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: EXOSOMES AND FERROPTOSIS: ROLES IN TUMOUR REGULATION AND NEW CANCER THERAPIES
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript entitled “Exosomes and ferroptosis: roles in tumor regulation and new cancer therapies” by Yixin Shi et al. comprehensively reviewed the roles of ferroptosis and exosomes in tumor pathogenesis. They summarized the regulations of exosome on ferroptosis, including fenton reaction, ferroptosis defense system, and lipid peroxidation. They also discussed exosome and ferroptosis-based anti-tumor therapies, including improving exosome-inhibited ferroptosis, delivering ferroptosis inducers in encapsulated exosomes, and developing exosomal inhibitors and ferroptosis inducers. This review will be a useful resource for scientists in the research field. The literature references were sufficient and authors provided sound field background. The article is well structured. The figures were presented nicely.
Experimental design: Not applicable.
Validity of the findings: Not applicable.
Additional comments: The manuscript will benefit from moderate language editing, including grammars and scientific writing.


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: EXOSOMES AND FERROPTOSIS: ROLES IN TUMOUR REGULATION AND NEW CANCER THERAPIES
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors addressed all concerns.
Experimental design: -
Validity of the findings: -
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: NEED FOR SPLIT: INTEGRATIVE TAXONOMY REVEALS UNNOTICED DIVERSITY IN THE SUBAQUATIC SPECIES OF PSEUDOHYGROHYPNUM (PYLAISIACEAE, BRYOPHYTA)
Review round: 1
Reviewer: 1

Output:
Basic reporting: This is a very solid study with a substantial amount of new phylogenetic and morphological information that illustrates how integrative taxonomy can help us to understand better bryophyte diversity. It also opens the door to new and exciting questions such as the intriguing difference in "behavior" of different organismal groups such as vascular plants and animals opposed to bryophytes regarding Rapoport´s rule (only one p), for example.  The methods are explained with great detail and the outcomes include a distributional map and graphs that highlight ecological differences in the habitat of the revealed lineages. Further, a key and diagnosis, as well as high quality images, drawings and tables of morphology are provided that can be used by a wide range of bryologists.
Experimental design: The laboratory and analytical methods are generally sound and explained in great detail. The results of the study are convincing and sufficiently supported by the data.
Validity of the findings: The sampling intensity within individual populations, and proximate sites is sufficient to draw conclusions about the population structures.
Additional comments: Overall, this is a valuable and interesting contribution and I recommend its publication.


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

Input:
Title: NEED FOR SPLIT: INTEGRATIVE TAXONOMY REVEALS UNNOTICED DIVERSITY IN THE SUBAQUATIC SPECIES OF PSEUDOHYGROHYPNUM (PYLAISIACEAE, BRYOPHYTA)
Review round: 1
Reviewer: 2

Output:
Basic reporting: This manuscript presents a good example of the hidden diversity that is frequently found in organisms with a relatively simple morphology, and then especially in environments where variable habitat conditions are likely to influence the phenotype. In addition, it highlights our poor understanding of the diversity of Asia outside the tropics. The latter no doubt reflects difficulties in accessing material from these enormous regions of Asia, something that was even more problematic in earlier times.

The English should be checked by a native English speaker. One example is the use or absence of the definite article (the).

Discussion: In general, I find the Discussion too speculative and difficult to follow. It consists of more than eight manuscript pages without a single break. I have a few suggestions to improve the Discussion in a way that will simplify for the reader to understand. First, I suggest that subheadings are used to introduce a clear structure. Second, I would suggest a clearer focus on Pseudohygrohypnum itself and less on general Holarctic diversity and species evolution. Third, I would take out speculation that does not relate directly to Pseudohygrohypnum, and make sure that the remaining speculation is backed up by relevant references. If these changes are made, my impression is that the length of the Discussion could easily be reduced by 20%.

In the text, Fig. 20 is cited before Fig. 12. On the other hand, I cannot really see the relevance of including Fig. 20 at all, and I suggest deleting this figure.

Figure 1: PP and BP values <0.95 and <0.67, respectively, indicate no support. This should be considered in the tree.

Figure 2: From what are the Bootstrap values inferred?

Figure 3: The colour of the samples does not seem to correspond with the descriptions. Are these old herbarium specimens?

Figure 8: Indicate which entities differ statistically significantly from each other using letters (e.g., a, b, ab, ac, c, d, and so on).

Figure 9: Why are the colours different from those for the same entities in Fig. 7?

Figure 10: The temperature curve is somewhat difficult to see.
Experimental design: The methods used to elucidate which species occur among aquatic Pseudohygrohypnum are in general highly suitable for the purpose. Besides a few unclear points (see 4. Additional comments), regarding the chosen methodology I only have a few comments regarding the MaxEnt modelling. First, I was happy to see that the data were adjusted for sampling effort. Secondly, it was a bit unclear if (lines 242 ff.) the authors first used the equivalency test. If this was the case, it should be noted that the equivalency test tends to excessively reject the null hypothesis of niche identity and should therefore be avoided (Broennimann, et al. 2012. Measuring ecological niche overlap from occurrence and spatial environmental data. Global Ecology and Biogeography 21: 481-497; Peterson. 2011. Ecological niche conservatism: a time-structured review of evidence. Journal of Biogeography 38: 817-827). If this test was used, this needs to be discussed and its use motivated.
Validity of the findings: As I explain below, I believe the Discussion needs to be shorter and better focussed so that the reader could understand which of the results are both significant and supported. At the moment, much of the Discussion seems to want to cover much more than the study itself justifies. One thing that strikes me as a contradiction that may be due to the authors' wanting to cover too much here is that the authors conclude that aridification is a driver for diversification. How can this be for organisms that are subaquatic and obviously stay subaquatic during the diversification?

This manuscript requires a clearer focus and structure, where the reader can easily follow the reasoning of the authors.
Additional comments: Several geographical names will be unfamiliar to most readers. Please consider if you can add some additional geographical information for those names that are potentially unfamiliar. (e.g., Bashkiria, Sikhote-Alin, Khamar-Daban, Badzal)

Lines 117 ff.: Please write out the complete names of the used molecular markers the first time they appear in the text. Also, note that in several places where the abbreviated molecular markers should be partly in italics, they are entirely in normal fonts.

Lines 123-124: There seems to be something wrong in this sentence.

Lines 163-164: I do not understand this sentence.

Line 172: What is meant by ‘considered in details’?

Lines 220 ff.: Many H. eugyrium specimens collected before 1970 were checked by Jamieson, who recognized H. subeugyrium. Thus, did you check older American H. eugyrium samples against the identifications cited in Jamieson’s thesis? If not, this could possibly expand the certain geographical distribution of H. subeugyrium in N. America.

Line 254: Is ‘(R-project)’ a reference? Normally, a reference to R looks different.

Line 261 (and elsewhere): Please explain acronyms, such as LSID, the first time they are used.

Line 320: What does ‘reasonably’ imply?

Line 322: Is the habitat used as an ‘informal’ character?

Line 341 (and elsewhere): Write out climate parameters, like BiO1 (BiO2, BiO7), the first time they are used in the text and if it helps to follow the text when they are brought up again much later. It is otherwise difficult to follow the text.

Line 367: Should Fig. 8 be cited in the second sentence?

Line 404: Should ‘valuable’ be changed to ‘significant’?

Lines 616 ff.: I believe it is relevant to concentrate on truly Amphiatlantic patterns (i.e., surrounding the Atlantic) and not W Eurasiatic-W North American patterns; the latter is what, for example, Huttunen et al. (2008) discuss. At least the difference between W and E North America needs to be seriously discussed here if such patterns should be considered. Homalothecium sericeum s.str. shows an Amphiatlantic distribution, like some Sphagnum species and Antitrichia curtipendula s. str. (with A. gigantea in W North America). At least some of these taxa most likely got this kind of distributions in connection with the Pleistocene oscillations rather than because of much older events.

Line 672: What is the evidence for bottlenecks and genetic drift in Pseudhygrohypnum?

Line 784: ‘shorter’ than what? (No length is provided under the first alternative).

Line 807 (and elsewhere): In what sense ‘peculiar’? This term means different things to different readers, so please describe how it looks instead.

Lines 819 ff.: This concerns all species descriptions. I found several discrepancies between the first two descriptions (which I checked carefully), regarding included characters, how characters are presented, and the order in which they are presented. Descriptions at the same hierarchical level need to be similarly arranged and to include the same characters to be fully useful to the users.

Line 860: What does ‘more developed’ mean?

Line 902: Why include Affinity here? This is a topic for the Discussion.

Line 1006: ‘red’ rather than ‘pinkish’?

Line 1103: ‘Tysslinge’

Line 1535: Either the central strand is absent or present. Thus, ‘clearly’ is redundant.

References: Authors are cited in at least three different ways. Capitalisation of titles also differ, and Latin names of species are mostly in normal fonts, but in italics in the last one. The references need to be formatted correctly for PeerJ.

Table 1: The shape of the leaf cells should be indicated for all species.

Table 2. In the legend, please explain what I and D mean (which tests ).


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: NEED FOR SPLIT: INTEGRATIVE TAXONOMY REVEALS UNNOTICED DIVERSITY IN THE SUBAQUATIC SPECIES OF PSEUDOHYGROHYPNUM (PYLAISIACEAE, BRYOPHYTA)
Review round: 1
Reviewer: 3

Output:
Basic reporting: I believe that the paper entitled 'Need for split: integrative taxonomy reveals unnoticed diversity in subaquatic species of Pseudohygrohypnum' is of great interest for the scientific community. The authors have performed a detailed study on the morphology, genetical variation and ecological affinities (especially regarding climatic conditions) for a representation of specimens of Pseudohygrohypnum that covers all the distributional area of this genus. The authors conclude that there is a higher diversity within this genus than that currently recognized and accordingly propose several new species. This proposal is sustained by clear genetical and ecological evidences, and includes a good morphological description of the studied taxa, including the newly proposed species. In addition, the authors include complete and clear illustrations of the considered morphotaxa, as well as a useful identification key that will help the bryologist to adopt their proposal. In my opinion, this is a very clear and good example of use of the integrative taxonomical approach.

