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: FOOD INSECURITY INCREASES ENERGETIC EFFICIENCY, NOT FOOD CONSUMPTION: AN EXPLORATORY STUDY IN EUROPEAN STARLINGS
Review round: 1
Reviewer: 2

Output:
Basic reporting: Excellent.
Experimental design: Potential order confound that needs explicitly discussing. Should not weaken main findings substantially.
Validity of the findings: Small sample sizes explicitly acknowledged. Findings are likely very robust to this.
Additional comments: Overall I really liked the work presented here. It addresses an important topic of broad interdisciplinary interest and generates novel, unexpected findings. Nevertheless I feel that the authors need to explicitly address a couple of issues to maximise the eventual impact of their work:

1. The authors discuss the potential importance of their work for understanding human obesity. However, I feel they do not acknowledge the limitations of using a small passerine in this context. In particular, since strategic body mass regulation is generally thought of as reflecting trade offs between the benefits of fat storage and the costs of carrying mass. In birds such costs are likely to be much more instantaneous than in terrestrial mammals (getting heavier will likely immediately impact flight performance) and so details of mass regulation dynamics are not likely to be easily generalisable to humans. I feel the authors need to acknowledge this explicitly in their discussion of the implications of their results for understanding obesity in humans.

2. As far as I can tell there is an unacknowledged order confound in their experimental design since all 6 male starlings they used went through experiments 1-3. We need details of the ordering of the experiments on these 6 birds (did they perform the experiments in numerical order? how long between experiments? etc). Since all 6 birds went through the three experiments together there is the potential for an effect of the sequence in the experimental series to impact their results. Although I can't see any way this would undermine their headline results, it needs to be acknowledged as it may help explain some of the differences among the experiments in the dynamics of the effects observed.


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: FOOD INSECURITY INCREASES ENERGETIC EFFICIENCY, NOT FOOD CONSUMPTION: AN EXPLORATORY STUDY IN EUROPEAN STARLINGS
Review round: 2
Reviewer: 1

Output:
Basic reporting: Good.
Experimental design: Good.
Validity of the findings: Good.
Additional comments: Great paper.


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

Input:
Title: FOOD INSECURITY INCREASES ENERGETIC EFFICIENCY, NOT FOOD CONSUMPTION: AN EXPLORATORY STUDY IN EUROPEAN STARLINGS
Review round: 2
Reviewer: 2

Output:
Basic reporting: Fine
Experimental design: Fine
Validity of the findings: Fine
Additional comments: I'm fine with the revised MS now.

Nevertheless, I didn't appreciate the overly dismissive treatment of the issues I raised:

1. On the issue of using small birds as models - they attacked criticisms i didn't make. It's not that i don't believe that humans suffer mass-dependent costs (I'm sure they do). Rather, the dynamics of the marginal costs of being heavier are likely to differ fundamentally between flying and other forms of locomotion. For non-flyers mass-dependent costs are less instantaneous: being a little bit heavier is unlikely to impact your ability much to move efficiently/accurately if you are swimming/walking.. So the costs of carrying additional mass are likely to be deferred over longer time periods (you accumulate more damage, are less able to sustain long bouts of activity, you accumulate chronic physiological burdens associated with being fat). This will make the direct generalisation of insights from fine-scale dynamics of strategic body mass regulation from starlings to humans difficult. Despite missing this nuance in their rebuttal, the section they've added at least acknowledges there is a controversy and deals with the low-hanging fruit criticisms.

2. Just because none of us can think of an explanatory/independent variable that was impacted by the order in which the experiments were conducted doesn't mean there isn't some hidden/unmeasured carry over influence. As I understand it, this is included in discussions of "confounds" in experimental design. But the fact that they give the reader all the relevant info in the text now is sufficient.


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: EFFECTIVENESS OF VARIOUS BIOREACTORS FOR THRAUSTOCHYTRID CULTURE AND PRODUCTION (AURANTIOCHYTRUIM LIMACINUM BUCHAXM 122)
Review round: 1
Reviewer: 1

Output:
Basic reporting: This work is a nice study on the bioreaction engineering area concerning mass transfer in bioreactors. The manuscript is clear, and well constructed. The outcomes are valuable and worthy to be published.

My main critic is that this work is not one of neither optimization nor design, but one of analysis/study about how different available aeration devices affects the performance of different bioreactors on a particular cell growth and bio-product production. So, the title does not reflect the real matter of the work.

In the hand, while the experimental aspects are well described, as well as the outcomes, there is no a related state-of-the art regarding the particular microorganism in bioreactors. Therefore, the work is likely to seam just a report of experiments. I mean, the scientific value is lost since this kind of tasks are reported even in books (e.g. Bioreaction Engineering Principles. by Villadesen, Nielsen and Lidén, Springer).
Experimental design: The experiments are well described, as well as the outcomes.

Performing diverse levels on factors is pretty expense; but an improved explanation of how these were set is convenient as well.
Validity of the findings: I think it is convenient to report the saturation concentration of oxygen, as well as the oxygen composition of injected air.

Although the increase of reactor size implies an increase on the air injection, it seems to me the resulted values of the transfer coefficient are low. Indeed a reference points how the mass transfer coeffcient increases in a stirrer tank reactor, with values greater than the ones reported here.

I found valuable the comparison of performance between reactor types.
Additional comments: The work is valuable to be published, but in its own and fair field of study. I mean, the work analyses and compare devices of aeration and bioreactors for a particular microorganism, but it does not contain neither concrete guides of design nor optimization tasks.


