes H
L1099: daily production of methane from cattle is largely feed intake driven, larger animals consume more and thus have larger emission of methane.  The cattle used in the Leng study were quite small (80 to 100 kg) compared to typical animals in the US or Europe (>200 kg at weaning). 500 L of methane daily would be for larger (mature) animals
L1185: it is my understanding that the Erickson et al. (2011) article that you reference in lines 425-433 also used an acidified biochar product (acid-washed activated carbon product made from lignite coal)
L1261: it would be very useful to have a list of parameters that should be analyzed as a recommendation to future researchers.  As you point out previously, veterinarians and nutritionists are not steeped in biochar knowledge or vocabulary and a list of important aspects would be very helpful.
L1525: I attempted to look at the EBC, 2012 reference to see if the above information was contained there, but the link is not active/working—however the article was easy to find.  Is this the list of parameters that you would recommend?
L2053: I believe the Winders et al. 2018 article has been published as a complete article (Translational Animal Science, 2019 https://doi.org/10.1093/tas/txz027)
A very important point is the need for safe and more uniform biochar being produced if feeding it to livestock in order to prevent unintentional poisoning—appreciate how you address this in the final paragraphs.


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: LONG-TERM WARMING RESULTS IN SPECIES-SPECIFIC SHIFTS IN SEED MASS IN ALPINE COMMUNITIES
Review round: 1
Reviewer: 1

Output:
Basic reporting: Please see the attached file "revision_peerj_31956.pdf"
Experimental design: Please see the attached file "revision_peerj_31956.pdf"
Validity of the findings: Please see the attached file "revision_peerj_31956.pdf"
Additional comments: Please see the attached file "revision_peerj_31956.pdf"


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

Input:
Title: LONG-TERM WARMING RESULTS IN SPECIES-SPECIFIC SHIFTS IN SEED MASS IN ALPINE COMMUNITIES
Review round: 1
Reviewer: 2

Output:
Basic reporting: Dear Sir/Madam,
I read the Manuscript 31956v1, entitled "Long-term warming results in species-specific shifts in seed mass in alpine communities" submitted to PeerJ with great interest.
This long-term climate change study aimed to study the impact of warming and grazing on seed mass.
In general, I found the results interesting. As Im not a native English speaker (third language) I will not comment on the language, but I found it clear and easy to follow. However, I have some suggestions for improvement. My main concern is that it is unclear whether they studied effect on total seed or mean seed mass of the individual species (this need to be clarified throughout the text). Both are important. From my understanding, they are showing the results on mean seed mass, but as they counted the number of seeds, they could easily address both cases. In addition, the authors do not report the results on total seed number, this should be included as it was measured. In addition, I suggest that they analyze if the changes in abundance had any impact on total seed mass of the different species? Are species that are increasing also producing more seeds (in total number and total seed mass)? This could strongly influence vegetation dynamics under climate change.
I have also included some comments in the attached pdf.
Experimental design: no comments
Validity of the findings: see basic reporting and pdf
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: LONG-TERM WARMING RESULTS IN SPECIES-SPECIFIC SHIFTS IN SEED MASS IN ALPINE COMMUNITIES
Review round: 2
Reviewer: 1

Output:
Basic reporting: I am happy to see that the authors addressed all my comments.
Experimental design: No comments
Validity of the findings: No comments
Additional comments: I am happy to see that the authors addressed all my 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: LONG-TERM WARMING RESULTS IN SPECIES-SPECIFIC SHIFTS IN SEED MASS IN ALPINE COMMUNITIES
Review round: 2
Reviewer: 2

Output:
Basic reporting: Language is clear, although in a few cases too general. The details are in the attached PDF.
Literature is relevant (one cited item missing; see the PDF). The weakest point is placing the results in the ‘species abundance’ framework. This perspective is poorly explained and insufficiently grounded in the text. Moreover, none of the results regarding this is significant. I suggest to remove all parts referring to the effect of species abundance.
Figures and tables meet the standards except for fig 2 (A and B) in which sample sizes and error bars should be added. I suggest to move tables S4 and S5 from the  supplement to the main manuscript. The tables refer directly to the predicted and discussed relationships.
Hypothesis III is irrelevant or at least insufficiently justified in the context of theoretical reasons and expected results.
Experimental design: The goal is clearly identified Research question is well defined except for the reference to species abundance.
Methods are well described.
Validity of the findings: The sites are not replicated within grazing treatments (one site per treatment). It’s worth to realize the weakness, although the results are convincing. Statistics is adequate.
Discussion could be more developed. Limitations of the methods used should be discussed. The manuscript would be more complete if the authors gave some suggestions on why certain species increase seed size while others reduce. What do these groups have in common? Some speculations on the direct mechanism by which grazing may affect the response of seed size to warming would be welcome. The phrase ‘trade-off between mechanisms’ sounds like a catchall term and it calls for a wider explanation.
Additional comments: The result that the shift in seed size in response to increased temperature is species specific is very interesting. It conveys also an important general ecological message that we should not expect a universal pattern of relationships, consistent across species. An analogous result is reported by Olejniczak, Czarnoleski et al. PLoS ONE (2018, 13(6): e0199224) The authors might find this article helpful in placing their results in a broader context of intra-specific seed size variation.
I would recommend to publish the manuscript after major improvements that I suggested here and in the text.


