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: PURPUREOCILLIUM LILACINUM AND METARHIZIUM MARQUANDII AS PLANT GROWTH-PROMOTING FUNGI
Review round: 2
Reviewer: 1

Output:
Basic reporting: The manuscript was greatly improved.
Experimental design: The present work was organized logically, and the results obtained were reliable and persuasive.
Validity of the findings: A great deal of work was conducted here, and the results were very accurate and significant.
Additional comments: The authors have satisfactorily responded to all the raised concerns and comments by the reviewers. The manuscript is now suitable for acceptance from my point of view.


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: PURPUREOCILLIUM LILACINUM AND METARHIZIUM MARQUANDII AS PLANT GROWTH-PROMOTING FUNGI
Review round: 2
Reviewer: 2

Output:
Basic reporting: The English language of the article is now clear and technically correct.
The background provided is sufficient.
The in-text citation style follow that recommended by the journal.
The structure of the article conforms to the format of standard sections, and the headlines improve the clarity of the manuscript.
The figures are relevant to the content of the article. However, P solubilization graphic should be the Figure 4, then “In vitro IAA production...” should be the Figure 5, and the last should be the Figure 6. Probably, there was an error in the submission process.
Available raw data is correct.
Experimental design: Methods are now sufficiently described.
Validity of the findings: Discussion of the results is well stated, connected with the main question addressed by the research and limited to which is supported by the results.
The previously identified issues were correctly argued and clarified, with an exception: the authors mentioned a change in lines 476-478 (“probably by” instead of “in addition to”) that was not observed in the final version.
Additional comments: Table 1 (legend at the end): It should say “Table 1” instead of “Tabel 1”


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

Input:
Title: A NEW SPONGE-ASSOCIATED STARFISH, ASTROLIRUS PATRICKI SP. NOV. (ASTEROIDEA: BRISINGIDA: BRISINGIDAE), FROM THE NORTHWESTERN PACIFIC SEAMOUNTS
Review round: 1
Reviewer: 1

Output:
Basic reporting: fine.
Experimental design: fine.
Validity of the findings: yes.
Additional comments: The MS is well-written and presents the data clearly and with an excellent description for a difficult group of animals.

My only real issue is with the Discussion’s treatment of A. patricki’s association with sponges. Other than their use as substrate, I don’t see any the basis of why sponges should be considered any more closely associated with the brisingid species in question than any other substrate. The author clearly finds this to be an important relationship, going so far as to name the species after a cartoon character which is friendly with sponges.

But even the author outlines how there seems to be no apparent relationship between the two.. there is no feeding or gain by a specific mutualism.

I agree with and think positively of the notion that brisingids could be taking advantage of affected water flow created by the sponge as substrate..but that is more association with the general physical environment rather than a specific biological relationship which closely links this species of brisingid with the sponge. Brisingids are often found on corals or rock faces in current flow.  Was there a statistically or anecdotally higher incidence of this species on sponges??  This could be entirely an incidental occurrence.

It seems to me that there should be a stronger basis to imply such a hypothesis. It is plausible..but I believe wording for this notion needs to be scaled back. And while I have no issues with naming the species after “Patrick” it should be borne in mind that if there is no association, the species epithet is a bit misleading.


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

Input:
Title: A NEW SPONGE-ASSOCIATED STARFISH, ASTROLIRUS PATRICKI SP. NOV. (ASTEROIDEA: BRISINGIDA: BRISINGIDAE), FROM THE NORTHWESTERN PACIFIC SEAMOUNTS
Review round: 1
Reviewer: 2

Output:
Basic reporting: This study is about integrative molecular phylogeny and morphological taxonomy for a deep sea asteroid species with good English writing. The authors showed the existence of new species and this manuscript might be an interest to the readers of Peer J and the basic methodology of molecular phylogeny and taxonomic handlings are generally acceptable and support the conclusions of this manuscript. But I think the manuscript needs to be improved with more information and interpretation with concrete pieces of evidence in molecular analysis and morphological observations.
Experimental design: The molecular phylogenetic analysis based on five gene regions but only three genes were concatenated. Considering that the increased number of genes provides more accurate phylogeny, concatenated genes analysis for all five genes should be done.

The “Description” was presented as a morphological summary of all examined specimens. However, the taxonomic description basically should be based on the holotype and other “variation” sections should be established in the manuscript. The description of the holotype is important because it is the name-bearing specimen. Photos and illustrations well help readers to understand the morphology of animals in taxonomic descriptions. But in the present figures (especially in Figs 2C-E; 3D, E), no closed up photos which enable us to understand the tiny morphology of ossicles are provided and it prevents readers, especially non-asteoird specialists, from smooth understanding the asteroid’s morphological features.

Thus I recommend authors to analyze molecular phylogeny based on multiple genes and improve the description part.
Validity of the findings: The “Habitat association with sponges” seems to me unnecessarily. The concrete data is a personal data of examination of stomach contents of a paratype (L248, 249). Other parts are the only review of the literature. If this part is not provided in this manuscript, validation of the description of the present new species is unquestioned. I recommend omitting this part unless you discuss this with your more concrete data (for example your phylogenetic trees).
Additional comments: Please see the other comments on my PDF file.


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

Input:
Title: A NEW SPONGE-ASSOCIATED STARFISH, ASTROLIRUS PATRICKI SP. NOV. (ASTEROIDEA: BRISINGIDA: BRISINGIDAE), FROM THE NORTHWESTERN PACIFIC SEAMOUNTS
Review round: 2
Reviewer: 1

Output:
Basic reporting: My suggestions are all corrected and the manuscript is now improved.
Experimental design: Increasing molecular markers in the phylogenetic tree and additions of more detailed figures make clear the confidences of new species. No more problems have been found in the experimental design in this manuscript.
Validity of the findings: Discovery of such a very rare deep-sea new species is important to understand the deep-sea ecosystem.  Has a good scientific impact.
Additional comments: My only minor request is as follow:

L151, "Description" should be "Description of the holotype"


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: EFFECTS OF TEMPERATURE ON METABOLIC SCALING IN BLACK CARP
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comments
Experimental design: No comments
Validity of the findings: No comments
Additional comments: GERENAL COMMENTS

The manuscript entitled “Effects of temperature on metabolic scaling in black carp” looked at examine whether metabolic scaling in black carp changes with temperature and to identify the link between metabolic scaling and surface area at the organ and cellular levels at two temperatures (10 vs 25 oC). Authors have found that resting metabolic rate, gill surface area, and red blood cell size varied with temperature, but the temperature’s effect was opposite between resting metabolic rate and gill surface area, and red blood cell size. However, all the parameters scaled consistently with body mass across temperature. This is a well written manuscript that was easy to follow and, although limited in its scope, was conclusive in its findings, and has interesting implications. However, this manuscript needs to be clarified in main sections (e.g., materials and methods) that I concerned as follows.

SPECIFIC COMMENTS

Line 27, change ‘095%’ to ‘95%’.

Lines 53-59, it is too long due to their un-consistent meanings. I suggest authors cut into two sentences.

Lines 65-66, it seems not to be appropriate presented at the end of the second paragraph, or it needs to be rewrote.

Line 80, delete ’significantly’.

Line 98, please provide main content for diet if available.

Lines 103-104, it is not clear whether fish from each temperature treatment were all kept within one tank. Fish should be kept in at least two rearing tanks to avoid pseudoreplication.

Line 106, it is too short for fish temperature acclimation, but would be better at least four weeks.

Line 111, is it sufficient using 48 h for 10 degree? Cite reference if available. Fish would extend their digestion and absorption (i.e., SDA) period at low temperature.

Line 112, what’s size for these chamber used for fish of different size? Please clarify here.

Lines 122-124 and elsewhere: a dot above the ’M’ in MO2 is missing in most places.

Line 170, check F-value again since it is so large.

All figures are not vectors so authors should provide more clearer figures if available.