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: EFFECTIVENESS OF VARIOUS BIOREACTORS FOR THRAUSTOCHYTRID CULTURE AND PRODUCTION (AURANTIOCHYTRUIM LIMACINUM BUCHAXM 122)
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript aims to show the design of three types of low-cost bioreactors; stirred tank (STB), bubble column (BCB) and air-lift (ILAB) were evaluated for the cultivation and DHA production of Aurantiochytrium limacinum in combination with optimized sparger.
The manuscript is well written and use a clear and technically correct words.
The use of references is sufficient and relevant references are used.
The structure in general is correct and the authors might change some tables for figures, for example table 2 will be better presented as figure.
The manuscript perse do not present an hypothesis, due to the fact that is just a comparison of 4 bioreactors: Shake flasks, stirred tank bioreactor, bubble column and airlift bioreactor.
Some concern about experimentation are expressed in the next paragraph.
Experimental design: In the materials and methods the authors omit many details that should be taken into account so that future researchers can reproduce their results.
The presentation of the bioreactors is not complete, and in addition to Figure 1, the presentation of photographs of the same can be beneficial.
The Bubble Column Bioreactor is not really a bubble column and should be called something like Bubbled Bioreactor but not Column. The ratio of the fill volume height to the tank diameter does not meet the definition of a bubble column.
The authors do not present the type of electrodes they use to evaluate dissolved oxygen and evaluate KLa in shaken flasks and in the three types of bioreactor. It is vitally important to present the electrodes that were used and if those readings were made online and offline.
Did the authors always use the YSI dissolved oxygen meter that they report?
If all the dissolved oxygen measurements in flasks and in the bioreactor were done with YSI equipment, were these measurements offline?
If the YSI electrode was put in the shaken flasks, how to avoid the baffle effect of the same electrode?
How to avoid the problem that it had to be able to measure perfectly the oxygen changes in the flask?
What is the response time of the dissolved oxygen electrode ?,
Is this not affecting the Kla measurements, causing all the bioreactors to reach very close and similar KLa values ?.
Is not the similarity of the measured KLa an artifact of the electrode?
Why was the KLa not measured in the culture medium?, The authors of the manuscript are invited to do the Kla measurement in the culture medium and compare the differences and trends in each bioreactor when doing it only in water or with the culture medium. The relationship is not linear. The KLa measured in distilled water and in the culture medium (which is rich in nutrients and especially in a carbon source) must be completely different and also behave differently depending on the bioreactor used.
Validity of the findings: The validity of the results found in the work and the relationship of KLa with the growth of its micro-organism and the production results will depend on how the KLa measurements were made in the bioreactors. At this moment, it is difficult for this reviewer to be able to consider that the results are really related or not to mass transfer phenomena.

This section can be discussed when it is formally clarified how volumetric mass transfer coefficients were measured and calculated.
The comparison in figure 4 is not clear in the sense of why or how the experiments were done. Comparison of four culture systems under conditions. A fair comparison of the 4 culture systems should be for example at the same value of the volumetric oxygen transfer coefficient or for example at the same delivered gassed volumetric power. The authors are unclear, or at least to this reviewer why they made that decision.
The figure captions are poorly written. The authors should explain each figure caption in detail, for example figure 3 is not clear about the experimental conditions of these results.
In many of the kinetics of figure 4, standard deviations are not seen, were all the experiments done in triplicate?
Additional comments: Although the article does not present highly novel results for the biotechnology field based on the design of low-cost bioreactors, it is a well-prepared manuscript, but it omits details such as those previously commented. This particular journal does not evaluate the novelty aspects of a manuscript and therefore this reviewer has not commented on this.


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: EFFECTIVENESS OF VARIOUS BIOREACTORS FOR THRAUSTOCHYTRID CULTURE AND PRODUCTION (AURANTIOCHYTRUIM LIMACINUM BUCHAXM 122)
Review round: 1
Reviewer: 3

Output:
Basic reporting: In this article titled “Design and optimization of bioreactors based on oxygen transfer efficiency for biomass and DHA production of Aurantiochytrium limacinum BUCHAXM 122”, authors have fabricated three reactors (STR, BCB, ILAF) at low cost and chosen optimum aeration rate, sparger and agitation speed for kLa values. Then, the reactors were compared for maximum biomass and DHA production at two different aeration rates.

The following comments need to be addressed while revising the manuscript.

Basic Reporting

Author should look deep into the manuscript for grammatical errors and rectify it

Literature is well referenced in introduction and discussion

Structure is clear and conforms to PeerJ standards

In Abstract, line 4, Author has mentioned, “The bioreactors were evaluated for their influence on oxygen mass transfer coefficient (kLa) using various spargers, agitation, and aeration”. In case of BLB and ILAB, there will be no agitators which makes the statement ambiguous. Also consider modifying aeration with aeration rate.

In Abstract, line 12, author can consider the rephrasing “especially the reactors with air bubbles”.
Experimental design: Experimental Design

In materials and methods, the materials used for sparging could have been analysed for pore size through SEM for better understanding.

Line 108: The equation for determining kLa is missing closed parenthesis. Please give upper and lower limit for solving the equation.

As indicated in manuscript, DHA production is in late stationary phase. Hence author could have reduced oxygen level once it reaches stationary phase to have high productivity.

Authors should develop empirical model correlating Kla vs other operating paraments and need to validate.

Table 2 is cumbersome and difficult to interpret the data. Authors need to improve the presentation of data

Table 3 and line # 198, What is the rationale behind conducting the experiment with 0.1 vvm and in table data for all other time points are not provided. Authors should provide all the data

Power input calculations is missing. It is important to compare which process is economical.
Validity of the findings: In figure 3, the graph shows there is no significant difference between the Eight bundle super-fine air stone between control and reactors. But in line 258, author has mentioned agitator speed has positive effect on kLa values. How the authors validate the contradiction?

In Figure 4, A and B refer to shake flask experiment at 200 RPM. But Fig 4A shows has less residual glucose and more biomass than Fig 4B at 120 h. Can you elaborate on this figure?

During DHA production, please discuss on the incomplete consumption of glucose at the end of treatment?

Internal loop airlift bioreactor produces ~40 % DHA which is equivalent to shake flask yield. What is the key factor that makes IALF better than shake flask?
Additional comments: Line 68: The reactor models for STB, BCB, ILAB exists already. So, consider changing the contextual meaning of the statement.

Please mention the basis of selecting these three bioreactors in introduction.

Please mention the pore size of silicon tube sparger (control)

Line 130: PSU should be in capital letters

Line 149: Please mention the column specification and running condition for Gas chromatography. Please give reference if applicable.

In Line 257, the text is missing with the word “agitation speed” but has mentioned 300 and 600 rpm. Please include it.

Mention the aeration rate in figure 3 caption.

Significance level indicated in table with alphabets in superscript is not clear. Author should define each alphabet with their significance level.

In Table 4, Why there is difference in fermentation time for biomass concentration and DHA concentration. If it specifies individual maximum, please indicate in the referred text location.


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: EFFECTIVENESS OF VARIOUS BIOREACTORS FOR THRAUSTOCHYTRID CULTURE AND PRODUCTION (AURANTIOCHYTRUIM LIMACINUM BUCHAXM 122)
Review round: 2
Reviewer: 1

Output:
Basic reporting: In my review, my main comment was about the congruence between the title and the matter of the work, and that the manuscript presented valuable results on the field of bioreactor engineering. Also I release some additional comments to clarify some points.