My main concerns regarding the manuscript include format and stile improvements, as well as English expression. The manuscript would profit from a revision of the English text, and the authors should carefully revise the guidelines for authors, especially regarding literature references and text subdivision.
Experimental design: This is an original research that perfectly covers the scope and interests of this journal. The research question is clearly exposed and addressed, and the experimental design is appropriate to answer the question. The investigation has been rigorously performed and meets the currently desired technical standards. Methods are in general clearly described.
Validity of the findings: As indicated above, I think that the results of this paper are of general interest for bryologist, since the authors clarify the real diversity of the studied genus. In addition, I think that this paper constitutes an example of good praxis in taxonomy that can help researchers from all disciplines to approach taxonomical studies in an integrative frame that combines morphological, genetical and biogeographical evidences. In this sense, I believe that the conclusions of this study are in general terms very robust.
Additional comments: As indicated above, I think that the authors should address some changes, especially regarding the English stile to improve and clarify the manuscript. In my opinion, the authors should consider to send the paper to an English native speaker.
I highlight here some of the points that could be addressed, although as I said I think that a general revision of English expression is needed all along the manuscript:
-	Summary: the use of the words genus and species is confusing: one genus cannot be an example of species. Do they refer to ‘species complex’?
-	Line 90: ‘the unusual distribution of P. subeugyrium s.l.’ Which is this distribution?
-	Lines 105-117: the selection of specimens is not clearly described. I would suggest to change the order: The study is based on the analysis of Pseudohygrohypnum eugyrium and P. subeugyrium, for which XX accession are included that cover all their known distributional area plus the detected morphological lineages. This study is performed within a phylogenetical frame that includes all the species currently included in this genus (a total of xx accessions from xx species), plus an outgroup that includes xxxx.
-	Line 147. How did the authors decide which model to use? Please, specify.
-	Line 157: indicate reference for FigTree.
-	Line 164: it is not clear to me what has happened with some of the markers that the authors had amplified, such as the mitochondrial. Please, especify.
-	Line 226: indicate references for QGIS and ArcGIS
-	Results section: I miss a description of the lenght and varibility of the matrices. This information would be needed to understand the validity of the results and the incongruences observed among the plastid and ITS data sets.
-	Regarding the analysis of the environmental variables, I believe that there exists some correlation among the considered variables, such as Bio12 with Bio17, Bio19 and Bio19. Have the authors taken this into account?
-	Line 365: the authors refer to the warmest quarter. Please, indicate that this refers to BiO18
-	The explanation about BiO19 is missing
-	Discussion, lines 463-490. I find all this part confusing. I think that it would help to divide the discussion of each new species in a different paragraph.
-	Line 481: the authors indicate that type material has not been yet revised. Ideally, this should be done in advance to the present publication. If this is not possible, the authors should indicate why this has not been done.
-	Line 724: diversity of “this group” of bryophytes
-	Line 769: check italics for P. subeugyrium
-	Lines: 967-968. The authors indicate that P. orientale was illustrated and described by Czernyadjeva as P. eugyrium, Do they mean that this species was included within the variability described for P. eugyrium? Please, clarify.
-	Lines 1405-1406. The same cmment as above for P. subarcticum under P. subeugyrium.
-	Figure 10: I think that the text would be sometimes easier to follow if the names of the species were given (eg P. orientale, etc.)
In addition, I have detected several mistakes in the references list that should be corrected:
-	Lines 83 and 93: Czernyadjeva (2002) is 2003 in the list
-	Line 94: Afonina, 2009 is 2019 in the list
-	Line 142: Lanfear et al., 2012 is 2017 in the list
-	Line 181: Tamur should be Tamura
-	Line 628: Confirm whether is Grímsson or Grimsson
-	Line 638: Does Denk, Grímssn & Zetter refer to Denk et al 2013? Please, confirm.
-	Line 1066: check author (or editor) of Flora of North America and cite accordingly
-	Line 1068: check Czernyadjeva et al 2003 (only Czernyadjeva in the list)
-	Line 1905: Smith 1978, check if this reference is cited and in this case complete in the list
Finally, the authors should check the instructions for authors regarding the following points:
-	Use of italics for the phylogenetic markers used (check and correct all along the manuscript).
-	Use of ‘Appendix’ or ‘Supplementary material’
-	Use of ‘et al’ or list all of three authors (e.g. line 551 Gama et al, Moroni et al; line 659 Liu et al; line 743 Pärtel et al)
-	Separation in paragraphs. The format is very compact and this is sometimes not useful for the readers. For example, I would suggest to separate the identification key from the descriptions, and one description from another
-	References list: the authors should check the format required by the journal. Please, pay especial attention to: the position of the year of publication (sometimes indicated twice, such as Afonina 2019), the use of italics for the scientific names (generally missing in all the list), the position of the accessory letters a and b in the case of El-Gabbas & Dormann 2018, the use of capital letters (see for eg. Hall 1999 or Katoh & Standley 2013), avoid the names of the authors (use only the initial, for eg. in Huttunen et al 2012 or Ignatov et al 2020), check the use of coma, dot or parenthesis for the year.


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: NEED FOR SPLIT: INTEGRATIVE TAXONOMY REVEALS UNNOTICED DIVERSITY IN THE SUBAQUATIC SPECIES OF PSEUDOHYGROHYPNUM (PYLAISIACEAE, BRYOPHYTA)
Review round: 2
Reviewer: 1

Output:
Basic reporting: I was very happy to see this manuscript again, and that the authors have followed my suggestions to make the Discussion text much clearer. It is now easy to follow the Discussion and to understand their reasoning. I found just a few minor issues that require attention.

Line 130: ‘scored using the’

Line 132: ‘homoplastic’

Line 148’ frequency of one’

Line 156: ‘on the extended’

Line 180: ‘contradict the’

Lines 286-306, and Fig. 1: PP < 0.95, and BP < 0.67 are insignificant. Even if was claimed in the rebuttal that the cutting levels were changed, this is not so. I do not understand why the authors put arbitrary cut levels instead of using the relevant ones. This will affect some of the branches in the trees, and may affect the text in the Results / Discussion concerning this. The network really clarifies the situation nicely (a tree is actually a special case of a network, so this is not surprising).

Line 333: Change ‘scheme’ to ‘hypothesis’

Line 379: Delete the last ‘one’

Line 479: ‘from the ASAP’

Line 522: ‘Presumably, the’

Lines 567ff.: Here, it may be worth pointing out that the found niche differentiation likely reflects a primary physiological differentiation (which is as significant as a morphological differentiation).

Line 590: Change ‘represent a’ to ‘are’

Line 653: Change ‘amphipacific’ to ‘amphiatlantic’

Lines 688, 701, 703: Avoid teleological expressions (managed to, succeeded in)

Line 730: ‘our climate model’

Line 736: Delete the comma after ‘both’

Figure 2: From what are the Bootstrap values inferred? I cannot see that any information on how they were generated has been added.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: No additional comments


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

Input:
Title: RADAR REMOTE SENSING-BASED INVERSION MODEL OF SOIL SALT CONTENT AT DIFFERENT DEPTHS UNDER VEGETATION
Review round: 1
Reviewer: 1

Output:
Basic reporting: In general, the manuscript "Radar remote sensing-based inversion model of soil salt content at different depths under vegetation” reports on the prediction of soil salt content (SSC) at different depths under vegetation using four different models (PLSR, QR, SVM and ELM) based on radar remote sensing. The manuscript reports on valuable experiment and a significant number of output results.
Experimental design: Experimental design is good.
Validity of the findings: the novelty of presented research is not clear. Please make it clearer. Why you chose all these four models to predict SSC? The comparison of four models is your novelty?
The discussion part is a little bit short. Please check if you could add more details such as a comparison of four models.
A more organized “Conclusions” is needed with a description of your study and the main conclusions, which is also a reflection of your novelty.
Additional comments: Line26-27: “support vector machine regression (SVM)” is supposed to be SVMR for the abbreviation.
Line59-60: "Nurmemet et al. (Nurmemet et al., 2015; Isak & Nurmemet, 2018; Duan et al., 2018)”, did you mean a group of references or just one paper? Please double check.
Line 110: “140mm” and “2000mm” need blank space between number and unit.
Line 133: Please point out the units of distilled water and the processed soil with ratio 5:1.
Line 200: what kind of cross validation did you use for the PLSR?
Line 265-266: the sentence needs to revise “The larger the R2 and the smaller the RMSE, the better the model effect.”
Line 277: after statistical analysis?
Figure 2 needs more annotation.