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: EFFECTS OF TEMPERATURE ON METABOLIC SCALING IN BLACK CARP
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript reports on temperature effects on metabolic scaling, gill surface area (GSA) scaling and the scaling of red blood cells (RBC) in black carp (Mylopharyngodon piceus). The authors found that at high temperatures resting metabolic rate (RMR) was elevated, while both GSA and RBC size was reduced compared to the low temperature treatment. Li et al show a consistent scaling exponent with fish body mass of all tested parameters, independent of temperature. Generally, I think that the findings of the study are interesting, and the hypotheses are scientifically relevant. However, significant changes and additions need to be made in order to meet the publication standard of PeerJ.
1	BASIC REPORTING
Firstly, I think the abstract would benefit from a more extensive discussion of the presented results (currently only one sentence L29-31). An explanation on how RBC size and gill surface area offset the negative effect of temperature on the metabolic scaling exponent would be interesting.
In some parts I find the reporting language slightly confusing, as some sentences are not well structured and the terminology is not consistently used throughout the manuscript. The whole manuscript language needs to be revised.  Some examples below:
L53-59: the structure of this sentence is hard to follow and hence hard to understand. I suggest breaking it up into multiple sentences and adding some information.
L73-76: repeated word for word from abstract (line 15-18). Consider rephrasing.
L80-82: It is not clear what the authors are trying to say. This sentence needs rewording
L157-159: be consistent with the acronyms (SRBC vs SRBC) throughout the text.
L208-210: This sentence is hard to follow. I suggest breaking it up in two and provide more information.
L218-222: A very nested sentence, which doesn’t flow well. A lot of information is presented, but is hard to grasp in its current form. Phrasings like “surface area of the rate limiting metabolic structure (L219)” make it hard for the reader to follow the thoughts of the authors. This needs rephrasing.
L236-239: Again, a nested sentence which would profit from being broken up into two.
The introduction gives a good insight in the topic and prepares the reader for the data presented. It could be improved by a thorough language revision as mentioned above. I think the introduction would be advanced further by more background information on the species used (especially of the habitat and temperature window it occurs naturally). The end of the introduction needs to contain the hypotheses on the effect of temperature on the GSA and RBC size scaling, not only that of metabolic rate (L87-88). I also think the authors need to elaborate more on the importance of how the metabolic rate, RBC size and GSA of black carb scales with temperature.
Furthermore, neither in the introduction, nor in the discussion do Li at al mention the literature on the scaling of body mass and GSA with temperature from Pauly et al and the debates it caused  (e.g. Lefevre, McKenzie and Nilsson (2017); Pauly (1981); Pauly and Cheung (2018)). I think the manuscript would greatly benefit from including these publications and from discussing the results with this background. Also the sentence in L205-208 needs a reference to support it, but besides that the literature presented in this manuscript is well referenced and relevant.
The structure of the article is well chosen and conforms to PeerJ standards. The conclusion however, needs to be more comprehensive than it currently is, more on that below. Also the term Q10 (L196) is firstly mentioned in the discussion, but had not been explained in the introduction. Furthermore the manuscript lacks the method of calculation of Q10 (should be in the methods section) and the presentation of the results (should be in the result section). Please amend.
The figures presented in this study are mostly relevant. Figure 3 and 5 are not essential for the narrative of the manuscript and hence could be moved to the supplementary material, in case space is an issue. Furthermore the figure captions need to be more descriptive (e.g. include scientific name of species, treatment, incubation time, statistical methods and so on). The figures need be understandable without information from manuscript, which currently they are not. Also Figure 4 contains only open circles, but the caption states there are filled ones. Please amend the figures accordingly.
I commend the authors for supplying a set of neatly organised raw data.
Experimental design: As of my knowledge the research presented in this study is original primary research, well within the scope of PeerJ. As I mentioned above, I think that the hypotheses need to be extended to also include the scaling of GSA and RBC. The knowledge gap the authors attempt to fill is well defined, however the relevance to justify the research needs to be stated more clearly.
The methods of this manuscript need extensive work in order to be considered for publication.
Firstly, the origin of the fish is unclear. Where have they been caught? Or are they reared in aquaculture? The authors mention that they were fished (L92) and later on Li et al say that their specimens are farmed (L136). Please specify.
The rearing facilities are not well described. This however is crucial for the study in order to be replicable. A detailed description of holding tanks (among others the volume, flow-through or recirculating system, filtration system, and most important how many replicate tanks per treatment) needs to be added, so reviewers are able to assess the experimental design. If the temperature treatments have not been replicated, than this would be a faulty experimental design, and would require admitting the design error in the text and discussing the potential issues with that. Furthermore, the rearing density of each replicate tank needs to be mentioned, as well as the supplier of the commercial feed. Also, it is unclear from the manuscript if temperature, ammonia and oxygen levels were measured during the acclimation and the trial period. Please include measuring techniques, equipment, and manufacturers. Further it is ambiguous how the temperature treatments were set, i.e. was the water heated/cooled? If yes mention the equipment used and its set up. In general I find 2 weeks of acclimatisation a very short period of time. A longer acclimatisation time might have led to different results. In L110 you use the word “acclimation period” for the time the fish were at their respective temperatures. Earlier on (L96) you say the fish were “acclimated in the rearing system at their original temperature”. This is confusing and I would suggest using the word “experimental or trial period” for the former instead, in order to clearly distinguish between the different experimental stages.
The set-up of the respirometers is also ambiguous. The authors need to specify the volume of the respiration chambers, and to which flow rate and how they adjusted the water inflow. Further measuring the water flow rate using a beaker seems to be a rather rough estimate to me. I suggest using more precise methods in the future. And did those measurements disturb the fish? How much were the fish moving in the respirometers and how much did that affect the readings when the authors only got one measure per hour? Please see Fig. 2 in (Clark, Sandblom & Jutfelt 2013) for further information on the potential issues of measuring oxygen consumption infrequently and discuss your results accordingly. Did the beaker oxygenate the water during the measurements? That would reduce apparent oxygen consumption in the most heavily consuming fish (lower oxygen levels give higher diffusion gradient), and perhaps that was manly the warm group?
The sampling method is well described. The duration from when the fish were anesthetised until blood sampling is missing though. Studies (Iwama, McGeer & Pawluk 1989; Phuong et al. 2017) show that fish stressed by anaesthetics can show a swelling of red blood cells/ higher haematocrit, which would lead to an erroneous measurement of RBC size. Was this considered in the design of the study? Also what was the time frame between sampling the blood and preparing the blood smears? And what substance did you use as anticoagulant? Please specify.
The sentence L133-134 is slightly confusing. It sounds like the authors transferred the gills to 70% alcohol (ethanol) right before measuring the GSA. Please verify if that was the method used, or if you stored the samples in ethanol until the analysis (e.g. for several days). Further, it sounds like the authors took three lamellae from every filament on arch two. This would be a very tedious analysis. Please give a more detailed description on which part of the organ you analysed.  It is also unclear how you measured the bilateral lamellae area. Please provide details.
Validity of the findings: Most of the underlying data are provided by the authors (apart from data on Q10, see above), and hence the study is easily comprehensible. With the information given at hand, I am not able to comment on the statistical soundness of the present study. The missing information about the experimental design, especially concerning replicate tanks make a judgement of the scientific merit problematic. Generally, I find that the discussion has a very heavy focus on the scaling exponents, and therefore dismisses interesting results, such as the overall smaller GSA at high temperatures compared to the high RMR. I suggest to include a discussion of this point as well. The authors talk about how an increased growth rate can mask the negative effect of temperature on the scaling exponent of the RMR. If that would indeed be the case, upon termination of the experiment I would expect the high temperature group to have a larger mass than the low temperature group. Was this the case? If not, this might not be a valid explanation.
The data for the RBC size seem to have a very high spread, and therefore a low r2 of 0.221, which might indicate that there is only a weak correlation of RBC with body mass. This however is not mentioned in the manuscript. Furthermore, data of some specimens are missing in the raw data (GSA of LQY33 and RBC of HQY31), as well as GSA data from fish HQY34 does appear in the raw data, but does not seem to be in the Fig. 2. Please explain.
Finally, the conclusion should combine all findings, and not only those of the scaling exponent of the RMR. Please add a short paragraph about GSA and RBC.
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: EFFECTS OF TEMPERATURE ON METABOLIC SCALING IN BLACK CARP
Review round: 2
Reviewer: 1

Output:
Basic reporting: This revised MS is substantially improved, and the authors have addressed nearly all of the two reviewers' comments, as indicated by sufficient backgroud, umambiguous texts, more clear result descriptions and discussion presented within the MS.
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: EFFECTS OF TEMPERATURE ON METABOLIC SCALING IN BLACK CARP
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comments
Experimental design: no comments
Validity of the findings: no comments
Additional comments: The manuscript entitled "Effects of temperature on metabolic scaling in black carp" reports on temperature effects on the scaling exponents of resting metabolic rate, gill surface area, and red blood cell size.
The quality of the manuscript improved significantly throughout the review process and I believe, after minor amendments listed below, it will be well suited for publication within the journal PeerJ.

Specific comments:
L28-29 This sentence is unclear: do you mean GSA?
L57 I suggest writing exponent b instead of b value
L141 I suggest writing “a commercial diet”, instead of “commercial diets”
L142 Please write “residual pellets” or “residual feed”, not “residual diets”
L166 Please give a short description of the setup (how big the water bath was (e.g. dimensions), and if all chambers sit in the same bath, or if several water baths were used).
L192 Saying the “first four gill arches” implies that black carp has more than four gill arches on each side. Is that true? If not, please refer to the left four gill arches.


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: ROLE OF ECHOCARDIOGRAPHY IN SCREENING FOR PORTOPULMONARY HYPERTENSION IN LIVER TRANSPLANT CANDIDATES: A META-ANALYSIS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The article structure and language is confusing. For example, "POPH is rarely in clinic" and "POPH patients can happen to acute right ventricular failure".
Experimental design: Study is well designed but adds little to the field.
Validity of the findings: I disagree with conclusion that echocardiography can be used as a diagnostic tool as it is only a screening test. The findings from the study do not support the conclusions.
Additional comments: The introduction and discussion could be condensed for clarity and reviewed for grammar in order to be more readable.