In this corrected version, the title was changed in order to make it congruent with the work. I agree with the new title.
In other hand, I see that particular comments were clarified and corresponding information was included in the manuscript.

Then, I consider the manuscript as valuable to be published.
Experimental design: In this corrected version, there were included more explanations about the experimental design. I consider all of the as convenient, in such a way the purposes and procedures of experimentation can be followed.
Validity of the findings: In this corrected version, explanation of results are better backed with references, in such a way the conclusions are well stated and linked to results.
Additional comments: The manuscript is susceptible of improvement. I am not an expert in English language, so I do not dare to point sentences and paragraphs, but I felt that some lines could be better written.


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: EFFECTIVENESS OF VARIOUS BIOREACTORS FOR THRAUSTOCHYTRID CULTURE AND PRODUCTION (AURANTIOCHYTRUIM LIMACINUM BUCHAXM 122)
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors did their best to respond to the comments made by this reviewer.
Experimental design: The authors did their best to respond to the comments made by this reviewer.
Validity of the findings: The authors did their best to respond to the comments made by this reviewer.
Additional comments: The authors did their best to respond to the comments made by this reviewer.


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: FIRST BODY OF EVIDENCE SUGGESTING A ROLE OF A TANKYRASE-BINDING MOTIF (TBM) OF VINCULIN (VCL) IN EPITHELIAL CELLS
Review round: 1
Reviewer: 1

Output:
Basic reporting: English and layout mostly good.
Context of study fairly well described.
Weak written results section: it is very brief and does not sufficiently describe the findings.
No attempts at quantification or statistical analysis of any of the findings, meaning results are purely descriptive.
Experimental design: Comments on specific figures and how they could be improved below:
Fig1: good
Fig2a: Agree that most TNKS is in intracellular membrane pool. There is perhaps a weak band in the plasma membrane fraction. 2b, c and d agree with the fractionation; it looks like nearly all the TNKS is on intracellular membranes. 2d. Some of these arrows don’t seem to point to any discernible staining. Agree that there is faint staining on right-hand cell that could be PM. Would need a plasma membrane marker to confirm this, as well as suitable antibody staining controls (at a minimum secondary antibody alone as well as either: 1. siRNA depletion of TNKS to show specificity of staining. 2. An alternative TNKS antibody showing the same staining pattern. 3. An exogenously expressed, tagged version of the protein also displaying the same localisation).
Fig 3. Are these images all taken with the same microscope settings? The F-actin staining seems to vary markedly between the images, making them difficult to compare. Also, the results text description of figure 3 is extremely brief; simply saying that all the inhibitors effect the PAR belt, and yet the different inhibitor treatments look quite different. Can these be related to the potency/specificity of the inhibitors described in table 2? Also cells in C and H don’t look confluent and therefore probably couldn’t form cell-cell junctions, so authors must ensure seeding density is comparable between all conditions.
Fig 4a good.
Fig4c-s: Evidence for co-localistaion  of Par and vincluin not clear, although there may be some overlap. Can co-localisation analaysis on confocal be performed?
Figure 5. Very hard to determine how cells are fitting in monolayer (or otherwise) when neighboring cells not visible. In any case, the two cells expressing mutant vinculin don’t appear any more “irregular” or “mesenchymal” than the cell expressing the wildtype vinculin. I don’t think there is much value to this figure in its current form. Therefore, the inferences made about the function of this site in vinculin are, in my opinion, unjustified by this data.
Validity of the findings: Although based on some potentially interesting observations, the data presented is underdeveloped and, as noted in the first section, nearly entirely descriptive. A major point is that at least some of the data presented here need supporting with quantification. Also, in some cases experiments could fairly easily be extended to make them a lot more informative. For example, in the final figure, the authors have gone to the effort of designing, cloning, expressing and imaging a potentially important mutant version of vinculin and yet the data presented is very basic and therefore cannot support the hypothesis that there may be a functional role of the mutated sequence.
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: FIRST BODY OF EVIDENCE SUGGESTING A ROLE OF A TANKYRASE-BINDING MOTIF (TBM) OF VINCULIN (VCL) IN EPITHELIAL CELLS
Review round: 1
Reviewer: 2

Output:
Basic reporting: No comment.  Meet criteria.
Experimental design: Additional controls, as detailed in "general comments", will make the findings and claims more convincing.
Validity of the findings: no comment.
Additional comments: The authors took a logical approach to support the novel notion that TNKS-related PAR belt near AJ or colocalizes with vinculin at the plasma membrane in VERO cells, and that loss of TNKS-vinculin interaction may promote EMT of MCF cells.  Although the designs are well-thought out, some important controls are missing, and some findings are not clearly described.

Fig 2A: ideally, cells should be treated with or without TNKS and PARP1 inhibitors (simultaneously).  Bands that disappear upon inhibitor treatment are likely to be bona fide PARylated proteins (not just nonspecific binding to the detecting agents)

Fig. 3.  Please use red-channel image (actin alone) to specifical describe how TNKSi alters subcortical actin ring.  Does TNKSi make the ring “serrated discontinuous”.

Fig 3: please explain why TNKSi apparently failed to make the PAR belt disappear.

Fig. 3: Highly selective TNKS inhibitors like G007 is not expected to diminish nuclear PAR levels, which are primarily the product of PARP1.  Therefore, please explain why nuclear PAR staining intensity is much stronger in panel A than in other panels.  Were images acquired and processed using the same parameters?

Fig. 4A  The WWE resin, by binding directly to PAR, will precipitate PARsylated proteins as well as (indirectly) their associated partners (which may not be PARsylated per se).  This limitation should be included in the discussion.  In this figure, it would be very informative (linking vinculin directly to TNKS) to show that after TNKSi treatment, vinculin is no longer detected in the affinity-precipitates.

Fig 4, panels F-K, please use arrow heads to point out yellow pixels, indicative of VCL-PAR colocalization

Fig. 5: please show more cells (not just 1 cell compared against 2 cells) and specifically point out their differences.

Minor concerns:

Title page and line 1:	Change “evidences” in the title to “evidence”

Line 79: change loose to loss

Line 121, change to “two being canonical”

Line 260: please elaborate on how MABE 1031 works


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: FIRST BODY OF EVIDENCE SUGGESTING A ROLE OF A TANKYRASE-BINDING MOTIF (TBM) OF VINCULIN (VCL) IN EPITHELIAL CELLS
Review round: 2
Reviewer: 1

Output:
Basic reporting: This has improved a lot. My main concerns were a brief and weakly written results section and lack of quantitation but these have both been clearly addressed.
Experimental design: All my points addressed, as well as a significant new undertaking by the authors to quantify their cell morphology results and improve microscopy images and accompanying figure legends.
Validity of the findings: As mentioned above, findings now backed by significant amounts of quantitation, adding considerably to their validity.
Additional comments: Thanks for making the effort to respond so thoroughly to my initial suggestions. I hope you agree that the manuscript is now significantly improved.