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: RADAR REMOTE SENSING-BASED INVERSION MODEL OF SOIL SALT CONTENT AT DIFFERENT DEPTHS UNDER VEGETATION
Review round: 1
Reviewer: 2

Output:
Basic reporting: 1. The grammar and logic of this article need to be revised with a native speaker.
2. Please provide a bit more big-picture motivation of how soil salinity analyses threaten society or agriculture or ecology and how they have evolved over the past decade. The paper is missing a "big-picture" introduction with some references in my opinion.
3. The scientific issues in the abstract seem overreaching. Agriculture and soil salinization has been studied in various studies based on remote sensing technologies.
4. The introduction section missing two important elements i.e., (i) Hypothesis explanation, and (ii) Novelty statement. These two important mentionings will guide the reader towards the heart of the article.
Experimental design: 1. As mentioned by the authors of the study, the microwave remote sensing data inversion depths are topsoil (0-20), this is because the microwave signal penetration depth is limited, and additionally, the vegetation further reduces the signal. Therefore, it is meaningless to estimate deep soil properties using only the backscattering coefficient and its derived indices.
2. Why have these machine learning algorithms been selected? Even though machine learning models dependent variables using a large number of independent variables, the mechanisms of geographic or remote sensing must exist.
3. Lines 104-115: What are the soil types as per USDA soil taxonomy in this region? And vegetation type？
Validity of the findings: 1. Line 174: Why choose the best variables by linear methods and then use machine learning to estimate soil salinity? Two types of methods do not match.
2. Why is the validation set of some models higher than the modeling set? What are the reasons for this phenomenon? What effect does it have on the results?
Additional comments: 1. Vegetation affects the backscattering coefficient of soil and is usually removed through optical data. Did the independent variable mention reflect soil or vegetation? It was an indirect estimate? The author should clarify; else, the article's foundation would be lost.
2. In the comparison of models, it is not enough to compare the prediction accuracy, but also to comprehensively evaluate the stability of the model, the degree of dispersion of the results, etc.
3. Lines 48-49: What is the most effective basis? This progression does not sort out well the application of remote sensing technology to soil salinity.
4. The discussions section is terrible. Discussions are not critical writing, do not compare well with prior research, do not explain well the reasons for the results in the paper


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: RADAR REMOTE SENSING-BASED INVERSION MODEL OF SOIL SALT CONTENT AT DIFFERENT DEPTHS UNDER VEGETATION
Review round: 2
Reviewer: 1

Output:
Basic reporting: I have read the revised manuscript and the authors' responses to my comments. However, there are still some questions need to be answered.
1. For the discussion section, it has been improved comparing with the previous version. However, the comparison with prior research needs to be specific. Most importantly, there is no model performance comparison with other studies, like R2 and RMSE.
For examples, “Compared with linear model (PLSR), nonlinear model (i.e., SVM, QR and ELM) can achieve better performance in prediction accuracy and stability (Taghadosi & Hasanlou, 2021; Wang et al., 2021).”  (lines 459-461), it this your study conclusion or the references’ conclusions? Why don’t you give a detail descriptions about the references’ study since you cited?
Again, “This discovery has also been demonstrated by Tan (2020) and Hu (2006).”(line 477-478), what exactly they found? What are the conditions they had, e.g. soil type?
 2. The reference format needs to be checked again, different font and italic are found.
Experimental design: None
Validity of the findings: None
Additional comments: 1. Line 63: any abbreviation indicated for (HH2 + HV2)/(HH2-HV2)?
2. Line 90: double blank space before “Therefore”.
3. Line 194-195: actually I mean how many fold you used and how many samples you had for each fold.


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: RADAR REMOTE SENSING-BASED INVERSION MODEL OF SOIL SALT CONTENT AT DIFFERENT DEPTHS UNDER VEGETATION
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The authors have made changes to the issues raised, and this version is worthy of recognition. However, there are still some problems:
1. Add a literature review of machine learning to the introduction to explain the rationale for choosing these methods.
2. What are the soil types as per USDA soil taxonomy in this region? This question was not answered. USDA soil taxonomy indicates soil order, such as Aridosols, Molisols, and etc.
3. Discussion should highlight the significance of the study.


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: ANALYSIS OF PROFESSIONAL SOCCER PLAYERS IN COMPETITIVE MATCH PLAY BASED ON SUBMAXIMUM INTENSITY PERIODS
Review round: 1
Reviewer: 1

Output:
Basic reporting: English is not my mother tongue. I cannot judge the correctness of the English language used. On the other hand, the scientific language used by the authors is correct in my opinion.

The introduction to the subject is, in my opinion, on a good level. As in any article, you can indicate corrections resulting from your own subjective view of the problem under study. In this aspect, I want to highlight three aspects:
1) there is an extensive statistical method in the article and the purpose of the article is very simple. You can consider introducing brief information about the applied statistical method (hierarchical cluster analysis) to the target. It will inform you from the very beginning that these are not simple comparative analyzes.
2) due to the variety and number of the described parameters and factors, in the introduction, I lack information about the tests performed in the context of all variables used in the work (e.g. high metabolic load distance, etc.).
3) In my opinion, the discussion is conducted with little use of a variety of references. Duplicate results are a large part of the discussion. The results are discussed with little problem depth. (e.g. there is no discussion of mean metabolic power and others as an important parameter). In addition, Line 315-336 - this is a partial description of the method used, consider moving some of this information to the Methods section.

The structure of the article is correct. However, I believe that the description of the results could be shorter and some of the calculations, e.g. post-hoc, could be shown in the table.
Tables do not include units next to parameters (eg table 2, eg Duration Sprint in what units? Etc.). All abbreviations used in the tables should be clearly explained (e.g. table 3 - factor - st.?).
Experimental design: In my opinion, the methods should be supplemented with the definitions of the parameters used in the article. I understand that they are collected using specific hardware, but the reader needs to know what it means and how it is calculated, e.g. metabolic load distance and other indicators.

Moreover, in the current research describing positions on the pitch, it is no longer possible to show only 4 positions on the pitch. There is a big difference in the actions of different midfielders and they should be distinguished as different positions (side, defensive, offensive, etc.).
Validity of the findings: The article describes a topic often found in the literature, especially through the increasingly available modern external load testing tools. However, what's new is that the results are shown in the context of the SubMIPs zone (threshold of 85% MIP). Unfortunately, there is no detailed calculation of the threshold values and individual thresholds.
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: ANALYSIS OF PROFESSIONAL SOCCER PLAYERS IN COMPETITIVE MATCH PLAY BASED ON SUBMAXIMUM INTENSITY PERIODS
Review round: 1
Reviewer: 2

Output:
Basic reporting: Clear and unambiguous, professional English used throughout.
Yes

Literature references, sufficient field background/context provided.
Yes

Professional article structure, figures, tables. Raw data shared.
Yes

Self-contained with relevant results to hypotheses.
Yes
Experimental design: Original primary research within Aims and Scope of the journal.
Manuscript in the field of performance analysis, sports science

Research question well defined, relevant & meaningful. It is stated how research fills an identified knowledge gap.

Yes
Rigorous investigation performed to a high technical & ethical standard.
Yes

Methods described with sufficient detail & information to replicate.
Rather yes
Validity of the findings: Impact and novelty not assessed. Meaningful replication encouraged where rationale & benefit to literature is clearly stated.
The research brings interesting information to the literature

All underlying data have been provided; they are robust, statistically sound, & controlled.
Rather yes

Conclusions are well stated, linked to original research question & limited to supporting results.
Yes
Additional comments: The topic is not very revealing, but it is in line with the current trends in the analysis of performance in football
In the abstract, in the method section, line 37, there is no sentence informing you which statistical analysis you used to present the results. Please add
Introduction is well-written and justified
Methods. Line 91, Azerbaijan Premier League is the highest level of competition in this country?
Line 99, One of the biggest my concerns regarding division of playing positions. Midfielders have different task when playing in central or in wide side of the pitch. this can distort the data analysis
In this type of analysis, the most common division is into five playing positions, not four
Line 102-106 fine
Results. Line 159-161 First sentence of the results is misleading, I can not understand second part. Please clarify
Discussion. Line 241-248 I appreciate the complexity of the analysis, although the main findings are not very new, in part they were to be expected
Line 247 “attractor” this word is misleading, please change, the same situation with this word in last line of abstract
Line 252-261 Authors should not at all compare results across disciplines because it makes no sense. I suggest to look for other references or write that in football such an analysis has not yet been carried out and these results have no reference in the literature
Line 263-268 I expect this result to be better / more thoroughly discussed
Line 270-282 it seems reasonable to refer here to various tactical systems, e.g. that in the system in which the examined team played 5 playing positions were not specified
Line 329-345 I would suggest authors to combine loose thoughts into broader paragraphs to eliminate short paragraphs that consist of one sentence. This rule should not normally be used (e.g. see the last two sentences / paragraphs in the discussion)
Conclusion and practical application. Legible and a bit short as presented in the main findings in the manuscript


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

Input:
Title: ANALYSIS OF PROFESSIONAL SOCCER PLAYERS IN COMPETITIVE MATCH PLAY BASED ON SUBMAXIMUM INTENSITY PERIODS
Review round: 2
Reviewer: 1

Output:
Basic reporting: Ok
Experimental design: Ok
Validity of the findings: Ok
Additional comments: Ok


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: EFFECT OF STEADY-STATE AEROBIC EXERCISE INTENSITY AND DURATION ON THE RELATIONSHIP BETWEEN RESERVES OF HEART RATE AND OXYGEN UPTAKE
Review round: 1
Reviewer: 1

Output:
Basic reporting: The author conducted a well structured and organized study with professional language used to report findings. The tables and figures are appropriate for the information being discussed. The hypothesis and research aims were clearly stated. The only suggested reference additions to further highlight the importance of what the authors accomplished relates to the following:

Please consider changing the ACSM guidelines reference from the 10th ed to the 11th ed in order to highlight the current recommendations (does not impact the writing or interpretations of the study).

Line 80-83: This information is critical insight into the exploration of HRR and VO2R relationship. It is suggestion that more background be provided identifying the use of VO2R in previous research and within the ACSM guidelines that encourages the use of VO2max – 3.5 ml/kg/min for VO2R calculation. The resting VO2 value of 3.5 is acting as a baseline/zeroed value which contradicts this important line in the introduction and reemphasizes the importance of your research methodology to collect each participant’s resting HR and VO2. I would suggest including the following article and have not seen much, if anything in the literature addressing this concern with the use of VO2R and metabolic equivalents – Byrne et al (2005) Metabolic equivalent: one size does not fit all J Appl Physiol 99: 1112-1119
Experimental design: The methods and aims of the study were clearly identified. However, there are some suggestions/considerations to better understand the methodology:

Line 148: Was all aerobic activity included or were participants recruited based on running experience with treadmill use?