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: ROLE OF ECHOCARDIOGRAPHY IN SCREENING FOR PORTOPULMONARY HYPERTENSION IN LIVER TRANSPLANT CANDIDATES: A META-ANALYSIS
Review round: 1
Reviewer: 2

Output:
Basic reporting: Introduction can be improved
In lines 26,  27 and 28 I suggest to point that POPH reduce the survival rate of patients with end stage liver disease specially if they are not diagnosed. Early detection may improve the prognosis.

In line 28: I think it is better in this way: The objective of this study is to explore the diagnostic value of echocardiography on liver transplant candidates for diagnosis of POPH compared to RHC.

The conclusions in line 40 – 42 need to be clarified.
Since the objective of this study is not to find  a cut – off value of RSVP (well defined in the literature), this is not really a limitation of the study.

Re write lines 56 and 57,  can be described in more appropriate words.

Regarding lines 62- 64, echocardiogram is the gold standard for screening PAH, so you have to re think the way you want to propose the relevance of this study.

In line 68.  The proper way to point out the criteria for performing a RHC is “when the PASP is more than 50mmHg”, instead of “is not less tan 50”.

In line 70 it is not necessary the comment after the reference.

Comments for the first paragraph of discussion
RHC can be safely performed in POPH patients,
There are well defined  echocardiographic criteria for screening.
There is a general agreement between experts that echo is not accurate in the diagnosis of PAH, specially POPH. Again I would suggest to change the arguments for this study.

There is a lot of text without references in discussion.

Line 206,207. RCTs are not used to determine the efficacy of a diagnostic test.
Experimental design: Research question is valid.
Are you exploring the value of echocardiogram as a diagnostic tool or as a screening method?
If you review the table 1, all the studies have a lot of False Positives, this is not unexpected considering this echocardiogram is a screening method.
It is very risky to conclude that echocardiogram may substitute the RHC for diagnosis of POPH (or other types of PAH)
Validity of the findings: There is an heterogeneous cut-off value of PSAP in the studies, with your findings you cant draw a valid and robust conclusion. Also consider observations in methodology.
Additional comments: It is tempting to substitute the RHC with echocardiogram in practice, but there is bunch of data against that, all the studies you showed have a lot of false positives. Remember there are several hemodinamic profiles in cirrhotic patients (Pseudo pulmonary hypertension, fluid overload and true PAH), that can only be identified correctly by RHC.
So you have to be very careful in your conclusions.


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: ROLE OF ECHOCARDIOGRAPHY IN SCREENING FOR PORTOPULMONARY HYPERTENSION IN LIVER TRANSPLANT CANDIDATES: A META-ANALYSIS
Review round: 1
Reviewer: 3

Output:
Basic reporting: Use of English language needs to be checked again by the authors. Alternatively, a native English speaker should review the manuscript. Some examples where language can be improved include lines 26 (first sentence), 33, 56, 70, 95, 146, 160, 179, 183, 188, 211.

References are consistent, but need to be transformed according to the journal guidelines.

Figure legends need to be more explanatory for the reader.
Experimental design: The meta-analysis examined the diagnostic accuracy of pulmonary artery systolic pressure evaluation using TR signal and right atrial estimated pressure, not the diagnostic accuracy of echocardiography in general as other indices to evaluate pulmonary artery pressure and to differentiate precapillary and post-capillary pulmonary hypertension have also been proposed. (DesJardin et al Clinical Transplantation 2019, Dimitroglou et al Journal of Clinical Medicine 2019). Therefore in my opinion, the title of the manuscript should be more specific. Moreover, please discuss the need to evaluate alternative echocardiography indices for the diagnosis of POPH.
Validity of the findings: In the study by Colle et al, true negatives were 148 according to the authors. However, as authors clearly state in their manuscript, in 102 of 146 patients, TR jet was absent or inadequate. While, TR signals is commonly insufficient in patients with low ePASP, how the authors imply that those patients were real true negatives?

In the study by Al-Harbi et al right atrial pressure in 10mmHg for all patients. Therefore, not only ePASP values, but also method to calculate ePASP differs among studies. Please discuss this limitation.
Additional comments: It was a pleasure to read the meta-analysis by Yin et al in a topic with increasing clinical interest. Below there are some specific comments.

Abstract
Lines 28-30. Objective of the meta-analysis has already been written above.
Introduction
Line 63. While bleeding risk is cirrhotic patients is indeed higher, thrombosis is also more frequent and INR is not linearly related to coagulation function. Therefore, in my opinion the word ‘decrease’ needs to be replaced.
Meta-analysis
Lines 146-149. Should be rewritten giving number of spearman coefficient, as the reader cannot understand what the authors imply.
Discussion
Lines 192-194. Authors stated that they did not manage to identify all studies meeting the inclusion criteria. Which studies were not included? Could a different search approach identify those studies?


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

Input:
Title: ROLE OF ECHOCARDIOGRAPHY IN SCREENING FOR PORTOPULMONARY HYPERTENSION IN LIVER TRANSPLANT CANDIDATES: A META-ANALYSIS
Review round: 2
Reviewer: 1

Output:
Basic reporting: I congratulate the authors for the improvement in english language.

Literature is much more informative and discussion is more explanatory.

Figure legends have been improved.

Results are relevant to the hypothesis.
Experimental design: I thank the authors for their explanation.
Validity of the findings: Results are valid and conclusions are more clear. The way limitations are presented has been improved.
Additional comments: This version is much better and in my opinion should be accepted for publication.


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

Input:
Title: EXACT INTEGER LINEAR PROGRAMMING SOLVERS OUTPERFORM SIMULATED ANNEALING FOR SOLVING CONSERVATION PLANNING PROBLEMS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Well presented manuscript with good placement in the literature
Experimental design: Clear experimental design and description of study.
Validity of the findings: conclusions well supported with data and follow logically from the description of the study design
Additional comments: This study explores the performance of a new approach to solving the spatial conservation prioritization problem that currently is commonly currently solved with the simulated annealing approach in Marxan. Recent advances in integer linear programming (ILP) have enabled the scale of problem that Marxan can handle by approximating optimal solution to now be feasible with an ILP approach, which can find an optimal solution. An R package (prioritizR) provides this functionality to conservation planners and researchers. Here, the authors explore the variation in recommended conservation portfolio (suggested protected areas) and the time to solution for a set of conservation scenarios that are run in Marxan, prioritizR with the optimization performed with Gurobi or prioritizR with the optimization performed with Rsymphony (two different options that differ in their availability to practitioners vs academics). They find that on all fronts, the Gurobi based solutions were superior (met the conservation targets at lower cost) and drastically faster than Marxan. The Rsymphony based solutions also were cheaper and faster than Marxan, although slower than Gurobi.

This paper is well written and generates important evidence supporting a new spatial conservation approach that can outperform the current industry leading approach. I have provided several suggestions for improvement and small edits but in general I think this paper is well presented, clearly written, and nicely executed.

Broad comments:
1.	I would have liked to see a little bit more information in the introduction on what the advances in ILP were that now permit it to handle nonlinear constraints and penalties. I expect that this is covered in the Haight and Snyder, or Bayer references, but a sentence or two of the technical details would make a reader more convinced that the time is right for a move away from simulated annealing.
2.	It was not clear whether the broad range of cost savings that could be achieved from the ILP over Marxan approach was due solely to the variation in problem specifications or if there was also an aspect of Marxan potentially providing different solutions to the same problem across different runs (because of the simulated annealing). Please add a bit of detail that comments on the variation due to difference in approach as opposed to the variation due to marxan’s approximate solutions.

Editorial comments.
Lines 22-23: It would help the reader if you could specify that this reduction in cost was due to being able to calculate the optimal solution as opposed to an approximation.
Line 53: Clarify that it ILP “historically” was not well suited
Line 59: It would be good to detail here what an optimal solution would be for readers who are not well versed in the terminology (e.g., something like “optimal being the configuration of protected areas that delivers the desired benefits and the lowest cost”).
Line 77-78: As a reader I wanted to know here how the ILP inability to deal with spatially compact solutions had been resolved
Line 100: I’m curious how you dealt with the repeated measures aspect of the sampling locations that have been visited multiple times. Is this something that unmarked has a strategy for accounting for?
Line 111-112: it might be worth mentioning that the use of tax assessment values as an estimate of conservation cost is an underestimate because tax assessment values are often lower than market value
Line 162: I initially read this as species per features and was confused. I think you actually mean species (e.g. features)
Line 162: the numbers run together and make it seem like one really large number.
Line 163-167: Please explain here why you wanted to explore additional Marxan configurations. I think it was to try configurations that increased Marxan’s ability to approximate the optimal solution but not sure from the text.
Lines 174-182: How did you specify the equivalent problem (BLM modifications) in ILP?
Line 195: Was the reduction because the ILP solution was able to find cheaper parcels or less parcels?
 Figure 1: Are the results shown for Marxan from a single run? How much variation might we expect around this line if there were multiple Marxan runs? (I don’t think they are necessary, just a comment on the expected variability in Marxan solutions)


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: EXACT INTEGER LINEAR PROGRAMMING SOLVERS OUTPERFORM SIMULATED ANNEALING FOR SOLVING CONSERVATION PLANNING PROBLEMS
Review round: 1
Reviewer: 2

Output:
Basic reporting: Well-written, professional throughout. All sufficient in this section.
Experimental design: All fine, although see comments on the framing of the research question in comments below.
Validity of the findings: All fine, although see major comments 1&2 below which will affect the framing/wording of the findings.
Additional comments: This manuscript makes an excellent point – Marxan is used as the gold-standard in conservation planning, but it is often not the best choice of algorithm. The manuscript is well-written overall, however, at the moment some components of the manuscript are lacking in this current form.