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: FIRST BODY OF EVIDENCE SUGGESTING A ROLE OF A TANKYRASE-BINDING MOTIF (TBM) OF VINCULIN (VCL) IN EPITHELIAL CELLS
Review round: 2
Reviewer: 2

Output:
Basic reporting: No comments
Experimental design: No comments
Validity of the findings: Line 386: the authors stated that VCL was affinity precipitated by macrodomain resins, and the precipitation was abolished by G007.  However, a close inspection of Fig. 2S failed to reveal any VCL in the pulldown.  Please clarify.  Fig. 2S is critical to the claim that VCL is indeed PARylated by TNKS.
Additional comments: The authors have robustly improved the manuscript.  Congratulations.

Minor comments:

Line 73: missing a period
79: loss instead of lose
133: missing a parenthesis
184 “for” 2 hours
196: subjected instead of subject
212: vice versa instead of viceversa
230: remove extra parenthesis
238 typo: symmetry
259: TBM instead of TBD
289: 14.5 instead of 14,5
377: “a fraction of VCL” instead of “a VCL fraction”
396 typo
531: further instead of deeper

table 1: please confirm the concentration unit of DAPI
table 2: please define tcPARP, and reveal the concentration unit for IC50

127: define TBM-II at its first appearance in the text
245: please briefly introduce NMuMG cells
322: please point out the evidence of TNKS being detected in the plasma membrane fraction (as stated in the paragraph heading)
400: please elaborate on this sentence

Several figure legends contain multiple occurrence of mysterious words like <!--[if !supportLists]-->


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

Input:
Title: IDENTIFICATION OF KEY MICRORNAS AND THE UNDERLYING MOLECULAR MECHANISM IN SPINAL CORD ISCHEMIA-REPERFUSION INJURY IN RATS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript should be revised by a native English speaker.

Raw data was not shared.
Experimental design: The authors should inform when the Pubmed search was performed. Also, if any exclusion criteria were applied, this should be described.

Figure 4: Which rat strain was used?

Figure 4B-E: The authors should describe how the statistical analysis was performed. Which test was used? Which software was used? Were the normality of data and the homogeneity of variances assessed?

Table 2: the authors should include more information about the studies, such as the time points, the models of SCII, as well as more information about the animals (age, sex and strain).

Table 3: I suggest that the authors inform the number of times that each DEmiRNA was found in independent datasets. Moreover, considering that each miRNA was not evaluated in all datasets, it would be interesting to show the percentage of times that each DEmiRNA was found.

I also suggest to include a table showing the discrepant results (i.e., DEmiRNA that were upregulated or downregulated in at least 2 studies, but that appeared with a change of expression in the opposite direction in another dataset).
Validity of the findings: It is not completely clear why only miR-3568 was selected for the validation study. If other miRNAs were tested and there were inconclusive results, these results should be shown.
Additional comments: Figure 4b: the label of the y axis should be corrected.


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

Input:
Title: IDENTIFICATION OF KEY MICRORNAS AND THE UNDERLYING MOLECULAR MECHANISM IN SPINAL CORD ISCHEMIA-REPERFUSION INJURY IN RATS
Review round: 1
Reviewer: 2

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: This manuscript aims to interrogate differentially expressed miRNAs in rat models of spinal cord ischemia/reperfusion. First, they surveyed public repositories of miRNA expression in the field and found 19 miRNAs that were consistently altered in ischemic/reperfused rats versus controls. They next attempted to determine molecular pathways and networks that could be deregulated by DEmiRNAs. Finally, they attempted to experimentally confirm alterations in miR-3568 and target genes. Overall, this is a timely and interesting manuscript involving bioinformatics analyses and experimental data. However, there are some issues that need to be addressed before publication.

1) Given that 19 miRNAs were found to be deregulated in rat models of ischemia/reperfusion (as per public data), I strongly suggest that the authors use their experimental model to validate more of these targets, as this would be an important contribution to the field.

2) I feel that the current discussion and interpretation of the obtained data fails to shed light on potential mechanisms/targets for spinal cord ischemia/reperfusion. For example, it is not clear enough how mir-3568 or the KEGG pathways could specifically mediate in the pathogenesis of spinal cord injury. Authors should also revise their discussion to more thoroughly convey their story.

3) Detailed statistical information are missing for all figures and should be included (e.g. statistical tests and parameters, sample size, definition of N).

4) Figure 4B-E: data should be presented as a scatter dot plot (for transparency), and the number of samples per group should be disclosed. In figure 4B, the graph label shows mir-374-5p, which I believe is a typo. Please check it.

5) Does suppression of miR-3568 rescue spinal cord injury/reperfusion-associated phenotypes?


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

Input:
Title: IDENTIFICATION OF KEY MICRORNAS AND THE UNDERLYING MOLECULAR MECHANISM IN SPINAL CORD ISCHEMIA-REPERFUSION INJURY IN RATS
Review round: 2
Reviewer: 1

Output:
Basic reporting: ok
Experimental design: ok
Validity of the findings: ok
Additional comments: The authors did a great job and the article improved. I only have the following comments:

1) Please include the catalog numbers (or RRID, if available) of the antibodies used for Western Blotting.

2) Full Western Blots should be shown in supplementary figures.

3) Bar graphs should ideally be replaced with graphs showing the individual data points.


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

Input:
Title: IDENTIFICATION OF KEY MICRORNAS AND THE UNDERLYING MOLECULAR MECHANISM IN SPINAL CORD ISCHEMIA-REPERFUSION INJURY IN RATS
Review round: 2
Reviewer: 2

Output:
Basic reporting: This manuscript substantially improved after revision, with all requirements for basic reporting sufficiently addressed.
Experimental design: No comment
Validity of the findings: My final suggestion for the authors is that data on Figures 3, 7 and 8 are presented as scatter dot plots, so that readers can identify the whole datasets.
Additional comments: My comments were adequately addressed.