Line 150: Please clarify what was considered to be ‘2 months of treadmill experience.’

Line 171-172: Please clarify the measurement of maximal HR and VO2 – was this an assessment of true maximal values or the highest achieved submaximal values during steady state? Was a maximal test performed immediately following steady state exercise?

Line 245-267: Please elaborate and clarify on the testing methodology used, especially with the addition of the verification protocol, which was great to see. There appears to be discrepancies in identification in maximal values compared to what has been previously established in the literature. For example, the following statement “For the pre-SSE session, the highest values of V̇O2 and HR recorded during either GXT or VT were assumed to be maximal values if at least one V̇O2 plateau was identified or if the highest HRs recorded during the GXT and VT were within 4 bpm (Midgley et al. 2009)” suggests that a plateau noted in either the GXT or VT would identify a VO2max – How was it handled if a plateau was noted in the GXT, but maximal VO2 values in the VT were higher? What was the error measurement between GXT and VT trial that was acceptable? If plateaus in VO2 in the GXT and VT bouts, how closely did these values need to be to each other in order to confirm a maximal bout was achieved?

Line 255-258: How was the GXT performed after the SSE session? Did participants cool down, recover and then perform the GXT or were they taken from their steady state level to maximal with incremental speed increases? This information is not clear within the methods section and more information/insight is needed in order to truly grasp the significance of the findings.

Line 259-260: If a participant did not achieve ‘max’ following the SSE trial within the GXT and needed to repeat, was the entire SSE session repeated or just the GXT?

Line 292-294: Please provide rationale for the use of VO2R with the ACSM metabolic calculation for running but then used HRR for the exercise protocols. Wouldn’t the use of the VO2R from a standardized, one-size fit all equation encourage the use of different relative warm-up intensities which could impact the results of the exercise trial?

Line 312: RMSE was a great addition to the study!

Methods (general): Were ratings of perceived exertion not gathered? If they were, please report them since they would provide considerable insight into psychological considerations that may influence HR response rather than purely being a physiological response.

Methods (general): The authors are commended for obtaining resting VO2 values to use in assessing VO2R and for also completing GXTs following each SSE trial. However, if it is noted that changes in the exercise session may influence maximal values, would it not also then be important to identify that resting VO2 values on the days of the SSE trails may also vary which would alter the percentages of VO2R? This is something that should be noted, at the very least, as a limitation.
Validity of the findings: Please include the absolute values of speed during the testing to compliment the decrease in percentages.

With the inclusion criteria being 30 min of moderate, structured PA on at least 3 days a week, couldn’t this also impact the result found? If participants were not aerobically active beyond this 30 min for 3 days a week or did not maintain aerobic intensities above mod (80% HRR/VO2R being categorized toward the higher end of vigorous activity, wouldn’t this decrease is relationship at higher intensities for a longer duration be expected based on running economy and mechanical movement considerations?
Line 385-389: Possibly. However, with reductions in running speed averages of nearly 4 kmh, there will be considerable changes in mechanical differences due to speed alone which would impact tissue level O2 demand, especially in the upper extremity which would drive VO2 down rather than maintain. The findings of the current study may be due to familiarity of intensity level based on pre-trial training alone.

Perhaps the largest consideration that has not been addressed in the present study discussion is the evaluation that HRR and VO2R may not be a 1:1 relationship with prolonged steady state… but this assumption has been made on group average data rather than individually driven interpretations. It has been well established that physiological thresholds (VT/VT1, VT2/RCP, lactate) occur at a wide range of HR intensity values. Therefore, the longer duration 1:1 HRR to VO2R considerations may be an inappropriate group average assessment and may underpin the reason for needing to decrease treadmill speeds throughout the higher intensity, longer duration sse trial. The above is an incredibly important consideration that should be noted within the paper as the field is (slowly) identifying fundamental issues and concerns with standardize group approaches to the percentages of HRmax, HRR, VO2max, and VO2R to be used at the individual level. It should also be noted that measurement error of the metabolic system used was not considered, as well. No metabolic analyzer has a perfect measure, and the error may impact the results, especially due to the low population size. Not a flaw of the study, but a suggestion to consider mentioning.

The authors are strongly encouraged to include a limitations section to help strengthen their paper by acknowledging some of the larger considerations and to highlight changes/alterations in future research methodologies related to the topic.

The authors are urged to use caution with the language within the practical applications section. Specifically, “When HR is used, the exercise V̇O2, and thus intensity and caloric expenditure, could be lower than expected; this implies a less effective exercise stimulus.” Furthermore, including the usage of bike wattage in the subsequent sentence is an inappropriate application of the findings since HR and VO2 responses during leg cycling do not align with those seen during running/walking on a treadmill.
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: EFFECT OF STEADY-STATE AEROBIC EXERCISE INTENSITY AND DURATION ON THE RELATIONSHIP BETWEEN RESERVES OF HEART RATE AND OXYGEN UPTAKE
Review round: 1
Reviewer: 2

Output:
Basic reporting: Well written - no English concerns.
No grammar or other punctuation edits needed.
Experimental design: Robust design with gold standard and well-defined procedures.
Validity of the findings: Data is robust and authors are to be commended.
My main comments are three-fold:
1. What is the expected biologically variability in the measures obtained (i.e., %HRR and %VO2R)? In our experience, it can at least be 5%. As such, how does this potentially impact the interpretation of your findings?
2. What are the individual responses? Prescribing exercise is done for the individual and not group. Were some individuals more precise on the %HRR/%VO2R relationship, especially with longer durations? And vice versa, was there even greater disparity between the relationship in other participants?
3. Near the end of the discussion, the authors make the following comment: "unexpected modification in the HRR-V̇O2 relationships could have possible drawbacks, which can range from a less effective exercise stimulus to a higher risk of exercise-related events." My question is do we really have evidence to show that the difference in the HRR/VO2R relationship will actually contributed to these scenarios? I would argue no and unless the authors can demonstrate otherwise, I think this statement is too much conjecture.
Additional comments: Overall the authors are to be commended. The study is well-designed and executed. Extremely well-written. I respectfully ask the authors to consider my remarks in the previous section.


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

Input:
Title: INFLAMMATORY BOWEL DISEASE BIOMARKERS OF HUMAN GUT MICROBIOTA SELECTED VIA DIFFERENT FEATURE SELECTION METHODS
Review round: 1
Reviewer: 1

Output:
Basic reporting: • The writing is clear with a few small mistakes in places
• The literature review is missing some important papers in the area (details provided in following sections)
• Strong claims about elements of the microbiome causing disease are made (lines 17 – 18, 39 -42). For the majority of cases only associations have been found, direct causation is rarely confirmed. Strong claims about causation should have strong evidence provided.
• The aetiology of IBD is complex and many other factors are associated with IBD, not just the gut microbiome (lines 19 – 20). This should be made clearer.
• Figure 5 should have y axis labels and confidence intervals to allow proper comparison of models
• Figure 6 C is impossible to read and understand, it’s too small
• Figure 7 is missing x and y axis labels
• Figure 8 is missing y axis labels, so I don’t know what it’s measuring (relative abundance?). There are too many groups and it’s difficult to tell species apart. I recommend visualising only the 10 or 12 species most abundant and combining the others into an “Other” category
• Figure 9 A) B) is very difficult to understand from a PDF (it’s 3D). I recommend including an interactive 3D plot in the supplemental instead.
Experimental design: • The methods section doesn’t describe the process of processing raw MetaHit sequence data to create the metagenomics dataset. Were the sequence data quality checked with FastQC and multiQC? Were non-biological sequences (e.g. adapters) removed with trimmomatic? Metaphlan2 uses a read mapping approach to match against reference genomes, so removing non-biological sequences is important. It can’t be assumed that these steps were done by the MetaHit team that uploaded their data.
• The methods section doesn’t describe preprocessing done to the metagenomic data. Different samples have a different number of bacterial reads, how was this normalised? See doi.org/10.1371/journal.pcbi.1003531 and doi.org/10.1186/s40168-017-0237-y
• What criteria is used to define if a patient has inflammatory bowel disease? (E.g. the Montreal classification) Were the patients treated with antibiotics? This will change the structure of the microbiome significantly. Was IBD in an active or remissive state when the samples were taken? I think a more thorough description of patient demographics is required – perhaps a table?
• Unfortunately, from Figure 2 it appears the feature selection process is fatally flawed, causing all estimates of predictive power to be overstated. Feature selection is done on the entire metagenomics dataset, and models are trained on the reduced dataset using cross-validation. Feature selection should instead be included inside the resampling method to estimate the stability of the feature subset and avoid overfitting. Ambroise and McLachlan (2002; doi.org/10.1073/pnas.102102699) and Svetnik et al (2004; doi.org/10.1007/978-3-540-25966-4_33) showed that improper use of resampling to measure performance will result in models that perform poorly on new samples.
• Spearman rank correlation should not be used on metagenomic data because it is compositional (the sum of all bacterial proportions in each sample is constrained to 1), see 10.1371/journal.pcbi.1002687, doi.org/10.3389/fmicb.2017.02224
• Comparing model performance should involve analysing confidence intervals generated from cross-validation resamples and using a statistical approach
• I’m not sure what the unsupervised approach contributes significantly to the paper. From Figure 10 there are two small clusters but there are many confounders that could contribute to this (e.g. antibiotic use or other treatments). To understand the relationship between samples and species other methods could be used such as interpretable machine learning (e.g. SHAP arXiv:1705.07874)
Validity of the findings: • The flawed feature selection procedure used means all prediction estimates are invalid. They should be recalculated with the correct feature selection procedure, incorporating feature selection inside of the cross-validation resampling process
• Given the performance claims made about the models (AUROC approaching 1 in Figure 4) I think it is very important that the trained models are externally validated. There are many large metagenomic datasets available such as Gevers et al. 2014 which contains more than 1000 samples (10.1016/j.chom.2014.02.005). Previous machine learning work predicting IBD has used external validation to demonstrate the ability of trained models to generalise (10.1109/TCBB.2018.2831212). Qiita (https://qiita.ucsd.edu) is an excellent platform to browse and discover new microbiome datasets

When using external validation data it's important that the data can be harmonised across data sources (e.g. species or genus names match, and the same reference database is used)
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: INFLAMMATORY BOWEL DISEASE BIOMARKERS OF HUMAN GUT MICROBIOTA SELECTED VIA DIFFERENT FEATURE SELECTION METHODS
Review round: 1
Reviewer: 2

Output:
Basic reporting: This article is generally well written, uses a professional academic structure, and is relatively self-contained. The argument presented is clear and uses professional English.