First, the authors have stayed away from a mathematical description of the problems being solved here. I think that is a mistake, and explicitly including the mathematical formulations could clear up or shine a light on a few of my major issues below (points 3-5). Second, some language throughout is misleading, which I detail in points 1 and 2 below. Other major issues follow, but I will point out that I think all of these are fixable with some very careful reframing/rewriting, and some different choices in the results section.

I do not think any of these issues call into question the fundamental relevance of this work to the conservation planning literature, and I hope the authors can clarify these issues in the manuscript since I hope to see it published.

Major issues

1.	The dichotomy between ILP and SA presented here is incorrect, but I think it is just a terminology issue. Integer Linear Programs are a class of problem, with a specific canonical form. Reserve site selection problems largely fit this class. ILP itself is not a solution method/algorithm – in fact, SA is a valid (heuristic) solution method for ILPs. I think the comparison the authors are looking for here is not ‘SA vs ILP’, but ‘Solving ILPs with SA vs [an exact method -- branch and bound, CPLEX, or Gurobi etc]’.

2.	Relatedly, the manuscript sometimes uses SA and Marxan interchangeably. Marxan uses a specific problem formulation in order to capture the intended problem (specifically by adding their Boundary Length Modifier), but then uses SA to solve that problem. It’s not clear until fairly late in this manuscript whether the authors are comparing:
•	Simulated annealing vs an exact optimisation method to solve the same problem (same objective function, identical constraints); or
•	The Marxan formulation of the reserve site selection problem vs an ILP formulation of the reserve site selection problem solved using an exact optimisation method.
The first half of the manuscript feels like it is setting up the first comparison, but the latter is what ends up being done – but it is not clearly stated, the reader is left to figure it out. This manuscript compares two ILP solvers (Gurobi and SYMPHONY) with Marxan. Marxan is a specific implementation of a simulated annealing algorithm, and is built from a very specific problem formulation. I do not think it is fair to claim this is a comparison of ILP solvers and simulated annealing in general for conservation planning problems, it is a comparison between ILP solvers and Marxan.

3.	In Materials and Methods, it would be very useful to introduce the problem being solved in either the first or second section. The last parag of the introduction mentions a ‘systematic planning problem’, but it is not yet clear what this actually means in this context.

4.	The Spatial Prioritization section would be easier to follow with a mathematical description of the ILP being tackled (i.e. write out the min set problem, with all constraints), and the Marxan problem. This is related to my issues with the dichotomy elsewhere – this is where that could be cleared up by embracing the math!

5.	Is there an analogue to the BLM in the ILP formulation used? How does the ILP formulation take into account clumpiness? One of the big arguments for Marxan is the ability to control or explore how clumped the solutions are, so that needs to be addressed here.

6.	Much of the M&M section is written assuming a fairly good knowledge of Marxan. There are some terminology that need to be cleared up:

Species Penalty Functions
Boundary Length Modifiers
Calibration
Iteration
Maybe more that I have missed since I know Marxan, give it a good scour for other jargon.

7.	In Fig 2, the two objective functions are different for the two problem formulations. Is this deviation in the actual lowest objective function value (which includes a BLM penalty), or the cost of the optimal solution? The latter would be a fairer comparison. For example, the BLM of 100 performs much worse at a 60% target, but it is not clear to me whether that’s just because a larger penalty is being applied to the objective function.

8.	The two results figures use the 10^8 iterations version of the Marxan runs. That seems like a lot – is that standard? It may be a fairer comparison to examine the times at the median number of iterations tested, which would drop the times back a lot. Figure 1 could actually be cool with multiple lines for Marxan, showing multiple interation choices – I think with only a few, it wouldn’t be distracting.

9.	The same point can be made for the number of planning units. The discussion notes that the standard recommendation is not to use Marxan for more than 50,000, yet the results presented here are for more than three times that size. Do the general observations here hold true for a problem where Marxan would actually be considered useful? I’d like to see the results and the figures for a more realistic problem more representative of how Marxan is used in practice. If the message here is really that these other approaches can solve much larger problems than Marxan, then the messaging needs to be changed throughout the manuscript.

10.	Although I agree that this is strictly a conservation planning paper (which is fine), it would be good to look outside the field for similar discussions and analyses. The tradeoffs between SA and ILP, or even heuristic vs exact optimisation methods is not a brand new one. The comparison is certainly novel here in a field that so heavily relies upon SA, but a richer context could be drawn from other literatures.

Minor issues

Table 1 is not referred to in the text where it would be very helpful, and it is hard to parse.

Line 51 ‘performing runs’ is a little jargony for this early in an introduction – would be good instead to make it clearer at this point that SA is expensive because it performs many hundreds of simulations to determine the impact of different candidate solutions.

Line 53 – talking about the drawbacks (e.g. the structure of the problem and the time to solve) of ILP in past tense, can that be contextualised that more? Have these problems been solved and are no longer issues, or are you proposing that they are worth the tradeoff?

Line 58 – ‘highly suboptimal’ is confusing wording

I do not know what cadastral data means, sorry.

Line 110 mentions polygons – the context for these has not been set yet. I suggest adding this to the section I suggested at the beginning of M&M describing the problem.

Line 126 – is land cost a socioeconomic cost? I’d be more inclined to call this a financial cost, since there isn’t anything social being captured.

Line 191 – it is disingenuous to present results from uncalibrated Marxan runs, especially when it results in something so extreme as a cost saving of over four thousand percent. That would be a very powerful number if it came from the intended use of Marxan!

Line 197 I really like the comparison as illustrated here – how much more you could select in the ILP formulation if you had the same budget as the Marxan optimal solution. If there are other obvious places to make this comparison in the results, I’d love to see this illustrated again. It may not fit neatly anywhere else though, which is fine.


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: EXACT INTEGER LINEAR PROGRAMMING SOLVERS OUTPERFORM SIMULATED ANNEALING FOR SOLVING CONSERVATION PLANNING PROBLEMS
Review round: 1
Reviewer: 3

Output:
Basic reporting: All good.
Experimental design: All good.
Validity of the findings: All good.  I note some of the interpretations go beyond what the paper shows evidence of and have made some suggestions below.
Additional comments: Thanks for the opportunity to review this paper. Sorry for my delay.

The authors conduct a real- world analysis to support already established findings that ILP is faster and more efficient than Simulated Annealing when it comes to solving the minimum set problem- the most common problem definition in spatial conservation prioritization.

From a technical perspective, the authors present their case clearly. The analysis is robust and the findings do not deviate from previous examples where similar comparisons have been made (e.g. Beyer et al. 2016). To have a real-world example, rather than a simulation-based analysis, is useful for users and a nice contribution to the conversation about tools to underpin planning processes. As a technical document, I think this work is helpful, and having a discussion around open source vs proprietary ILP solvers will be useful for many people looking to use ILP in their analyses.

Where I do have some concerns is in the framing towards broader conservation planning guidance and recommendations.  While the authors use real-world data, I do not see clear real-world planning evidence offered in the paper to support some of these broader claims.

For example, Line 39: I would disagree that systematic conservation planning is about framing optimization problems. SCP is a ten step process, only one step is about framing and solving the problem for prioritization, the rest is about dialogue, communication, stakeholders, transparency, structure and policies. A SCP process can take 10 years- running a tool takes a week.  We should be careful to not over-emphasise the role of tools and algorithms in the much larger effort of SCP.  In the end, the algorithm is only a supporting application and the same can be said for the other planning softwares like Marxan and Zonation.

Lines 232: the authors encourage “making the switch to ILP as being advisable now that it is computationally feasible” to support SCP processes.  I think adding in a clause about “where technical capacity exists” would be prudent. Many decision-makers and technicians in the places where we desperately need to deploy SCP approaches and tools are not even computer or spatially literate, and so I am not sure “who” this message is for and “who” is advising this switch. Some clarification and evidence is needed if a grand statement such as this is going to be made. I think this paper will be of great interest for technically savvy people working on large optimization problems where optimality and speed are desirable- but this is a different target audience than mid-level practitioners in Tanzania or Solomon Islands working to support government decision-making through SCP.