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: LONG-TERM WARMING RESULTS IN SPECIES-SPECIFIC SHIFTS IN SEED MASS IN ALPINE COMMUNITIES
Review round: 3
Reviewer: 1

Output:
Basic reporting: The authors put a substantial effort into improving the manuscript. All reviewer’s comments have been taken into account and the authors responded in a satisfactory manner. The meaning of the manuscript is clear and deprived of distracting issues now. Fig. 2 has been corrected and meets the scientific standards. I hope it is legible after publishing.
Experimental design: The goal of the manuscript is clear and meaningful now. Methods are adequate and well described.
Validity of the findings: After including relevant corrections the manuscript constitutes a coherent report of reliable research. Methodological limitation is highlighted and discussed. Conclusions are clear and sufficiently grounded.
Additional comments: Making the manuscripts clearer disclosed few minor points to correct.
Lines 55-57 – species names in Italics;
Line 58 – ‘hirsuta’ not ‘hirsute’;
Line 83 – ‘as we all know’ – too informal, remove or replace with ‘To our knowledge, there is no…’
Line 155 – The ANOVA is important not only because the effect of warming is non-significant, but most of all, because the treatment × species INTERACTION IS significant. It proves that the effect of warming is species-specific and justifies analyses for separate species that follow. I suggest a stronger opening sentence, like: ‘We did not find a significant effect of warming on mean seed mass consistent across all species in both sites (Table 1), however, the significant treatment × species interaction indicates that the effect is species-specific.’
Table 1 – move ‘treatment×species’ one line up.


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: SHARED MYCORRHIZAE BUT DISTINCT COMMUNITIES OF OTHER ROOT-ASSOCIATED MICROBES ON CO-OCCURRING NATIVE AND INVASIVE MAPLES
Review round: 1
Reviewer: 1

Output:
Basic reporting: The study compares and contrasts patterns of root associated microbes of seedlings of two co-occurring maple trees (native and non-native comparison).  Though the study is descriptive (i.e. compares associational patterns), the authors spend a great deal of space attempting to interpret the likely functional importance of these patterns.  I think they should devote most of their lines to describing the patterns and not trying to interpret functional importance (e.g. how microbes might influence Norway maple's invasion).  For example, I noted that their conclusion statement includes much too much on functional interpretations which are beyond the scope of the study.  There are also concerns that the maple-to-maple variation might be typical of tree-to-tree variation among resident tree species.
Experimental design: A general description of the sampling design is provided (L185-91) but there is not a clear description of total number of collected seedlings per species.  Did you collect just one of maple spp. seedling specimen per transect per period (n=1)?

A potential limitation is that don’t we expect species to species variation in compositional associations?  In other words, is the variation between the native vs. non-native maples actually greater than simply the variation between sugar maple and an average other co-occurring resident woody species?  By having only one native and one non-native species, it is difficult to interpret whether the variation in associational similarity is typical for differences between any two species or something that is somehow special/atypical.
Validity of the findings: See reply to #1 & #2 above.
Additional comments: Abstract- Discussion points are highly speculative (e.g. L45-6).  Your study describes patterns of just two host species and includes no experimental manipulations that are necessary for comparing soil biota effects differences for the two hosts.

L88 I think the Klironomos citation is technically wrong.  John’s study did various things.  One was to compare abundant exotics to rare natives, which found differences in effects.  But those differences were confounded by plant abundance.  Anacker et al. (2014) re-analyzed John’s data (large community dataset) and found no differences between feedbacks of natives and exotics when controlling for abundance.  In other words, rare plants tended to have negative feedbacks and abundant plants tended to have neutral to positive feedbacks regardless of invasive status.

L105 It seems you might want to draw on the plant pathology literature here.  I don’t think these papers actually directly isolated and measured pathogenicity.