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

Input:
Title: IDENTIFICATION OF POTENTIAL GENE SIGNATURES ASSOCIATED WITH OSTEOSARCOMA BY INTEGRATED BIOINFORMATICS ANALYSIS
Review round: 1
Reviewer: 1

Output:
Basic reporting: NA
Experimental design: NA
Validity of the findings: NA
Additional comments: In this manuscript “Identification of potential gene signatures associated 1 with osteosarcoma by integrated bioinformatics analysis”  Jia et al. have identified the differentially expressed genes in osteosarcoma patients and performed functional enrichment analysis to identify certain genes with prognostic value. Authors have performed multiple bioinformatics analysis but biological insights gained from the analysis is not discussed. Some of the major comments that need to be addressed are:

1. It is not clear why autjors have relied on microarray data when TCGA has RNA-seq information of almost 100 osteosarcoma patients. Reanalysis with RNA-seq data will show the robustness of the results.

2. Authors have used different datasets from GEO. How these different datasets were batch corrected and normalized together and  differentially expressed genes were identified?

3. What does color represent in Figure 1. Font of the text in the figure is very small and unreadable. What does these network diagrams in panel B represent? How these top genes cluster together? A heatmap representation of top DEGs could help in understanding the variations in the expression levels of these genes acorss various patients.

4. Are these identified DEGs enriched to be hub genes in protein interaction map analysis? STRINg includes many other interactions than just experimentally validated interaction data. Similar analysis across various other datasets (e.g. IrefWeb, HumanNet) could give a clear picture of the interaction patterns of these DEGs.

5. Figure4: Indicate the p values of significance. Also, show the number of samples in each boxplot. Instead of relative expression level, absolute expression level can clearly show if the differences in expression are robust in an independent dataset.

6. ROC analysis is not informative as numbers of samples are very low. Does any of these genes are predictive of survival status in osteosarcoma patients? Are these genes useful to predict drug response in these patients?

7. There are spelling mistakes at some places in the manuscript and English needs some correction.


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

Input:
Title: IDENTIFICATION OF POTENTIAL GENE SIGNATURES ASSOCIATED WITH OSTEOSARCOMA BY INTEGRATED BIOINFORMATICS ANALYSIS
Review round: 1
Reviewer: 2

Output:
Basic reporting: 1. Relevant prior literature has not been appropriately referenced. For example,
Identification of potential crucial genes and key pathways in osteosarcoma PMID: 32665038
This study is very similar with the paper I mentioned above.
2. In Figure4, why the authors show box plots of seven differentially expressed genes, why not other DEGs? and no statistical analysis was used to compare, this data is of no meaning.
3. The quality of Figure 5 is poor.
Experimental design: This work aims to 20 screen novel potential gene signatures associated with OS using bioinformatics anaysis.
1. The analysis and design is very traditional and lack of novelty.
2. The authors downloaded four datasets from the GEO database, however, the detailed information of these datasets has not been provided. Are these datasets from the same platform?
3. The authors should make it clear how did they processed the four datastes?  If these datasets come from different platform?  How did they combining these datasets? Has batch correction been down? I did't see the quality control data in this manuscript
4.  What is the cutoff value of Fold change of DEGs were not mentioned .
5. What is the significance of hierarchical clustering analysis of top 100 DEGs?
6. What is the meaning of top15 nodes? The authors should make it clear how and why they choose top 15 , but not top 20? or top 10? top 5? they should also clearly define how the top 15 nodes were choose?
7. Why the top 20 up- and down-regulated genes were regarded as the candidate genes? Why not 10 up- and down? What is the reason.
8. In Validation of DEGs and ROC analysis section, the authors state they validate the DEGs using ROC, I do not agree this is validation. It is just another kind of show of the DEGs. It is not a validation.
Validity of the findings: 1.  Expression of microarray data is used to be correlated with bias, and the sample used in this study is very limited. If possible, the authors should use other independent datasets to validate their results.
2. One major issue is the batch effect between the four datasets.
Additional comments: 1. In fact, this paper is very similar to published articles.
2. The authors should clearly point out what contributions they did ? The potential genes? this data is not very solid.


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

Input:
Title: IDENTIFICATION OF POTENTIAL GENE SIGNATURES ASSOCIATED WITH OSTEOSARCOMA BY INTEGRATED BIOINFORMATICS ANALYSIS
Review round: 1
Reviewer: 3

Output:
Basic reporting: English language should be improved. There are some parts that were unclear and ambiguous. Also, there contain some grammatical errors and typos such as:
- High-throughout sequencing ...
- Sun et al evaluated difference ...
- ... biomakers for OS ...
- ... is a antimicrobial peptide gene ...
- ... which may be as a potential ...
- ... these two gene were both ...
- ...
Therefore, the authors should re-check and revise carefully. It is better to be checked by a native speaker or English editing service.

The authors should provide more background in the abstract and introduction. Now the motivation or objective of the study is unclear.

Quality of figures should be improved.
Experimental design: What is "porgn"?

The authors should explain clearly how they considered the inspection and removal of batch effects when merging five sets of data from GEO.

The authors used R to implement the analyses. It is important to provide the source codes to help reproducing the results.

ROC curve & AUC have been used in previously biomedical works such as PMID: 31277574, PMID: 31921391, and PMID: 32613242. Therefore, the authors should refer more works into this description to attract broader readership.
Validity of the findings: What is the cut-off value for GO enrichment analysis?

In the Fig. 5's legend, the authors mentioned x‑axes and the y‑axes show specificity and sensitivity, respectively. However I think x-axes should be "1-specificity".

The authors should compare the performance between 7-genes signature and all-genes signature in terms of ROC curve or AUC, or pathway analyses.

How about prognostic biomarkers for these genes?

The authors convinced some different genes from previous works on OS, then they should compare the performance results with the previous works and to see which gene set is really significant in this disease.
Additional comments: No comment


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

Input:
Title: IDENTIFICATION OF POTENTIAL GENE SIGNATURES ASSOCIATED WITH OSTEOSARCOMA BY INTEGRATED BIOINFORMATICS ANALYSIS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The same with previous review.
Experimental design: The same with previous review.
Validity of the findings: The same with previous review.
Additional comments: The authors failed to response to my review comment carefully.

All comments should be considerd carefully instead of making a perfunctory effort.


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

Input:
Title: IDENTIFICATION OF POTENTIAL GENE SIGNATURES ASSOCIATED WITH OSTEOSARCOMA BY INTEGRATED BIOINFORMATICS ANALYSIS
Review round: 2
Reviewer: 2

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: My previous comments have been addressed satisfactorily.