While reading this paper- the following analogy came to mind:
I find this paper analogous to being told to choose between a Porsche (ILP) and a pick-up truck (SA), and then being told the Porsche is what I should buy because on all counts, it performs much more efficiently than the pick-up truck. It will get me where I need to go faster than anything else. But what if my objective is to build a house? Would I still be told to choose the Porsche? According to the logic of this paper, the answer is yes, because speed and efficiency are the only things driving the choice. In reality, there will be a suite of external factors influencing the choice of car that go far beyond performance efficiency. The “one is superior to the other” message that continuously emerges in the paper, and which is solely driven by the technical efficiency of the algorithm, feels a bit myopic when the authors move beyond the algorithmic comparison to discuss its applicability for integration in broader planning processes.

Prioritzr, Zonation, and Marxan all aim to serve the same role: to support and improve transparency in decision-making for planning. They all have pros and cons and can be deployed in different ways for different users in different political and planning contexts. I would encourage the authors to remove statements suggesting the world transition to ILP because of its superior performance,  if their only evidence for this claim is efficiency. To demonstrate otherwise would require a different comparison that is beyond the scope of this paper.

However, the ILP-based Prioritzr is a promising new addition to the suite of tools we have at our disposal for planning and I think with a minor tweak to some of the framing, this paper will be well-received amongst those individuals looking for more powerful solvers.

It might also be useful to suggest how ILP and SA can compliment each other in broader planning processes if the authors do want to bring the discussion up to a higher level than technical comparisons.


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: EXACT INTEGER LINEAR PROGRAMMING SOLVERS OUTPERFORM SIMULATED ANNEALING FOR SOLVING CONSERVATION PLANNING PROBLEMS
Review round: 2
Reviewer: 1

Output:
Basic reporting: Excellent
Experimental design: Excellent
Validity of the findings: Excellent
Additional comments: I have read through the revised version and the author's response and am happy with how they have addressed my and the other reviewer's comments. I have no additional comments to add and hope to see this paper published soon.


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: EXACT INTEGER LINEAR PROGRAMMING SOLVERS OUTPERFORM SIMULATED ANNEALING FOR SOLVING CONSERVATION PLANNING PROBLEMS
Review round: 2
Reviewer: 2

Output:
Basic reporting: Good
Experimental design: Good
Validity of the findings: Good
Additional comments: Thanks for the opportunity to review this manuscript again. While some issues have been addressed well, some concerns remain after being insufficiently addressed in the revision and response document.

1.	Remaining issues with the language:
Throughout, there are still places where ‘SA’ is referred to, but the authors specifically mean Marxan (i.e. it is not a generalizable statement about Simulated Annealing):
Line 276
Line 280
Line 281
Line 312
Line 321
Line 330
Line 387
Throughout, “EILP” is used to mean ‘Exact Integer Linear Programming’, but this should be followed by the word ‘solver’ in most cases. The ‘exact’ thing is the solver.

Likewise, in some places EILP is referenced but I believe the authors are referencing a specific implementation of ILP (probably prioritizr, but not clear):
Like 364, 372, 377

2.	The Introduction is still unclear on why EILP is suddenly useful. It’s alluded to in the tail end of two paragraphs, but what are the changes that mean EILP has overcome all the issues it had that allowed the meteoric rise of SA? Without this motivation this paper reads as if the authors have just heard of EILP and thought “why not have a crack” – which would be fine, except it implies that the use of SA over the years has somehow been blind to the existence of EILP. This is a naive implication, and as currently written the paper seems so evangelically in favour of EILP that its drawbacks cannot be touched on, which makes the reader (maybe incorrectly) suspicious of the findings. The addition of the sentence at line 76 since the previous review is insufficient to mitigate the appearance of this attitude.

3.	Methods section has no description of the problem – what are you solving? This relates to my point in the first review:
‘4. The Spatial Prioritization section would be easier to follow with a mathematical description of the ILP being tackled (i.e. write out the min set problem, with all constraints), and the Marxan problem. This is related to my issues with the dichotomy elsewhere – this is where that could be cleared up by embracing the math!’
In my opinion, adding a section to the Supplementary Information is not sufficient – it is not clear from reading the Methods section what that objectives or constraints of the problem are. The readership is limited to those who deeply understand the underlying Marxan and reserve site selection formulations.

4.	The lacking objective function definition also makes the response to my comment about Figure 2 insufficient – the y axis is being measured by a function that is not in the main text of the paper, and therefore is very opaque.

Minor: Line 104 ‘minimum set’ has not been described yet. Use a different, less jargon-laden description.


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: EXACT INTEGER LINEAR PROGRAMMING SOLVERS OUTPERFORM SIMULATED ANNEALING FOR SOLVING CONSERVATION PLANNING PROBLEMS
Review round: 2
Reviewer: 3

Output:
Basic reporting: The revision is a much improved version of the manuscript.
Experimental design: The scenarios are well developed and the testing is rigorous and comprehensive.
Validity of the findings: The findings reconfirm what is already known.  The results are presented clearly and make sense. The license versus open source EILP comparison remains a nice, new addition to the topic of discussion.
Additional comments: I think the previous review process was very helpful. Particularly the attention paid to differentiating  ILP as a class of problem and the EILP solvers used in the analysis.

I think the paper is acceptable to publish and have no further comments.


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

Input:
Title: BIOINFORMATICS PROFILING IDENTIFIES SEVEN IMMUNE-RELATED RISK SIGNATURES FOR HEPATOCELLULAR CARCINOMA
Review round: 1
Reviewer: 1

Output:
Basic reporting: English should be carefully checked by native English speakers. Figures and Tables (Results) support their hypothesis well. Literature summary regarding the impact of expression of CFHR1, APOA2, IL27, FCGRT, CD1D, CTSE, and NCOA6 should be done and discussed.
Experimental design: In this paper, research question is well defined, and it is significant in the research field. The knowledge gap being investigated is well explained and the authors clarified how they contribute to filling that gap. Methods are well described in this study with adequate information and references.
However, I strongly recommend them to perform functional analysis using cell lines (and siRNAs) to validate accuracy the markers (CFHR1, APOA2, IL27, FCGRT, CD1D, CTSE, and NCOA6) they identified as immune-related signatures for HCC in this study. If this proposed experiment is hard, the authors should compare the expression level of these genes in different differentiation stages of liver cancer cell lines (differentiated vs undifferentiated).
Validity of the findings: This study provides reproducible analysis and its impact is important for the field of liver cancer research. The conclusion is well illustrated, and it is closely related to their hypothesis.
Additional comments: The author bioinformatically analyzed the transcript sequencing data of liver cancer to find an immune gene marker to predict the clinical outcome of HCC using TCGA database. As such, they concluded 7 immunogenic genes as novel prognostic markers for predicting survival in patients with HCC. This paper is well written, and their analysis is clear. However, functional studies (as suggested) should be performed to validate the bioinformatic analysis.


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: BIOINFORMATICS PROFILING IDENTIFIES SEVEN IMMUNE-RELATED RISK SIGNATURES FOR HEPATOCELLULAR CARCINOMA
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript is clear and well structured. English written is OK, and if get more improvement will better
Experimental design: The results of this paper are potentially interesting and it appears that the data are in agreement with the conclusions of the authors. The bioinformatic analysis method and statistical analysis used appropriately and described with sufficient detail.
Validity of the findings: Conclusions are well stated, linked to original research question .
Additional comments: The results of this paper are potentially interesting and it appears that the data are in agreement with the conclusions of the authors. The bioinformatic analysis method and statis used appropriately and described with sufficient detail. However, the discussion of manuscript does need strengthen, what are the relevant and meaningful among 7 immune genes? Any possible suggestions in clinic from authors? if authors concluded them as biomarkers for diagnosis? Though authors could not provide the experimental validation for 7 immune signature found in the current study, more related report in liver cancer or other type of cancer of those 7 genes should be discussed in Discussion.


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

Input:
Title: DESCRIPTIONS OF FIVE NEW SPECIES OF THE SALAMANDER GENUS CHIROPTEROTRITON (CAUDATA: PLETHODONTIDAE) FROM EASTERN MEXICO AND THE STATUS OF THREE CURRENTLY RECOGNIZED TAXA
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comments
Validity of the findings: No comment
Additional comments: Brief review of: Descriptions of five new species of the salamander genus Chiropterotriton (Caudata" Plethodontidae) from eastern Mexico and the status of three currently recognized taxa

I have carefully read this paper and think it is an extremely well-written and reasoned contribution.  It makes a huge step in understanding the diversity of one of the most difficult plethodontid genera that has been a challenge to researchers or many years.  These small salamanders are speciose and often morphologically conservative.  The authors are well suited to tackle this problem and are among the foremost systematists of Neotropical salamanders.

Information available previously showed that a number of undescribed species were present in the mountains of eastern Mexico.  Therefore, this is a welcome follow-up to previous work that demonstrated that the genus contained a number of cryptic species.  This paper summarizes previously known information and adds considerable additional molecular and morphological (i.e., CT scans) data.