Further supporting information is required for the claims made in lines 38-40.

There are some minor spelling and grammatical errors including:
Line 60: referencing style should be kept consistent throughout.
Line 297: Euclidean (E not e).
Line 308: Remove ?
Lines 325-326: Grammatical error "applying a careful feature selection".  Please rephrase.

There are some difficulties with the following Figures:
Figure 4: Please use . instead of , for the decimal point. Please also add (%) as units to the header of the Accuracy columns. Increasing font size would be desirable here even if it means increasing the table sizes and significantly reducing the white space between them.
Figure 5: Colours are difficult to differentiate for those that are color blind. Please consider differentiating the bars with different fill textures/patterns. Please also add a label for the y-axis on each subfigure and please include confidence intervals.
Figure 6: The text in all three sub-figures is illegible. Note the x-axis label for (a) has been cut off by sub-figure (c). The numeric values in (c) are completely illegible and should be removed to aid clarity.
Figure 7: Please label y-axis. lease increase font size on x-axis. Please adjust the y-axis so that the numeric values are consistent on both.
Figure 8: Please label y-axis and increase the font size of the text on the right-hand side.
Figure 9: Please adjust the yellow coloring as it is difficult to see with the white background.
Figure 10: Perhaps this is best presented as a whole page image in landscape format?
Experimental design: The aims of the paper clearly fall in the remit of the journal.

The research question is well defined, relevant and meaningful. The authors have described how they feel the results address the deficiencies of the knowledge base.

There are however some concerns over the scientific methodology.
- The feature selection approach in its current form is at risk of leading to overfitting of the classification models. The feature selection process should be carried out as part of the resampling method. The methodology should be revised and the experimentation repeated once this change has been implemented.
- More information is required on the pre-processing of the raw MetaHit sequence data. Are there any further preprocessing steps conducted to refine the metagenomic data?
- How were patients classed as having (or not having IBD)? How homogenous was the IBD patient cohort (and indeed the control cohort)?

- Line 218: How were the parameters optimized?  In general, please provide parameters and where appropriate the names of functions used from toolboxes.
- Line 220: Please confirm if the train/test split was consistent across all models.
- Line 222: The intersection of features would also be interesting to examine as these are the features that consistently came up - are they the most useful?

Some minor points:
Lines 126-127: please include a citation to justify the claim "commonly used as a feature".
Lines 128-139: Please link back to the three types of features discussed in lines 122-124. Lines 122-124 are introduced but appear to be unused in the later discussion.

Please clearly explain what the 3302 columns in Figure 1 represent. I would also recommend in Figure 1 moving the "no of sick/healthy samples" from the right-hand side to the left-hand side so that we can see the 328 samples are split into 148 and 234. Currently, it looks like the text is associated with the 1st and 2nd row of data explicitly.
Validity of the findings: The primary concern here follows from the risk of overfitting caused by the methodological approach (as described in my comment above). The results should be revisited once the methodology is revised. There are some general comments which would be beneficial for the authors to consider in their revision.

Whilst the sample size is reasonable for this study, any findings from this work (with the revised methodology) would be significantly strengthened by validating the findings on a larger open-sourced dataset. Whilst this is substantial work it will result in more robust findings, support model comparisons in the future, and ease reproducibility, which will hopefully see the author's efforts rewarded through increased community adoption of their approach.

Line 250-251: Please comment on the biological relevance of the features contained in the intersection of all feature selection methods. Is XGBoost good because it identifies particularly interesting species or are the really interesting species contained within the 10 species of the intersection?
Line 270: Please elaborate on how the values 12 and 7 were identified. They are not immediately obvious from Figure 7.
Lines 263-281: Please comment on the role the isolated species are known to have in biology similar to the discussion presented in lines 258-262.
Line 298: Why cut off at the top 24 species? This seems arbitrary without further explanation.

Discussion Section:
Lines 325-326: The description of "careful feature selection" is perhaps misleading. The authors apply a number of feature selection methods, but what is the take-home message for the reader? Which feature selection method should be used? Is it important to select a particular feature selection method depending on the classification model to be used? Should a number of feature selection methods be used and the final features are determined by the union/intersection?
Lines 330 and 334: "unprecedented performance/results" - This is a bold statement and should be revised. The conclusion "feature selection can improve classification results" is a well-established finding so there is precedence that your results should be higher than previous studies. The performance increase is certainly good but I don't think unprecedented is the appropriate description.
Line 339: "leads to a framework" - perhaps the rephrase along the lines of  "provides the foundation for a framework to be developed in future work" would be more appropriate.
Line 341: "would imply a potential for narrowing" - I don't think it is clear what is meant here. I think a rephrase would be helpful for the reader.
Line 352: "provides a blueprint". I think one of the following may be more appropriate: "provides evidence" or "provides justification" or "provides motivation" or "provides support".
Additional comments: The paper is well written and I would compliment the authors for some good practices, such as Figures 2 and 3 which clearly and concisely illustrate the concept outlined in the main discussion. The paper presents promising results however, further experimentation should be conducted to ensure the classification models are not inadvertently overfitted as a result of the feature selection approach outlined in the current manuscript.


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

Input:
Title: INFLAMMATORY BOWEL DISEASE BIOMARKERS OF HUMAN GUT MICROBIOTA SELECTED VIA DIFFERENT FEATURE SELECTION METHODS
Review round: 1
Reviewer: 3

Output:
Basic reporting: The reporting of this article is, to describe it briefly, unbalanced. It is written in clear, professional English, and there are sections of well developed and explained text, both in Materials and Methods and in the Experiments (according to PeerJ standards it should be named Results), but there are other sections that are clearly missing content, or the one written is contradicting itself. As an example, we do not need to look further than the Abstract, where we can find a claim that dysbiosis in the cause of IBD and other gastrointestinal illnesses, to a few lines later read that we do not have a clear picture of how this dysbiosis affects IBD, just that they show a strong association (this is also repeated in the Introduction). Claims of causality should be avoided unless evidence of this relationship is abundant, and with the gut metagenome we are just not there yet.
Getting into the Introduction, I find that some general description of what we know about the relationships between IBD and dysbiosis gut environments would be beneficial to highlight the dire need for this kind of diagnosis approaches. I would add a brief description of the most common symptoms of IBD and the relationship of between this illness and the gut to lines 51 to 57. The mention of the high comorbidity of IBD with other dysbiosis related illnesses, as depression, anxiety or obesity could also highlight the relevance of this research.
Regarding the figures, I do not have any major complaints. The tables in Figure 4 may be too small in some screens, so maybe they should be independent supplementary tables. Other than that, they are clear, well described and easy to interpret.
Raw data was supplied through the project’s website. This should still be disclosed on the paper.
Experimental design: The experimental design follows the trend of using different Machine Learning algorithms for classification of different atypical markers of illnesses. This is a powerful approach both to understand better which alterations in the microbiotic environment are most influential on IBD, and for clinical purposes as diagnostic and treatment.
The research question is well defined and relevant, and the investigation is, as far as I can see, performed according to technical and ethical standards.
In the description of the methods we find, again, this unbalance that we previously saw in the introduction and abstract. The first part of that section (lines 122-144) and the Feature Selection descriptions are good. Model Selection and Unsupervised Learning, however, are too brief and shallow. There is no description of what any of the classifiers used, nor a justification of their use. In fact, the Model Construction section is composed mostly of a description of the performance metrics used to analyze how well those algorithms performed, instead of why those algorithms were used and how they work. Something similar happens with the Unsupervised learning, where the description is so brief that a description of the algorithms has been included in the Experiments (Results).
Validity of the findings: To my knowledge the results presented in this paper are novel and relevant. I would like the authors to take a bit of time to try to link them to other research in IBD and metagenomics, mostly on the topics I already mentioned on my comments on Basic Reporting. IBD is a complex, multifactorial and comorbid illness, and that should be addressed both in the introduction and description of the issue, and on the discussion and interpretation of our results, even if the scope of our analysis does not include all those factors or comorbid conditions.
I must admit that I do not like to see some of the results, as the most relevant species for IBD diagnosis according to your analysis, firstly mentioned on the Discussion. Maybe restructuring the article according to PeerJ Standard Sections template would help avoiding this. I understand that the focus of this paper is on the use of Machine Learning as a diagnostic tool, but if you are going to mention it at all, it still should be mentioned in the Results section.
Additional comments: No additional comments


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

Input:
Title: INFLAMMATORY BOWEL DISEASE BIOMARKERS OF HUMAN GUT MICROBIOTA SELECTED VIA DIFFERENT FEATURE SELECTION METHODS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The use of "our", "we" throughout the manuscript makes the writing quite informal and I would recommend that this be changed throughout. The manuscript is the presentation of a scientific investigation. It is not owned by, nor belongs to, any set of authors as the same study can be proposed and implemented by others. Therefore, it is more appropriate to refer to "The study", or "the results" and what explicitly occurred during the investigation, rather than "Our study" "Our dataset", "we applied" etc..