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: BRACON WASPS FOR ECOLOGICAL PEST CONTROL–A LABORATORY EXPERIMENT
Review round: 1
Reviewer: 1

Output:
Basic reporting: The paper is written in clear and professional English. There are some sentences that I believe should be reworded, which I noted in the annotated pdf. Otherwise, the paper is well written.
Most of the relevant literature is reported however, I believe two papers on the temperature effects on B. brevicornis, which I think should be cited since they are very similar to the study conducted.
Rao, B. R. S., and S. S. Kumar. 1960. Effect of Temperature and Host Density on the Rate of Increase of Bracon brevicornis Wesmael (Hymenoptera: Braconidae). Beiträge zur Entomol. 10: 872–885.
Thanavendan, G., and S. Jeyarani. 2010. Effect of different temperature regimes on the biology of Bracon brevicornis Wesmael (Braconidae: Hymenoptera) on different host larvae. J. Biopestic. 3: 441–444.
The article is well structured, and the hypotheses are clearly stated and supported by data.
I only have two comments on the raw data provided.
Supplemental data:
Appendix 1 Strains: Specify where the B. brevicornis was caught with a hand net if possible
Appendix 2: Unclear what ges stands for.
I have a few comments on the figures and tables, which I noted in the annotated pdf. My major comment is that I do not believe both tables and graphs should be included in the main paper as they represent similar data.
The paper is self-contained, and the results are relevant to the hypotheses.
Experimental design: This is original primary research on the B. brevicornis and B hebetor parasitoids reared on Ephestia kuehniella. However, to improve the introduction  and conclusion clearly emphasize why E. kuehniella is important, and why temperature and host density are important for parasitoid rearing.
The methods are described in sufficient detail and information to replicate. Methods are also typical of those found in other parasitoid rearing papers. The investigation had sufficient replication and was performed at a high technical and ethical standard.
Validity of the findings: All underlying data have been provided, and statistics are appropriate for this study. Conclusions are well stated and linked to original research questions.
Results support the majority of the discussions and conclusions, however, there is some speculation on how these results can be used for the rearing of the Bracon species on other hosts. This final speculation on lines 331- 336 is not identified as being such.
Additional comments: I made suggestions for minor improvements in the annotated pdf.


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

Input:
Title: BRACON WASPS FOR ECOLOGICAL PEST CONTROL–A LABORATORY EXPERIMENT
Review round: 1
Reviewer: 2

Output:
Basic reporting: The paper perfectly falls within the scope of the journal and deserves publications, provided some improvement. I will recommend getting the English of the manuscript thoroughly edited.
I have made some suggestion in the PDF but they are not exhaustive.
The introduction should cover some basic on the two parasitoid wasps biology such as temperature requirement and life table. When referring to host species of Bracon hebetor you should include Corcyra cephalonica which is widely used in Africa and India. It is a bit surprising you missed C. cephalonica while Ostrinia nubilalis which is not among most common host species is cited.
As a general rule, make sure that when referring to different organisms for the first time in the manuscript to use the recommended nomenclature (e.g. Bracon hebetor Say). It will also be good to mention that Bracon (= Habrobracon) hebetor.
-A short description of the two Bracon species is needed as B. hebetor and B. brevicornis are sometime being confused.
-Regarding Tables and Figures, I’ve had the impression that the data use to generate table 1 where the same used in Figure 1. Why do you need to present total number of emergence in figure 1 and them break it per sex in table 1 while presenting the sex ratio in the same table. This is redundant. The only new data in Table 1 as compare to Figure 1 is the sex ratio. Please discard table 1 and present the sex ratio in the text while commenting the result on offspring emerging from single host-larva parasitism. The same comment may apply to Table 4. Please check other tables to avoid duplication of data presented in figures.
-Figures and tables captions must be revised as one should be able to understand what the Figure/table upfront. I have made some suggestion (see attachment)
Experimental design: In the experimental section it is stated that after five days, the generation was frozen and newly hatched wasps were used. This is unclear, please explain clearly what you meant.
The experiment on reproductive success of female wasps on a single host larva at different temperatures is not well described. It is unclear for how long Bracon was given the host larva to parasitize. It is also unclear what the replicate stands for, is it 1 female of a given species at a different temperature. You should mention clearly the number of replicates you used while starting the experiment. I can understand that at the end some replicates are not considered for one of the reasons that you mentioned (survival rate), but I do not understand why you discarded some replicates because of delayed in the parasitoid developmental cycle.
While testing Bracon with different host larvae, It is unclear how many replicates were used for each species and strain at a given host-ratio (1:5; 1:10; …1:25) and given temperature (12 to 36°C). It would have also been good to know the rationale behind the number of host larvae (5 to 25) that you choose to test. Likewise, what is the rationale for confining host larvae with Bracon parasitoid for 4 days, why not 48hrs. These questions (host exposure duration and host-parasitoid ratio) could have been avoided if enough background information was given in the M&M section while describing the two species
-In Figure 3 & 4 it appears that you didn’t test the same number of host-larva at different temperatures (5,15, 25 larvae at 12C and 27C and 10, 20 larvae at 20C and 36C). This should be explained in the Material and Method.
 -I do not understand why parasitism of Bracon in figure 5 is not expressed as percentage. This should be corrected.
Validity of the findings: The findings are good but not always well presented.
-There is a need to discuss the findings in relation to previous one reporting 200-400 offspring produced per Bracon hebetor female in other settings with different host species (see Kabore et al., 2019; Nikam and Pawar 1993; Yu et al. 1999; Chen et al. 2011). Likewise, when comparing the performance of Bracon with other studies it should be good to consider the diet on which the host-larva was reared as this could influence parasitism and progeny production (see Amadou et al. 2019). By the way this is missing in the M&M section.
In the conclusion it is suggested to test the usefulness of Bracon wasps in controlling insect pests in field situation. This should not even be questioned given that Bracon hebetor is already used in augmentative biological control program in Asia and Africa for several insect pests (search literature on Bracon hebetor against millet head miner in Africa and Bracon hebetor against Carob moth in Iran).