Most of these new species of salamanders, along with previously known species, are microendemics.  This paper elucidates the composition of the genus and provides distributional data that will be useful to a wide audience, including biogeographers, evolutionary biologists, and conservationists.

A helpful overview of the history of research is given.  Solutions are provided to several long-standing taxonomic problems of the group, such as the identities of Chiropterotriton orculus and C. chiropterus, by designating neotypes for each.  A more complete description of C. lavae is also given

Descriptions are thorough and follow previous formats making comparison among species easier.  Especially welcome are the detailed diagnoses (i.e., "Comparisons") of these new species with congeners.  Redescription of previously described species and their comparison with other species is necessary in view of the current complexity of the genus.  This kind of systematic review is labor-intensive and necessary understanding the diversity of this neglected and previously poorly understood group.

It might be noted that, despite the admirable effort expended by the authors in this research, they indicate that the last word on Chiropterotriton diversity has not been said, and they identify several other yet-to-be-described populations that undoubtedly will be part of subsequent papers when additional data become available.

The figures are well executed.  I strongly endorse 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: DESCRIPTIONS OF FIVE NEW SPECIES OF THE SALAMANDER GENUS CHIROPTEROTRITON (CAUDATA: PLETHODONTIDAE) FROM EASTERN MEXICO AND THE STATUS OF THREE CURRENTLY RECOGNIZED TAXA
Review round: 1
Reviewer: 2

Output:
Basic reporting: Please see attached
Experimental design: Please see attached
Validity of the findings: Please see attached
Additional comments: Please see attached


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: EFFECTIVE CONNECTIVITY DIFFERENCES IN MOTOR NETWORK DURING PASSIVE MOVEMENT OF PARETIC AND NON-PARETIC ANKLES IN SUBACUTE STROKE PATIENTS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Please see General reporting
Experimental design: Please see General reporting
Validity of the findings: Please see General reporting
Additional comments: Comments to editors

I thought that this was an excellent submission. It applies dynamic causal modelling to the sensorimotor system of people with paretic and nonparetic stroke. There were a couple of technical issues in their application of dynamic causal modelling that need to be addressed. However, I think that these can be resolved with a minor revision – and that they will not change the conclusions of this study. I have tried to suggest how the authors could rationalise their analysis in my comments below.

I hope that these comments help you in your evaluation.

Comments to authors

I enjoyed reading this concise and compelling report of a dynamic causal modelling study of subacute stroke. In many respects, I thought that this report was exemplary. Your motivation for analysing effective (directed) connectivity in this clinical setting was clear. I also thought that the description of your analyses was excellent. Having said this, there are some technical issues that you need to resolve. I suspect that this will require a reanalysis of your data; however, it should not take too long. Perhaps you could consider the following:

Major points

The main problem with the current analyses is that you have included modulatory effects (parameterised by the B matrices) in your DCM. This is not necessary and introduces a redundant parameterisation. The reason that you do not need the modulatory effects is that there is only one experimental factor in your design (namely, the active versus inactive block conditions). You only need to use modulatory effects in multifactorial designs. Put simply, this means that there is only one thing perturbing your distributed neuronal responses and therefore you only need to estimate the endogenous connectivity (the A parameters) for each subject. This will not make any difference to your results (note that you go got no significant differences between the B matrices).

The second analytic issue is the fact that you did your model comparisons within each (paretic and nonparetic) group separately. When testing for differences in connectivity between subjects, it is important not to do anything that is specific to each group before you do the final comparison. This means that you should combine all of your subjects (from both groups) in the same (random effects) family wise model comparison. Looking your results, you will get exactly the same family as you have currently used.

Finally, although the original description of DCM used the label "intrinsic" to refer to endogenous connectivity, it is now used to refer to within-region connectivity (i.e., self-connections). Can you replace "intrinsic" with "extrinsic" throughout the manuscript? When you first mention extrinsic connectivity introduce it with: "… extrinsic (i.e., between-region) directed connectivity"

In summary, these changes should simplify your analysis. First, simply omit any reference to modulatory effects in the introduction and results (and tables). Second, you only need to describe one set of family wise Bayesian model comparisons (for both groups combined).

In the discussion, I think you should include a qualification of how you analysed your between subject effects. I would recommend adding:

"In this work, we have used a conservative approach to compare extrinsic connectivity between the clinical groups. This nonparametric approach to classical inference at the between-subject level is conservative because it tests for differences in each connection separately (using false discovery rate to adjust for multiple comparisons). We could have used a multivariate test (e.g., canonical covariates analysis). This would have been more sensitive and would have highlighted significant group differences. However, we would not have been able to assign a unique q value to each connection. Finally, it should be noted that more recent analyses of between-subject effects on effective connectivity in dynamic causal modelling would normally use parametric empirical Bayes (Friston et al 2016, Kass & Steffey 1989). However, our classical inference is sufficient for our purposes – and represents a simple way of accommodating between-subject random effects."

Minor points

Abstract: please say: "in extrinsic connections of the MNW and to explain the haemodynamic…"

Replace "collected by" with "collected under"

Replace "and the stimulus modelling" with and input stimulus modelling"

I would also add the following to the abstract or introduction:

"Note that in contrast to most DCM studies, we leveraged the ability to make inferences about neuronal and haemodynamic coupling. This is particularly prescient in the context of stroke research, where lesions can produce both neuronal and neurovascular disconnections."

Line 61: please delete "and cortical activation"

Line 122: change "during the upper" to "during upper"

Line 126: I would say: "which provides evidence for one model over other based upon evidence ratios (i.e., Bayes factors) or differences in log evidence."

Line 133: change "top" to "to"

Line 139: delete "algorithm"

Line 216: replace "interconnected or subdivided" with "coupled"

Line 232: replace "connection system" with "connectivity architectures"

Line 242: say: "a model space comprising 4x3 = 12 model variants.… which were arranged into a matrix form, to emphasise the factorial structure of this model space.

Revise analysis of the modulation effect paragraph. Please remove any reference to modulatory effects.

Line 258: I would replace "further statistical…" with "Statistical analysis of between-subject (i.e., group) effects was performed under the winning model using (nonparametric) classical inference. The winning model was identified by pooling the evidence for different models over all subjects studied."

Line 266: remove "that are modulated by experimental variations"

Line 268: please revise your description of family wise model comparison (please see above)

Line 306: please replace "stimulus binding" with "stimulus driving"

I hope that these comments help should any revision be required.


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: EFFECTIVE CONNECTIVITY DIFFERENCES IN MOTOR NETWORK DURING PASSIVE MOVEMENT OF PARETIC AND NON-PARETIC ANKLES IN SUBACUTE STROKE PATIENTS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The main goal of the present study is interesting. However, there is insufficient background provided. The methodology section lacks clarification and there is missing information (see pdf attached for more information).  Finally, the reading of the paper is tough and not easy to follow (see my comments in the pdf document).
Experimental design: The methods presented in the study are not described with sufficient detail (see pdf document for more information).
Validity of the findings: Conclusions fall beyond the findings presented in the manuscript. Authors should try to discuss their results from a biological point of view instead of looking for validations in other studies in which the methodology conducted is enough different to draw firm conclusions (see pdf document for further details). Besides, limitations should be addressed: a small sample and the election of the brain regions of interest...
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: EFFECTIVE CONNECTIVITY DIFFERENCES IN MOTOR NETWORK DURING PASSIVE MOVEMENT OF PARETIC AND NON-PARETIC ANKLES IN SUBACUTE STROKE PATIENTS
Review round: 1
Reviewer: 3

Output:
Basic reporting: 'no comment'
Experimental design: 'no comment'
Validity of the findings: 'no comment'
Additional comments: The paper describes some novel findings regarding directed functional connectivity in patients with subacute stroke using a passive movement task.

Major concerns:

- small sample size (n = 10). The patient cohort is pretty inhomogeneous (NIHSS ranges from 1-3). Did the authors consider the paresis severity in their DCM computations as well as in the “standard” GLM fMRI analyses?
- Clear hypotheses are missing
- some patients are probable able to move their ankles, as the NIHSS is only 1. Why did the authors involve a physiotherapist? I guess it is to control the frequency of movements. If so, how was the frequency of the CPM controlled. Would the authors expect a different (GLM or DCM-wise) result, for self-controlled ankle movements?
- Using S1 as DCM region makes only sense in case of touch-related tasks, as done in the present study (i.e. a physiotherapist is moving the ankle). However, this is not a realistic scenario, in case a patient is undergoing normal physiotherapy training. Thus, the observed BMC results (S1 -> M1 and S1 -> PM), is most likely only present in case of external sensory input (e.g. externally triggered CPM). Alternatively, is the selected S1 region corresponding to the feet region in the brain?
- What is an expected probability value of 0.433 and exceedance probability of 0.63 (Figure 2a/b) telling us? Is this a strong or a weak effect (what are the limits, can we relate that to an effect size)?
- DCM still suffers (except of newer versions such as generalized DCM) from the fact that a ton of models can be defined by “knowing” somehow the winner model a priori. How did the authors limit the likelihood of overfitting (as the authors tested 12 models)?