The section "Validation on external data" includes the use of , instead of . for decimal points and should be changed.

References are generally appropriate and in place. Page 9, websites should be referenced properly alongside the other references (including the date the website has been accessed on since webpages can change).

Figure 1: No of sick/healthy samples should be No. of ... or # sick/healthy samples.

Table 1: There are a lot of values. Perhaps it would be helpful to highlight in bold the highest accuracy/recall/specificity etc. in each sub-table and the lowest std. dev. .

In the Dataset and Preprocessing Steps section (and elsewhere as appropriate) all weblinks should be properly referenced in the list of citations including date on which the content was accessed since the contents of the weblinks can be changed.

Changes in Figure 4 mean that the units for Accuracy are no longer %.

Please clarify to which dataset the results in Figure 5 belong to.

Figure 7: "Relative abundance" ....relative to what? Y-axis now has values but remains unlabelled and without units.

Figure 8:Y-axis is still not labelled/no units provided.

Figure 10 is still difficult to read although is more legible from the original submission. I think the authors should reflect on the "so what?" question here: What do you want the reader to take away from this figure? There is a lot of information presented in Figure 10. If you feel the reader needs all of this information in order to understand the conclusions drawn then please make the figure larger and more legible. Perhaps highlight specific regions of the figure and label with (a), (b), (c) etc to assist with your discussions. Alternatively, the authors may wish to revise the figure to present the key information (i.e. less information) more clearly to the reader so that they can interpret the results and follow the conclusions discussed in the paper. You can generate a Hierarchical clustering map...this doesn't mean you should.
Experimental design: Line 442: "Very similar preprocessing"...How similar? What was the same? What was different and why are the differences insignificant?

In the validation on external data RF is isolated however the initial findings from Table 1 would suggest LogitBoost would be equally as strong or appropriate to use. The authors may wish to reiterate why they focus on RF explicitly.

The inclusion of the independent dataset has improved the rigour of the study.

The research is original and of interest to the audience of the journal.
Validity of the findings: Line 424 "As shown in Figure 5..." These findings can also be found in Table 1.

Table 1 has a lot of values. Perhaps it would be helpful to highlight in bold the highest accuracy/recall/specificity etc. in each sub-table and the lowest std. dev. .

In Table 1 it is worth commenting that CMIM, FCBF and MRMR show signs of poor fitting across all models with low accuracy and high recall. (re lines 411-413).

Results Section (from line 419-...) RF is singled out here but it could be argued LogitBoost performed best (or as good as RF) when all 1331 features are used.

Line 431-432: "diagnosis could be performed with 88% accuracy" - so what? Is good enough for clinical adoption? At present this sentence just repeats what is in the table but doesn't really make a statement. The subsequent "only analysing the amounts of 14 specific species" - More appropriate to say "14 features are sufficient" to accurately classify IBD compared with using all 1331 features. (Note the typo: "by checking only analyzing".

Line 433-434: I think the final sentence of this section needs to be rephrased, perhaps purpose rather than proposed. Also the subsequent Validation on external data seems to conflict with this point since it appears that only 10 species are required. If line 434 is a hypothesis then test this on the independent external dataset (which turns out to not be fully possible - hence explain why and possibly revise under which circumstances people would use the 14 species). Furthermore given that 10 species are highlighted in the validation dataset it might be appropriate to leave any conclusion/propositions until after all the results are presented.

Validation on External data section: There are references that the results are shown in Table 1 but I was unable to see these findings - perhaps a table is missing?

Line 455-459: This appears to be contradictory. 10 selected species yielded higher performance metrics in the validation than exploration. Therefore the 4 additional features are beneficial? Please clarify - are you saying, that the 10 species from the validation cohort considered in isolation in the exploration cohort are less useful than 14 features in the exploration cohort? Furthermore, using the 10 selected species in both cohorts it was found that the exploration cohort was easier to classify than the validation cohort - in which case why might this be the case?
Additional comments: As a side note, it would be appreciated if the authors could cross-reference where the changes can be observed in the revised manuscript in their responses.


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

Input:
Title: A DNA BARCODE SURVEY OF INSECT BIODIVERSITY IN PAKISTAN
Review round: 1
Reviewer: 1

Output:
Basic reporting: In this study Ashfaq provide the first DNA barcode library for insects for a South Asian country. The library includes almost 49,000 DNA barcodes that were uploaded to BOLD and assigned to 6,590 Barcode Index Numbers. This study bears evidence for a long and comprehensive, careful work. Finally, it is another proof of that DNA barcoding represents an effective molecular method to identify specimens. Without doubt, the topic of this manuscript is interesting and appropriate for “PeerJ”.
Experimental design: The research question is well explained, the used methods represent state-of-the-at approaches to analyze DNA barcode data, using the BOLD workbench and offered tools. All data are open to the public and not deposited in local data bases.
Validity of the findings: As already pointed out, I think that the topic of this manuscript is very interesting and appropriate for “PeerJ”. It represents an important step in building-up comprehensive DNA barcode libraries for Asia which. Such libraries represent the backbone of any modern biodiversity studies using high-throughput sequencing technologies (e.g., “meta-barcoding”, eDNA analysis). Furthermore, it highlights the necessity of on-going taxonomic studies and efforts to document the insect species diversity of Pakistan. It is hoped that the present sequence data will become the basis for new species descriptions.
Additional comments: If possible, it would be nice to add some images of selected insect species in order to document the enormous diversity of the insect fauna of Pakistan.

Furthermore, please check the uploaded pdf for additional comments.


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

Input:
Title: A DNA BARCODE SURVEY OF INSECT BIODIVERSITY IN PAKISTAN
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript is well written without any obvious grammatical or other language problems. Many relevant references are cited.
Figures:
Figure 2:  colours not possible to interpret - please add taxon names to the actual pies
Figure 3:  these simple Excel figures are fairly ok, but not particularly elegant. In (a), what is a 'species' - as identified by BoLD search?
Figure 6: better to rank countries by their insect faunal similarity

Raw data are available in BoLD.

It is not immediately clear how the hypothesis on Pakistan diversity can be tested with massive DNA barcoding.
Experimental design: For the purpose of the study the experimental design is fine. Data obtained are trusted and handled correctly and possible to inspect in BoLD. Sample size is very large.

There are some questions of interest being tested and answered such as how many more samples are needed to cover the near entire fauna of insects (rarefaction and similar measures).

The main problem with this work is the obviousness of the data. Yes, the main holometabolan orders were the most prevalent in the sampled specimens; yes, the diversity is underestimated, but estimates are fairly close to the tallied numbers from the literature; yes, the BoLD (and mirrored GenBank) databases are not densely populated for the Pakistani fauna; yes, many bins have no names (because taxonomists are not invited or take years to get an answer); yes, more bins are related to neighboring countries than far away, even though there are a certain proportion of widespread species, often pest species.

So then - the 1000$ question - what is the point of doing all the work? It certainly does not provide any new information about the Pakistani fauna! The BoLD (and GenBank) database is propagated with sequences from unidentified specimens. In most cases these data do not provide any useful information and it will clog the system in the end. A kind advice for the future is to focus on fewer target groups, and connect with good taxonomists long before the study is initiated. Then you will get highly informative data, including necessary corrections to the current taxonomy and at the same time you will get estimates on diversity and sampling properties.
Validity of the findings: See above. Impact and novelty marginal, but data are clean, and properly curated.
Additional comments: Line 224:  coupling of morphological inspection with barcode matches - how exactly did it facilitate the provision of BINs as stated in line 225?

Line 227: 'parameterization' - meaning what? - do you mean propagation?

Line 241: Here you suddenly mention Malaise traps - any particular reason for that? Were Malaise traps the major collection method? - not mention in the methods ...

Line 252: No - a barcode study cannot say which group is the more diverse! It depends on sampling strategies and time. Beetles differ tremendously from flies and wasps because they are on average very specimen poor and rarely occur in such large numbers as do flies and wasps.

Line 257: endemism - yes, partly true, but remember that neighboring countries are also poorly investigated and the Pakistani fauna is largely shared with Iran and north to the Caucasus areas etc. The fauna in Pakistan is not so endemic as the data in this barcode study may indicate.


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

Input:
Title: A DNA BARCODE SURVEY OF INSECT BIODIVERSITY IN PAKISTAN
Review round: 1
Reviewer: 3

Output:
Basic reporting: I enjoyed reading this paper, which presents an impressive DNA barcode dataset on insects from Pakistan. It is written in clear and unambiguous English with citations to relevant literature. The article is well organized and figures and tables presents results in a professional manner.
The raw data is available in an open dataset in BOLD. The results are discussed appropriately in context with similar studies and present valuable new knowledge about the insect diversity in Pakistan.

The introduction gives proper background to the field, but I think the reader would appreciate a brief reminder of situations where DNA barcodes do not work so well to delimitate species (e.g. reference to Wolbachia, recent speciation, incomplete lineage sorting, etc.). Moreover, the BIN system has been found to overestimate diversity in certain insect groups such as Sciaridae and Chironomidae (e.g. https://doi.org/10.1139/gen-2018-0100, http://dx.doi.org/10.1371/journal.pone.0138993). The actual reason for this is unknown, but it is not unlikely that large effective population sizes over long evolutionary time periods could be one explanation. I do not think these facts affects the general and new results presented here, but I think it is worth mentioning in the introduction and in the discussion since it might affect the species diversity estimates for Diptera.
Experimental design: The article presents original primary research and is within the scope of the journal.
The research is performed to a high technical and ethical standard.