L110-3 Del- “Klironomos (2002) showed that invasive plant species in eastern Canada had neutral feedback to soil communities while a negative feedback was observed in native plant species suggesting that invading plant species may have escaped from their natural soil pathogens.”  See comment above for L88.

L110-6 I think you could have drawn on better literature for these comparisons (e.g. Reinhart and Callaway 2004, Reinhart et al. 2010, Halbritter et al. 2012).  Though you may want to limit content on the functional importance of microbes since this is not the focus of your study...

L122 A couple of papers have reported the opposite (Reinhart and Anacker 2014, Veresoglou and Rillig 2014).

L141 “Rinehart” check spelling

L151 try- and/or

L167 “deceased”

L170 “cooccurring”

L199 “microtubes tubes”

L252 “demuliplexed”

L323 “comminutes”

L353 This isn’t very compelling evidence for differences in enemy impacts between the two maples.

L396 “low in with”

L422-5 These sentences are on the functional importance of microbes but this was not directly measured in your study.  Shouldn’t your ms stay focused on differences in microbe associational patterns?

L428 Your comparison is extremely limited.  What I mean is that you compare one native maple to one non-native maple.  Might you see the same differences if you compared co-occurring red and sugar maple?  In other words, shouldn’t we assume that some associational variation can be detected even among closely related species?  Yet, this does not tell us about popn dynamics of the two species…  The comparisons are so limited (one native sp. vs. one non-native sp.) that it is difficult to gauge if such small compositional differences (e.g. ordinations with lots of overlap) truly tell us much about invasion processes.

L440 Highly speculative.

L443 try- “compared to native maples”

L459 detrimental to what (tree or microbes)?  Richness topic is extremely vague and not necessarily grounded in theory.  For example, I don’t think disease outbreaks are typically related to microbial richness as opposed to pathogen prevalence/presence.  While I recognize that a community may contain microbes that also protect the host from the pathogen, this is not necessarily a direct attribute/effect of richness.  One strong pathogen (or antipathogen) could have huge impacts that are largely independent of richness.

L470 try “Acer” or maple

L505 Conclusion statement is heavily focused on perceived importance of root association patterns which is beyond the scope of the study.  It seems it should summarize the main patterns and minimize interpreting these patterns.

Literature cited

Anacker, B. L., J. N. Klironomos, H. Maherali, K. O. Reinhart, and S. Y. Strauss. 2014. Phylogenetic conservatism in plant-soil feedback and its implications for plant abundance. Ecology Letters 17:1613-1621.
Halbritter, A. H., G. C. Carroll, S. Güsewell, and B. A. Roy. 2012. Testing assumptions of the enemy release hypothesis: generalist versus specialist enemies of the grass Brachypodium sylvaticum. Mycologia 104:34-44.
Reinhart, K. O., and B. L. Anacker. 2014. More closely related plants have more distinct mycorrhizal communities. AoB Plants 2014:doi: 10.1093/aobpla/plu1051.
Reinhart, K. O., and R. M. Callaway. 2004. Soil biota facilitate exotic Acer invasion in Europe and North America. Ecological Applications 14:1737-1745.
Reinhart, K. O., T. Tytgat, W. H. Van der Putten, and K. Clay. 2010. Virulence of soil-borne pathogens and invasion by Prunus serotina. New Phytologist 186:484-495.
Veresoglou, S. D., and M. C. Rillig. 2014. Do closely related plants host similar arbuscular mycorrhizal fungal communities? A meta-analysis. Plant and Soil 377:395-406.


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: SHARED MYCORRHIZAE BUT DISTINCT COMMUNITIES OF OTHER ROOT-ASSOCIATED MICROBES ON CO-OCCURRING NATIVE AND INVASIVE MAPLES
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript is well written and presented in an easily understandable language.  The introduction list several mechanisms and theories regarding the role of microorganisms for invasive plants, but this is not really the aim of the manuscript, as the aim only is to see if there are differences in the microbial communities of invasive and native plants.
Experimental design: I have serious concerns regarding the experimental design. The survey is conducted within one 40 x 60m plot. In that sense, it is pseudo replicated, as more sites should have been studied to conclude that the two plant species differ in their microbial communities. Moreover, the host species only explained 3.6% of the bacterial variation and 8.4% for the fungal communities, indicating that environmental variables could be more important. This could be different in another area.
Validity of the findings: I do not agree with the conclusion that the difference in communities are due to the invasiveness of the Norway maple. Such differences could probably also be found between two native plant species. It would be highly unlikely that the two species should have exactly the same communities given their difference in growth pattern. If the differences are due to the invasive characteristics of the Norway maple, this should also been seen at other plots
Additional comments: No additional comments