Abstract:
- The word “lateralized” needs clarification
- the term “signal decay” needs a short introduction
- The conclusion is speculative. To what extend is a DCM-based result informs us anything about any appropriate rehabilitation procedure?

Introduction:
- first sentence (line 50): The terms “directly” and “indirectly” are confusing. For example, are they related to functional or anatomical connections?
- line 61/62: In which brain regions?
- line 78: This section needs to be slightly extended, so that the reader can understand what is modeled on the neuronal level by DCM.
- line 93: this needs more explanation. Why can we exclude a cerebral hemodynamic impairment?

Figure 4: The last sentence is difficult to understand, i.e. what is meant by “the location of external stimulus”? In addition, the twisted arrows are not explained. Please provide the p-value of the results.

Methods:
- line 270: What is the difference between the winning or best model? In this paragraph, authors need to explain the measures D, T, and E in more detail.
- line 274: What is a model inversion?
- line 290: what is the assumption behind the idea that self-inhibition = negative values. What is meant with self-inhibition?

Conclusion:
The first part of the conclusion rather belongs to the Discussion section. Critically, either the results do inform about “during” or “after rehabilitation”, as this is cross-sectional study. Please specify. The last sentence is a limitation. A limitation section should be added.

Discussion:
Lines 346-363 are somewhat redundant as the authors repeat results. Please shorten this part.


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: EFFECTIVE CONNECTIVITY DIFFERENCES IN MOTOR NETWORK DURING PASSIVE MOVEMENT OF PARETIC AND NON-PARETIC ANKLES IN SUBACUTE STROKE PATIENTS
Review round: 2
Reviewer: 1

Output:
Basic reporting: Please see below
Experimental design: Please see below
Validity of the findings: Please see below
Additional comments: Many thanks for attending to my previous comments – and congratulations on interesting 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: THE COMBINED EFFECTS OF LIGHT INTENSITY, TEMPERATURE, AND WATER POTENTIAL ON WALL DEPOSITION IN REGULATING HYPOCOTYL ELONGATION OF BRASSICA RAPA
Review round: 1
Reviewer: 1

Output:
Basic reporting: 1.	When binary comparisons described, the order of comparison is not clear. If all binary comparison were done in the same order as in lines 188, 192, 195 this should be noted in the text. In addition, the statistical test which done base on these comparisons is not clear. e.g.  Line 228: “The three environmental factors significantly influenced the elongation of epidermal …”, what statistical test used and what are the parameters?
2.	In the results section, some of the measurements are described twice, once in the figure/table and a second time is in the main text. My general suggestion is, if the measurements are clearly described in the figure there is no need to specify it again in the text.  These will make the reading more fluent. e.g. lines 177-184.
3.	When OE, IE and CO used in the results section for the first time (line 247) the full name needs to be added.
4.	In Fig. 3 adding letters will help to clarify it. My suggestion will be to add A for the OE panel and B for the CO and IE panel. Describe the different letters in the figure legend.
5.	In table 4, need to describe what is TCW (column 6).
6.	In the paragraph starting at line 318, it’s not clear which comparison was done in order to get the fold change. For example: Is the increase of 1.92 in response to low light is the average of all the low light conditions (LlThWh, LlThWl, LlTlWl,LlTlWh) relative to all high light conditions (LhThWh,LhThWl,LhThWl,LlTlWh)? This need to be clarified.
7.	 I couldn’t find the legends for all the Sup. Fig. They need to be added.
8.	In Fig S4 there is no title for the y-axis.
Experimental design: Most of the cell wall measurements come from the middle of the hypocotyl. This could raise some concern that it does not reflect the whole hypocotyl. Since the author clearly described it, and explain the rationale of doing it there, it can stay as it is.
Validity of the findings: 1.	I have a concern regarding the author conclusion “ Light intensity had significant effects on cellulose, hemicellulose, and pectin deposition, but high temperature and high water potential only had significant effects on pectin content”. This conclusion was based on results from Table S2. However, the analysis done in this table is unclear, showing the formula used for the Anova test will be helpful. In addition, temperature and water potential had shown some effect on cellulose and hemicellulose in some tested conditions: e.g. cellulose and hemicellulose were affected by water potential when LlTlWl compared with LlTlWh.  and by temperature when LlTlWl compared with L1ThWl. Therefore this conclusion should be phrased differently or explained in a more convincing way.
Additional comments: This manuscript by Wang and Shang showed a detailed phenotypic analysis of hypocotyl elongation, cell wall thickness and composition, under combined effects of light intensity, temperature and water potential. This approach reveals that these three environmental factors are effecting hypocotyl cell elongation independently and in combination. The authors suggest that low light represses wall deposition by influencing the accumulation of cellulose, hemicellulose and pectin while, high temperature and high water potential effects pectin accumulation only.
The authors collect an extensive amount of high quality and valuable data, the question addressed is clearly outlined and the manuscript is generally written in a logical order.
There are a few points in which clarity are needed as described above.


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

Input:
Title: THE COMBINED EFFECTS OF LIGHT INTENSITY, TEMPERATURE, AND WATER POTENTIAL ON WALL DEPOSITION IN REGULATING HYPOCOTYL ELONGATION OF BRASSICA RAPA
Review round: 1
Reviewer: 2

Output:
Basic reporting: The outline of the article is clear and easy to follow. However, the text has spelling and grammatical mistakes even though an editorial certificate has been provided.
Sufficient context is provided to understand the article.
The figures and tables and their legends are self-explanatory.
Experimental design: 1. Line 103 - The authors study the middle segment of the hypocotyl based upon previous studies that showed that the cells at the mid- and apical-segment contributed to hypocotyl elongation. It would help to know if the environmental conditions in this study affect hypocotyl elongation in a similar way before assuming that the mid-segment is the only part of the hypocotyl affected.
2. Line 122-136 - It is very difficult and confusing to understand the measurements here. It would help to define the transverse wall, and the outer and the inner epidermal cell wall here.
3. Furthermore, a supplementary figure showing the parameters of the cell wall (i.e. length, breadth, perimeter, etc.) taken into account for the calculations of the volume.
4. What is the meaning of "the number of IE/OE"? Are they counting the number of walls?
Validity of the findings: 1. Are the effects of the environmental factors significant under high and low conditions? It would help to provide the p-values in all such comparisons. For example, how significant is the effect of temperature on the hypocotyl elongation, irrespective of the light intensity and the water potential? And likewise for the other comparisons. From the text, it seems like the authors have concluded the significance by eyeballing.
2. Line 303-314 - The expression of the genes included in the text do not follow the trend in Figure S8. For example, the expression of the genes CSCL and MUR3 are not inhibited and GAUT7 was not induced at high temperatures. The same goes for the rest of the genes.
3. Where are the statistics showing the effect of water potential/temperature/light intensity on the genes' expression?
4. Line 375 - Gene name - YUCCA or YUC
Additional comments: No additional comments


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

Input:
Title: THE COMBINED EFFECTS OF LIGHT INTENSITY, TEMPERATURE, AND WATER POTENTIAL ON WALL DEPOSITION IN REGULATING HYPOCOTYL ELONGATION OF BRASSICA RAPA
Review round: 1
Reviewer: 3

Output:
Basic reporting: Figure 1, 2, and 3: The legends should include what the L, T, W, and subscript h, i are representing.

Figure 4: For clarity makes the bar color white and black for the lowest and highest hypocotyl elongation, respectively. Then follow a gradient of pattern for all other bars. That will help to co-relate things immediately.
Experimental design: The knowledge gap and the research question are not clearly mentioned in the abstract. Moreover, the abstract is loaded with redundant information that is causing confusion to understand what the main observation and conclusion are.
Validity of the findings: No Comments
Additional comments: Thank you for the nicely done and well-written manuscripts.