Here is a list a reference that you find useful:
Yu S-H, Ryoo MI, Na JH (1999) Life history of Bracon hebetor (Hymenoptera: Braconidae) on Plodia interpunctella (Lepidoptera: Pyralidae) on a dried vegetable commodity. J Asia Pac Entomol 2: 149–152

Nikam PK, Pawar CV (1993) Life tables and intrinsic rate of natural increase of Bracon hehetor say (Hym., Braconidae) population on Corcyra cephalonica Staint. (Lep., Pyralidae), a key parasitoid of Helicoverpa armigera Hbn. (Lep., Noctuidae). J Appl Entomol 115: 210–213

Chen H, Opit GP, Sheng P, Zhang H (2011) Maternal and progeny quality of Habrobracon hebetor say (Hymenoptera: Braconidae) after cold storage. Biol Control 58:255–261

Amadou L, Baoua IB, Ba NM, Muniappan R (2019) Develop- ment of an optimum diet for mass rearing of the rice moth, Corcyra cephalonica (Lepidoptera: Pyralidae), and pro- duction of the parasitoid, Habrobracon hebetor (Hy- menoptera: Braconidae), for the control of pearl millet head miner. J Insect Sci 19:1–5

Kabore, A., M. N. Ba, C. Dabire-Binso, A. Sanon. 2019. Towards development of a parasitoid cottage industry of Habrobracon hebetor (Say): optimum rearing and releases conditions for successful biological control of the millet head miner Heliocheilus albipunctella (De Joannis) in the Sahel. Int. J. Trop. Insect Sci. doi: 10.1007/s42690-019-00005-w
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: BRACON WASPS FOR ECOLOGICAL PEST CONTROL–A LABORATORY EXPERIMENT
Review round: 2
Reviewer: 1

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


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

Input:
Title: BRACON WASPS FOR ECOLOGICAL PEST CONTROL–A LABORATORY EXPERIMENT
Review round: 2
Reviewer: 2

Output:
Basic reporting: The Manuscript has been significantly improved and reads better. My recommendations have all been taken into consideration. I have however made few suggestions in the PDF for

 -As a general rule always give Order and Family of organism when citing them for the first time in the text (e.g. Ostrinia nubilalis Hübner (Lepidoptera: Crambidae); Orius insidiosus Say (Hemiptera: Anthocoridae). Please check for other organisms in the attachment.

-Regarding Tables and Figures, I’ve had the impression that the data use to generate table 4 where the same used in Figure 5.

-Check attachment for further suggestions
Experimental design: -It is important to indicate initial number of replications thought some were discarded due to failure.
- I do not agree with the method use for computing parasitism. Unless you have supporting reference, a parasitized larva is a host larva on which the parasitoid lay eggs. Bracon can sting and paralyze the host larvae without laying egg on it (see Kabore et al., 2019). The host-larvae can eventually be killed as a result of host feeding but not due to parasitism. In your case host feeding may have been important given that parasitoids were not given any food (honey, sweet solution…)
Validity of the findings: My previous comments have all been addressed. However I am suggesting within the discussion to consolidate all the section related to sex ratio in one paragraph.
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: NEW INSIGHT INTO THE DIVERGENT RESPONSES OF PLANTS TO WARMING IN THE CONTEXT OF ROOT ENDOPHYTIC BACTERIAL AND FUNGAL COMMUNITIES
Review round: 1
Reviewer: 1

Output:
Basic reporting: 1. The manuscript uses clear and unambiguous English. However, there are some sentences that need improvement. I recommend a native English-speaker to proofread the text for minor editing, but some examples of sentences to be corrected are:

 L. 26-27: The gap this study addressed is not clearly stated in the abstract. Perhaps the issue here is the structural problem of the sentence. Please, review this sentence to improve comprehension.

L. 33-35: Rewrite this sentence to improve comprehension. My suggestion is: "Both elevation and host identity significantly affected the composition of root endophytic bacterial and fungal communities."

L. 306: insert a “to” before “host”

2. The references cited in the manuscript provide the necessary background of the study.

3. Raw data were shared and it is accessible.

4. All figures are relevant for the study, but a few corrections should be carried out to improve comprehension:

Figure 1, please, add the statistics used to find differences between groups.

Figure 2, I was a bit lost regarding letters A, B, C, D etc. One suggestion is to label A and B (and so the others) in the horizontal line of the figure to improve comprehension.

Figure 4: what are the yellow circles? Please, include this info in the figure legend.

6. Structure of the manuscript:

L. 109-111: I believe this sentence fits better before introducing the model system of study (in L. 102).

L. 223-226: These sentences fit better at the beginning of the results section (L. 215).

The abstract is clearly written for the most part. I was a bit frustrated at the end of the abstract because there is no general conclusion of the study. Thus, to improve even more the abstract, a general conclusion could be added at the end of the abstract. In this conclusion, try to link it with the gap of the study.
Experimental design: The manuscript brings original results of environment effects on the diversity and composition of plant-endophytic communities. The research question is well defined and fills the gap in the current literature (i. e. linking adaptations of host plant to environmental stress with changes in root-endophyte communities. In order to help readers understanding the experimental set up, please consider the following:

L. 129: How many plants were sampled per plot? Were the data of different plant species averaged considering the same plot? I did not find the actual number of samples (roots and plant individuals) analyzed in this study. Because of that, interpretations of figures and tables are somewhat difficult. It appears from the text that three samples were analyzed at each altitude per plant species. This should be clear stated in the manuscript.

Also, why choose the 20 TOP OTUs and not the 10 TOP OTUs? What was the criteria for this selection?
Validity of the findings: All underline data was provided. The conclusions are well stated. My only concern with the conclusions is how the sampled sized used in the experiments is sufficient to support the conclusions. See my comments above about the sample size.
Additional comments: This is an interesting manuscript that explored the association of host environmental adaptation to stress conditions and changes in endophytic microbial communities. I commend the authors to explore this link, because this is indeed a gap in the literature. When reading this manuscript, I had the following thoughts that I think it is worth to bring and I hope they can be useful for strengthening the message of this manuscript.

1.	In their 2005 review, Barbara Schulz and Christine Boyle state that endophytes do not necessary establish harmonious symbioses with plant hosts. Is there a particular reason to focus only on the "good" side of endophytes? Please, check the review in 10.1017/s095375620500273x

2. L. 61-62: Are the endophytic communities responding to the plant stress or are the plants responding to the changes of endophytic communities?

3. The manuscript brings that “…greater abundance of genes associated with nutrient absorption and oxidation resistance in warmer environments (lower altitude) might contribute to the growth of E. nutans under global warming (Fig. 6).”

I would suspect the genes “associated with nutrient absorption and oxidation resistance” should be abundant not only in E. nutans but also in K. pygmaea bacterial microbiome, because they are also necessary to survive in cold conditions.


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: NEW INSIGHT INTO THE DIVERGENT RESPONSES OF PLANTS TO WARMING IN THE CONTEXT OF ROOT ENDOPHYTIC BACTERIAL AND FUNGAL COMMUNITIES
Review round: 1
Reviewer: 2

Output:
Basic reporting: Wei et al. studied the response of two plant species and their endophytes in relation to warming condition. Authors studied both bacteria and fungi. The article is generally well written.