Please provide reference to the stand-alone version of the RESL algorithm used and where this tool can be accessed.
Validity of the findings: The findings are sound and valid and conclusions are appropriate. However, please see comment under section 1.
Additional comments: Two formulations should be changed:

Line 172: I suppose you mean the "remaining eight" orders, not the "other".
Line 200: Please reformulate because 2684 BINs are not shared with 199 other countries, but with at least one of 199 other countries.


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

Input:
Title: A DNA BARCODE SURVEY OF INSECT BIODIVERSITY IN PAKISTAN
Review round: 2
Reviewer: 1

Output:
Basic reporting: No more comments.
Experimental design: No more comments.
Validity of the findings: Noe more comments.
Additional comments: > Keywords: Change "cytochrome oxidase I" to "cytochrome c (in italics) oxidase subunit I"
> Be aware to write Wolbachia in italics (It is a genus that contains various species)


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

Input:
Title: SPATIAL VARIABILITY OF MICROZOOPLANKTON GRAZING ON PHYTOPLANKTON IN COASTAL SOUTHERN FLORIDA, USA
Review round: 1
Reviewer: 1

Output:
Basic reporting: I could find only a few corrections needed for spelling/grammar and basic reporting:

1) check if the label and order of Fig. 5 should be changed to Fig. 4 and vice versa (because the first mention of Fig. 5 is on line 221, and Fig. 4 is later on line 229)

2) line 250: change to "were"

3) line 290: maybe "data" should be treated as a plural, in which case change to "these data"

4) line 297: change to "tends to be"

5) Figure 3: blue, black and red colours can not really be discerned on the reviewing pdf.

6) Table 1 caption: delete "for" in last sentence.
Experimental design: The data here are used mostly for basic description of differences between two regions, and among several stations within those regions, concerning environmental parameters and traits of plankton biology including primary production, respiration and grazing. The introduction describes how the knowledge gap filled using this experimental design is to learn more about plankton variability in an "understudied" region. Although there are many other comprehensive studies on these plankton traits from other regions and the experimental design here is basic, I think it achieves its stated aim and will be useful for informing future related studies in this region.

My only comments are:

line 191: Please provide details of the tests of assumptions of ANOVA, such as Levene's or Cochran's tests, for testing homogeneity of variances.

Table 1: It seems here like only the environmental parameters were statistically compared among regions. For me it was unclear why such comparisons were not extended to the grazing and respiration rates, which in the same way could also be statistically compared among regions. The authors could consider making this addition.
Validity of the findings: no comment
Additional comments: No additional comments


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

Input:
Title: SPATIAL VARIABILITY OF MICROZOOPLANKTON GRAZING ON PHYTOPLANKTON IN COASTAL SOUTHERN FLORIDA, USA
Review round: 1
Reviewer: 2

Output:
Basic reporting: The English used by the authors is professional and the manuscript is generally easy to follow. I provided minor linguistic comments here and there for clarity. The Introduction was simple yet good enough to demonstrate the problem at hand. I would suggest making the objectives a standalone paragraph for clarity but otherwise, it provides context, is well referenced and relevant. I have added some structural points to my comment where needed. The general quality of the figures is not great and makes it hard to distinguish some data points from others, particularly in fig. 4. I suggest removing the back grid in all figures, using black colour for the axis lines and all characters within the figure (instead of light grey as it is now). Labelling is fine unless specifically mentioned. The raw data was not supplied (i.e., Chlorophyll a values versus dilution level) apart from the example figures in Supplementary Information. I do not think that this is an issue as this data is only useful when growth and grazing coefficients are determined. And these are shown unambiguously.
Experimental design: The experimental design was not perfect as, for example, community compositions could have been determined. However, from personal experience, the workload involved in the current experiments and the time required to process and analyse all the samples were already massive as it was. Thus, it would be extremely naïve to ask the authors for more data as their work is commendable. I did highlight a few things on the Methods, though. In addition, I found the Methods to be extremely detailed and believe that the length and detail could be shortened.
Validity of the findings: Even though I did ask for a few tweaks on the author’s analysis that will likely affect the results directly, I think that the fundamental conclusions are not wrong and won’t be affected. Some statistical details are necessary. I think that the conclusions might need a reassessment after addressing the issues that I mentioned in the Discussion.
Additional comments: The Methods are extremely detailed whereas the Discussion could benefit from added information. Aside from a few suggestions in the Methods that will affect the calculations of some figures and the values displayed in the Results, most of my comments focus on the Discussion of the manuscript, which I found a bit insufficient. I will go through each section individually to ensure that each of my comments is easy to understand.

ABSTRACT
The Abstract uses the terminology “Everglades” and “Florida Keys”, whereas the rest of the manuscript compares “SWF” to “FK”. My opinion is that the words are better than the abbreviations as they make the text easier to follow. Also, the words oligotrophic and mesotrophic are used in the Abstract and Introduction but not retained through the rest of the manuscript, namely in the Discussion as this is certainly an important difference between the two regions that must be further highlighted.

INTRODUCTION
Line 57: The abbreviation DOM is never used again; there is no need to abbreviate it.
Line 63: add parenthesis to NPP
Line 72: delete (Fig. 1); you should mention it only in the Methods, I think
Line 74 and 75: I do not see the point of adding results to the Introduction. Delete “Significant (…) locations”.

METHODS
line 81: remove the word “and” before the word “nutrient”
line 78-89: I suggest moving the sentences “Typically (…) since 2014” to different sub-headings of the methods, where environmental data and grazing experiments are described, respectively. The words “at five stations (Fig. 1)” can be moved to the end of the first sentence of this paragraph for clarity.
Line 94, 118, 153: remove the word “from”. Although I must say that it feels odd to repeat this entire sentence three times when one would suffice.
Line 98: re-write as “samples were frozen and stored at -20 (…)”.
Line 101, 106: “Nitrate plus nitrite” should read NOX as per the abbreviation in line 95
Line 105: replace “above” with “previous” – one never knows if it will be located above a given line once the text is formatted to its final form.
Line 107: write the full-name of NaDTT
Line 117: Carpenter (1995) does not mention primary productivity in his paper, nor does he mention the light-dark bottle method. This reference is not suitable to cite here. If something, it should be re-located to line 127 as it specifically addresses the Winkler titration. I could suggest Wielgat-Rychert et al. (2017) as a semi-valid option although there are certainly older works that are more suitable to cite here.
Line 132-134: what is described is the photosynthetic quotient. A more recent estimate (Wielgat-Rychert et al., 2017) suggests that this value is ca. 1.28, not 1 as used by the authors. In addition, I believe that there should be a distinction between respiratory and photosynthetic quotients. Williams and del Giorgio (2005) suggest using an average respiratory quotient of 0.89 after taking into consideration the multiple C sources that can originate CO2 in the cell. Considering that the authors do not know the composition of their communities, I would suggest using these values in their calculations. This implies that GPP must be calculated after converting NPP and R into C-specific values (with the photosynthetic and respiratory quotients, respectively). This modification will affect the results and amends throughout the text and figures will be required.
Lines 150-152: Did the authors use nutrient-amended bottles for their dilution experiments? Not clear and in the methods (although assumed since the original research by Landry and Hassett, 1982 is cited). This is critical for the interpretation of the results (see Calbet and Saiz 2018)
Line 151 and 152: despite being fundamentally correct, displaying grazing and growth coefficients simply as (d-1) is not useful. What the authors are measuring are Chl a-specific coefficients (i.e., Chl a Chl a-1 d-1). I do know that the units nullify but chlorophyll-specific rates are different from cell or C-specific rates, for example (see Ferreira et al., 2021). It is better to write the null units anyway, for clarity. Particularly because GPP is shown in C, not in Chl a.
Line 155: replace “from here on in” with “hereafter”
Line 159 and 160: 20L and four 1L flasks, correct?
Line 161: re-write as “incubated on deck, on a flow (…)”
Line 185: “negative values of g were changed to zero”. Despite having references that support this procedure, I do not agree with it. In fact, a much more recent study by Latasa (2014) specifically recommends not doing so. Negative grazing rates are nothing more than a beneficial influence of the predators over their prey which could be the release of nutrients or the removal of competitors, for example (see Ferreira et al., 2021 and references therein for details). The presence of mixoplankton (see Flynn et al. 2019 for the nomenclature) can also cause these “abnormal” results (see also Ferreira et al., 2021) and, as consequence, these values shouldn’t be neglected nor modified.

RESULTS
Line 201: just add “(Table 1)” after “five stations” and avoid it throughout the rest of the paragraph.
Line 208, 231: remove “amount of”
Line 209: replace “was” with “were”; delete the word “and” before NOX and add a “,” after.
Line 210-211: delete the sentence “However, average NOx concentrations at station 57 and 54 211 had a larger range of variability compared to MR, 21/LK, and WS (Table 1)”. It can be inferred from the table.
Line 211: do not start a sentence with “(Fig. 2)”. Connect the following sentence with the description of the table to improve the readability of the text.
Line 225: add the word “that” before “we ran”.
Line 227 and 228: delete “The three negative (…) not changed (SI Table 1,2)”.

Line 230: this is the first time that accumulation rates are mentioned. The authors use the abbreviation “acc” in Table 1 and in SI but used always the entire words in the manuscript. I suggest changing it to read “accumulation rate” everywhere.
Line 234-235: delete “At all three stations in the coastal region of Florida Keys, there was no linear relationship between g and μ (Fig. 5a).”
Line 243-244: delete “At both station, there was a significantly linear relationship with g and μ, suggesting that μ and g increase together (Fig. 5b).”