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

Input:
Title: THE COMBINED EFFECTS OF LIGHT INTENSITY, TEMPERATURE, AND WATER POTENTIAL ON WALL DEPOSITION IN REGULATING HYPOCOTYL ELONGATION OF BRASSICA RAPA
Review round: 1
Reviewer: 4

Output:
Basic reporting: The present manuscript is the revised version of the previously submitted manuscript. The authors have substantially improved the language of their reporting and the content is easy to follow and understand. They have provided sufficient references and background for their work. The authors have provided the shorthand of their treatments for example HhLlWh which is easy to understand. However, they have not added these acronyms to when they are explaining their data leaving the readers to guess which data (for example see lines 222-227, page 6) corresponds to what treatment. So, it would be appropriate if the authors state somewhere in the manuscript as to the order of the treatment that they are going to follow when they are reporting their data or provide the acronyms as they describe their data.
Experimental design: The authors have provided the statistical analysis for most of their data reported which is important to show the significance of the data.
The authors have now provided in detail the formula that they have used to measure the cell wall volume which is helpful in knowing the approach they have taken in the measurments. The formula suggests that the authors are reporting the cell wall volume for the entire hypocotyl length as they use the number of cells in the hypocotyl in their analysis for example: the formula of calculating cortical cells (CO) is perimeter of the CO by its number, by the thickness of the CO, and by the hypocotyl length.. It would be helpful if the authors provide the number of cells for cortex and inner epidermal wall columns in Table 4.
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: THE COMBINED EFFECTS OF LIGHT INTENSITY, TEMPERATURE, AND WATER POTENTIAL ON WALL DEPOSITION IN REGULATING HYPOCOTYL ELONGATION OF BRASSICA RAPA
Review round: 2
Reviewer: 1

Output:
Basic reporting: In the raw data file, the tab “cell wall extension” has some words which are not in English.
Experimental design: no comment
Validity of the findings: Making the changes below will make it easier to understand:

-	Line 302-304, clarify this part by splitting it into 2 sentences, and refer to Table S2 after: “The influence of temperature on pectin deposition reached significant level…”. Also refer to Table S2 after line 297 “Low light significantly repressed cellulose, hemicellulose, and pectin deposition…”
-	Line 307-309: When described the effect of water potential “…and the effect on pectin accumulation did not reach significant level under the background of high light × low water potential (Table 3; P < 0.05; Duncan’s multiple range test).”  Since it does reach a significant level overall (Table S2), as also mentioned in the abstract, adding a sentence that claims it, like done for the influence of temperature is necessary.
Additional comments: The revised version of the manuscript “The combined effects of light intensity, temperature, and water potential on wall deposition in regulating hypocotyl elongation of Brassica rapa”, addressed all the points from my first review. They now described their findings in a better way especially in explaining the statistical analysis they did.


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

Input:
Title: THE COMBINED EFFECTS OF LIGHT INTENSITY, TEMPERATURE, AND WATER POTENTIAL ON WALL DEPOSITION IN REGULATING HYPOCOTYL ELONGATION OF BRASSICA RAPA
Review round: 2
Reviewer: 2

Output:
Basic reporting: The language needs to be corrected. It can be difficult for a non-native speaker but it would get it corrected professionally.
Experimental design: Thank you for making a figure to explain the volume measurements. It makes it easy to understand the article and its finding better.
Validity of the findings: The statistical question has not been answered yet.
The statistical analyses do not prove that the effect of one stimulus. They represent the significance of the combinatorial effect. Without proper statistical tests, the conclusions that the stimuli regulate cell wall thickness in isolation does not hold. Probably going through some R-based programs to do the statistical tests would help. But this is important.
Additional comments: No additional comments


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

Input:
Title: THE COMBINED EFFECTS OF LIGHT INTENSITY, TEMPERATURE, AND WATER POTENTIAL ON WALL DEPOSITION IN REGULATING HYPOCOTYL ELONGATION OF BRASSICA RAPA
Review round: 3
Reviewer: 1

Output:
Basic reporting: Clear English used.
Enough literature provided to help the paper.
The supplementary figures and tables do not have legends to explain the content.
Experimental design: No comments.
Validity of the findings: I thank the authors for taking care of the statistical analyses. It helps strongly support the results of the experiments included.
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: CHARACTERIZATION OF THE BACTERIAL MICROBIOTA COMPOSITION AND EVOLUTION AT DIFFERENT INTESTINAL TRACT IN WILD PIGS (SUS SCROFA USSURICUS)
Review round: 1
Reviewer: 1

Output:
Basic reporting: English used in the article is at a satisfactory level, the whole manuscript was written technically correct. First part of the paper explains in a clear way recent state of knowledge in the field of Sus scrofa ussuricus microbiomes. Notwithstanding, the authors did not avoid some minor misprints in this part:
•    Line 25 – “behavior”
•    Line 86 – “fecal”
•    Line 95 – “micobiota”
All of presented research to show the background of the study is well referenced by articles published in reputable journals. Introduction leads us through the microbiome research across all vertebrate species. Text is clearly divided into separate sections, which make it easier to read and understand.
According to figures supplied:
•    Figure 1 – these images are readable only when opened as a separate file. Fonts in axes descriptions are too small to be readable. Legend with rank-abundance on the right is completely invisible and makes it unclear without zooming in.
•    Figure 2 -  description of the figure is cut in the middle. There is no information about panels C and D.
•    Figure 3 -  description of the figure is cut  in the middle. Panels do not have assigned letters (A and B) which are referenced to in the part of the explanation.
•    Figure 4 – it is hard to estimate percent values for specific groups with this type of scale on the y-axis. The authors should consider adding visible values to the diagram separately.
•    Figure 5 – In the description the authors mentioned that diagram presents bacterial genus comparison while in the vertical row bacterial orders and families are placed. Position of Spirochaetes is doubled.
•    Figure 6 – description cut in the middle.
•    Figure 7 – description is missing and not adequate to presented diagram.
•    Figure 8 –“KEEG” is misspelled, description cut off in the middle. Vertical row without any commentary is completely unclear.
According to raw data supplied:
•    Raw sequences were uploaded correctly to PeerJ server and are possible to reanalyse.
•    It was impossible to access raw sequences via GeneBank with the attached number and submitted link. It shows that “The requested page does not exist”.
Experimental design: Research conducted by the authors meets Aims and Scope of PeerJ. Research question is clearly described in the methods section.
Line 106 - Experiment was designed to use only three animals, which may not be enough to draw far-reaching conclusions. What is more, only female individuals were used. Can the authors provide more information about the process of choosing the quantity of animals and their gender?
In lines 106 – 117 the authors describe sample preparation process. Luminal contents seem to be an appropriate material for microbiome research in general. In this specific study, to compare different parts of the intestinal tract, this approach can be questionable. Obtained luminal content contains DNA not only from the specific point of the tract that it was taken from. It consists of remains of undigested food from the previous parts of the gastrointestinal tract as well as dead bacterial cells which were living along the digestive system. Isolation of DNA and further 16S rRNA gene sequencing does not provide any distinction between identified living and dead bacterial fractions. It can lead to false positive results as sequencing can show mixed fractions from different regions of the gastrointestinal tract because of dead bacterial cells accumulation in luminal contents along the entire length of the intestine. Can the authors explain why they used contents instead of a different type of material e.g. specimens?
In line 123 the authors mentioned which 16S rRNA regions were amplified to conduct the study. Why did the authors choose only V3-V4 for this analysis? Can the authors provide any reference materials about previous studies with this type of investigation?
In line 135 the authors start to describe their library preparation process, but the information about final DNA input is missing.
In line 141 the authors showed that an outdated, no longer supported version of QIIME was used. Was there any reason to not use the most up to date QIIME2 compilation?
Sequence analysis was performed according to the previous well referenced pipeline. Unfortunately, in line 150 there is no information about Greengenes Database version used in analyses. Can authors provide this information?
Validity of the findings: Line 194 – As mentioned before, higher OTU number can be a result of accumulation of bacteria from the preceding sections of the gastrointestinal tract.
Line 265 – the authors mentioned that 3 more enriched pathways were detected in cecal samples, among others – “Epithelial_cell_signaling_in_Helicobacter_pylori_infection”. How was it possible given that H. pylori is not able to infect pigs naturally. I did not observe any H. pylori presence in the samples. Can the authors provide any information about this specific point of analysis? Definitely the part with functional analysis of the gut microbiota along the intestine tract should be extensively explained in the discussion section.
Conclusions were written clearly, according to obtained results. In 399-401 line it is suggested that “This indicates that in the process of pig evolution, the intestinal microbes in vivo also evolved, and changes in the intestinal microbial diversity is also the main driving force for the evolution of pigs.”. There are not enough samples to draw such conclusion. A group of three individuals, all representing only one gender, is too small to extrapolate results for the whole species. More research aimed at different groups should be conducted.
Additional comments: To sum up, this submission is an examples of a well-prepared study designed with the use of latest technologies that can help extend the knowledge about the role of microbiome in Sus scrofa ussuricus . Nevertheless, I had some questions about the research methods that I mentioned above. Most importantly, I would like to know why the authors used luminal contents instead of local specimens. One of the issues is also to properly share the raw data from sequencing via GenBank. The link is misleading, hence the upload may need additional action.

Thank you for reading my review. I hope it will help you make your manuscript even better.


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: CHARACTERIZATION OF THE BACTERIAL MICROBIOTA COMPOSITION AND EVOLUTION AT DIFFERENT INTESTINAL TRACT IN WILD PIGS (SUS SCROFA USSURICUS)
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: Article is technically sound.
Validity of the findings: Finding and presentation of result is satisfactory.
Additional comments: Yang et al. have presented an article "Characterization of the bacterial microbiota composition and
evolution at different intestinal tract in wild pigs (Sus scrofa
ussuricus)" where V3-V4 region of 16 S rRNA is used for preparation of metagenomic library. Upon analysis it is found that 6513 OTU of 17 phyla that include 163 genera of bacteria resided in to intestine if wild pig. Among all Actinobateria were found to be predominant in test condition. Authors emphasized that during course of evolution intestinal bacterial community changes and it happened in pig too. Article is well written and I would recommend this to be published in Peer J.