DISCUSSION
I believe that the topic of your Discussion that starts on line 285 should be the sentiment that leads the entire Discussion. I’m not saying that the authors should start the Discussion here but the overall sentiment should be directed towards the community analysis. I know that it was impossible to have a detailed community analysis (and I am not criticising it), but it could be a determining factor to explain the differences that are mentioned in the Results. For example, if the FK is dominated by diatoms, microzooplankton grazing rates will certainly decrease as they often display a negative selectivity towards diatoms (e.g., Burkill et al., 1984; Suzuki et al., 2002; Calbet et al., 2011; Ferreira et al., 2021). Nevertheless, you discard this option at the beginning of the Discussion, when diatom blooms are mentioned only in the SWF area.
I also believe that mixotrophy could be part of the reason why some dilution experiments yielded negative or zero grazing rates though I haven’t seen any possible explanation for these events in the discussion (whether related to mixotrophy or not). Are there any studies that show the proportion of mixotrophs in this area? If so, are they dominant? Fig. 2 and 3 haven’t been mentioned at all in the Discussion or Conclusion. If they are earlier figures in the Results, they should be individually discussed before Fig. 4.
Line 250: chlorophyll a is not the same as phytoplankton biomass. It is a proxy for phytoplankton biomass (Landry and Hassett, 1982). And not necessarily a good one due to inter-species differences among other factors (Kruskopf and Flynn, 2006; Ferreira et al., 2021).
Lines 251-252: reference for nutrient concentrations? Is it table 1? There is a clear difference in SiO2 (table 1) but not in NOx, DIN:DIP or in PO4. NH4+ is tendentially higher but the differences are not completely region exclusive (e.g., 21/LK = 54 and 57). Suggest re-writing for clarity.
Line 279: I do not understand the connection with benthic grazers here. You state in the methods that the samples were filtered through a 200µm mesh. There couldn’t be any benthic grazers in your experiments. I do understand and follow the rationale through the rest of the paragraph, but I would be tempted to suggest (without any evidence to support it) that if benthic grazers are such important members of the community, upon getting rid of them, microzooplankton would seize the opportunity and feed more than usual. See the general comment on the Discussion as a community analysis could partly resolve this issue.

CONCLUSION
After applying the suggestions I made to the Methods, Results, and Discussion, I believe that the Conclusion will require a bit of polishing. As I do not know exactly where how will the authors develop the ideas of the manuscript, I am afraid that I cannot make very specific and detailed comments on this section.

Line 310: versus?
Line 310-312: This is a very poetic sentence (e.g., “avenue of mortality”) but is hard to understand. Suggest re-writing it.
----------------------------------------------------
Fig. 1.
I believe that the quality of Fig. 1 can be severely improved. I think that the reader would benefit from having a wide perspective (say of the entire Gulf of México) with a close-up of the sampling area with all 4 sampling stations marked in the close-up perspective. This strengthens the fact that the study was conducted in an under-sampled location. Also (although just an opinion), I would suggest making it look like a drawing instead of a Google Earth snapshot as in Batista et al. (2014) or Gutiérrez-Rodríguez et al. (2015), for example. Colours can still be used if desired.
Fig. 2.
I would suggest keeping only panel a instead of having both. The information is the same and the reader can always go back to Fig. 1 to confirm the region where a given station is located. Also, the method used to determine the statistical ellipses is not mentioned in the methods. If a standard bayesian ellipse was used and its area compared, I would suggest referencing Jackson et al. (2011). This paper dwells on isotopic niches but the statistics should be similar. Regardless of the statistical method employed, this information should be added to the Methods.
Fig. 3.
Must be re-calculated considering respiratory and photosynthetic quotients as I suggested before.
Fig. 4
Is Ave = average? It is not defined in the legend. I would delete panels a and b and make a table instead (see my comment on Tables SI 1 and 2).
Fig. 5
Delete. The information is already shown in figure 4 and nothing new can be inferred from this figure. If necessary, the significant linear fits can be added to figure 4a and 4b. But, as mentioned before, a Table would suffice.
----------------------------------------------------
Table 1.
In the legend, where statistical differences are mentioned, the statistical test and the exact P-value should be referred. The letters µ and g are italicized many times in the manuscript but not here. Revise it. Statistical differences are typically highlighted with lower case letters, not upper case ones, if I am not mistaken.
----------------------------------------------------
Fig. SI 1-4
Examples are ok to show but are not very useful because the Methods have a very thorough explanation of what is a standard dilution result and what isn’t. If the idea is simply to show the different slopes as an example, a single figure with 4 panels is enough. Each panel could depict one scenario instead of having four 4-5 panelled figures.
Fig. SI 4. These are typical responses to food saturation. In these cases, the growth and grazing coefficients should be determined as follows: phytoplankton growth rates (µ, Chl a Chl a−1 h−1) are determined from the interception of the linear regression with the most diluted treatments. The grazing coefficients (g) are then calculated as g = µ−K where K (Chl a Chl a−1 h−1) is the apparent growth rates obtained in the undiluted bottles according to Gallegos (1989) and Dolan et al. (2000). This information should also be added to the Methods under the heading “Microzooplankton grazing and phytoplankton growth”.
----------------------------------------------------
Tables SI 1 and 2
I believe that this information should be included in the main manuscript (negative values included and specified – Latasa 2014) as a single table. Ultimately, one can extract this data from Fig. 4a and 4b but it is easier if a table is provided instead.

Good luck,
Guilherme D. Ferreira

REFERENCES CITED
Batista, M.I., Henriques, S., Pais, M.P., Cabral, H.N., 2014. Assessment of cumulative human pressures on a coastal area: Integrating information for MPA planning and management. Ocean Coast Manag 102, Part A, 248-257.
Burkill, P.H., Mantoura, R.F.C., Llewellyn, C.A., Owens, N.J.P., 1987. Microzooplankton grazing and selectivity of phytoplankton in coastal waters. Mar. Biol. 93, 581-590.
Calbet, A., Saiz, E., 2018. How much is enough for nutrients in microzooplankton dilution grazing experiments? J. Plankton Res. 40, 109-117.
Calbet, A., Saiz, E., Almeda, R., Movilla, J.I., Alcaraz, M., 2011. Low microzooplankton grazing rates in the Arctic Ocean during a Phaeocystis pouchetii bloom (Summer 2007): fact or artifact of the dilution technique? J. Plankton Res. 33, 687-701.
Dolan, J.R., Gallegos, C.L., Moigis, A., 2000. Dilution effects on microzooplankton in dilution grazing experiments. Mar. Ecol. Prog. Ser. 200, 127-139.
Ferreira, G.D., Romano, F., Medić, N., Pitta, P., Hansen, P.J., Flynn, K.J., Mitra, A., Calbet, A., 2021. Mixoplankton interferences in dilution grazing experiments. Sci. Rep. 11, 23849.
Flynn, K.J., Mitra, A., Anestis, K., Anschütz, A.A., Calbet, A., Ferreira, G.D., Gypens, N., Hansen, P.J., John, U., Martin, J.L., Mansour, J.S., Maselli, M., Medić, N., Norlin, A., Not, F., Pitta, P., Romano, F., Saiz, E., Schneider, L.K., Stolte, W., Traboni, C., 2019. Mixotrophic protists and a new paradigm for marine ecology: where does plankton research go now? J. Plankton Res. 41, 375-391.
Gallegos, C.L., 1989. Microzooplankton grazing on phytoplankton in the Rhode River, Maryland: Nonlinear feeding kinetics. Mar. Ecol. Prog. Ser. 57, 23-33.
Gutiérrez-Rodríguez, A., Selph, K.E., Landry, M.R., 2015. Phytoplankton growth and microzooplankton grazing dynamics across vertical environmental gradients determined by transplant in situ dilution experiments. J. Plankton Res. 38, 271-289.
Jackson, A.L., Inger, R., Parnell, A.C., Bearhop, S., 2011. Comparing isotopic niche widths among and within communities: SIBER – Stable Isotope Bayesian Ellipses in R. J. Anim. Ecol. 80, 595-602.
Kruskopf, M., Flynn, K.J., 2006. Chlorophyll content and fluorescence responses cannot be used to gauge reliably phytoplankton biomass, nutrient status or growth rate. New Phytol. 169, 525-536.
Landry, M.R., Hassett, R.P.l., 1982. Estimating the grazing impact of marine micro-zooplankton. Mar. Biol. 67, 283-288.
Latasa, M., 2014. Comment: A potential bias in the databases of phytoplankton growth and microzooplankton grazing rates because of the improper formulation of the null hypothesis in dilution experiments. Limnol. Oceanogr. 59, 1092-1094.
Suzuki, K., Tsuda, A., Kiyosawa, H., Takeda, S., Nishioka, J., Saino, T., Takahashi, M., Wong, C.S., 2002. Grazing impact of microzooplankton on a diatom bloom in a mesocosm as estimated by pigment-specific dilution technique. J. Exp. Mar. Biol. Ecol. 271, 99-120.
Wielgat-Rychert, M., Rychert, K., Witek, Z., Zalewski, M., 2017. Calculation of the photosynthetic quotient (PQ) in the Gulf of Gdańsk (Southern Baltic). Balt. Coast. Zone 21, 51-60.
Williams, P.J.I.B., del Giorgio, P.A., 2005. Respiration in aquatic ecosystems: history and background, in: del Giorgio, P.A., Williams, P.J.I.B. (Eds.), Respiration in Aquatic Ecosystems. Oxford University Press, Oxford University Press Inc., New York, pp. 1-17.