I feel there is still some scope for the improvement of the manuscript. My suggestions are following in other sections.
Experimental design: Authors selection of two plant species is good enough to answer their research question. The methods used are very standard in the plant endophytes research.

Authors have not mentioned that how many plants (roots) were sampled and how many of them were sequenced. Number of samples per plot or per plant species and total number samples should be clearly explained in the materials and methods section.
Validity of the findings: I highly appreciate that the authors shared the raw data of this study. The standard statistical methods are used in the manuscript.
Additional comments: Line 111 to 114: The introduction is more general. I feel detailed explanation of their hypothesis citing relevant literatures is necessary to make the readers understand the scope the of the work well. The different plant species can have different endophytes just because of their physiology. Please explain it in detail still how do you prove your hypothesis in that case.

Line 150, 186 and in abstract: Please use the terms same throughout the manuscript. For example, 16S rRNA gene,16S rDNA and 16S amplicons are used in the manuscript. It should be corrected as 16S rRNA gene through out the manuscript.

Line 150 to 154: Authors should cite the publication relevant to the primers they have used.

Line 150 to 151: I hope the 16S rRNA gene PCR amplification results in mitochondrial as well as bacterial amplification. Could you give some details about how did you overcome this problem?

Line 157: ......RNA amplification....? please correct it. It is not clear.

Line 297 to 299: How the results consistent with your hypothesis? It is just partially consistent as the bacterial diversity is different? Change in fungal diversity does not mean that plants are adopted due to that.  Please give more explantation.


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: NEW INSIGHT INTO THE DIVERGENT RESPONSES OF PLANTS TO WARMING IN THE CONTEXT OF ROOT ENDOPHYTIC BACTERIAL AND FUNGAL COMMUNITIES
Review round: 1
Reviewer: 3

Output:
Basic reporting: See below.
Experimental design: See below.
Validity of the findings: See below.
Additional comments: This paper has analyzed whether and how elevation influences fungal and bacterial composition  in  two contrast plant species, one more adapted to high elevation (K. Pygmaea), the other more adapted to low elevation (E. Nutans ) in Qinghai-Tibet Plateau.
ITS and 16S meta-sequencing analysis conducted in this study suggest the following results.
1. Plant genotype influences both fungal and bacterial composition. This seems to be consistent with the previous studies.
2.  Diversity of fungal communities in E.  Nutans significantly decreases in a negative correlation with elevation.  This might be because E. Nutans grow poorly in high elevation.
3.  Fungal or bacterial species specifically enriched in either plant species are identified.
4.  K. Pygmaea associates with bacteria with genes related to Cold resistance. In contrast, E. Nutans associates with bacteria with genes related to nutrient acquisition.  This seems to be reasonable results considering their habitat ( K. Pygmaea more grows in colder elevation than in E. Nutans).

Over all, this paper contains useful information for researchers studying microbiome and ecology. The method itself seems to be approbate. However, due to poor English throughout the manuscript, I felt difficulties to catch the contents.  It is essential for the authors to submit this paper to a professonal English editing service.

Minor points

: In material and method,  I don’t fully catch how the authors collected the samples from two different plant species grown in various elevation. It is necessary to show this in a single picture.   I think modifying figure 6 would be useful for this purpose.
: Please describe how many plant samples are used per one conditions.


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: NEW INSIGHT INTO THE DIVERGENT RESPONSES OF PLANTS TO WARMING IN THE CONTEXT OF ROOT ENDOPHYTIC BACTERIAL AND FUNGAL COMMUNITIES
Review round: 2
Reviewer: 1

Output:
Basic reporting: Authors improved the manuscript in response to all the reviewers comments.
Experimental design: The experimental design is very standard in the manuscript.
Validity of the findings: All the data are available.
Additional comments: Thank you for your response to my comments.


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

Input:
Title: NEW INSIGHT INTO THE DIVERGENT RESPONSES OF PLANTS TO WARMING IN THE CONTEXT OF ROOT ENDOPHYTIC BACTERIAL AND FUNGAL COMMUNITIES
Review round: 2
Reviewer: 2

Output:
Basic reporting: This version is significantly improved. I have only pointed out a minor point as described below.
Experimental design: Looks fine.
Validity of the findings: This should be fine.
Additional comments: The authors have mentioned that pants belonging to Brassicaceae recruit another fungus, Helotiales, under phosphorus-limited conditions (Almario et al., 2017).  To mention this,   it would not be appropriate to cite Hiruma et al., 2016 that show that A. thaliana recruit an endophytic fungus Colletotrichum tofieldiae, under phosphorus-limited conditions.
Although it is not essential, this paper would be significantly improved if the authors have cited appropriate papers related to plant-microbe interactions.


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: ONLY TWO OUT OF FIVE ARTICLES BY NEW ZEALAND RESEARCHERS ARE FREE-TO-ACCESS: A MULTIPLE API STUDY OF ACCESS, CITATIONS, COST OF ARTICLE PROCESSING CHARGES (APC), AND THE POTENTIAL TO INCREASE THE PROPORTION OF OPEN ACCESS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript of White et al is a clear and well constructed description of the state of Open Access in New Zealand for articles published in 2017.

The literature review covers the main topics well and discusses issues with the measurement of open access levels. The authors may want to consider updating the literature with two recent pieces of work (Robinson-Garcia N, Costas R, van Leeuwen TN. 2020. PeerJ 8:e9410 https://doi.org/10.7717/peerj.9410 and Huang et al 2020.BiorXiv https://doi.org/10.1101/2020.03.19.998336, acknowledging that our recent piece is a preprint at this point, currently undergoing peer review).

The tables are clear and well laid out and the conclusions are justified by the evidence presented. I note that the data, and the code is made available. I have some queries below with respect to data analysis and provide my own attempts at replication.

The replication is conducted in a Jupyter notebook which is available at:
https://github.com/cameronneylon/review_white_2020

and as a PDF via attachment to this report.
Experimental design: The experimental design is well thought through and described with some caveats on the clarity of the analysis procedure. The data is provided and code is also provided that generates the output data.

I have not attempted to run the code as I do not have a Sherpa Romeo api key. I have however conducted some elements of a conceptual replication and find the results consistent (below).

I have two questions for clarification on the details of the analysis. These are based on my replication of the reported analysis using the dataset provided.

Details of the analysis and filtering

The provided datafile has 12,931 lines, not 12,600 as described in the article text. In addition when I filter down to articles with 20 or fewer authors I get 12,226 not 12,016 articles. Similarly there are small differences in the numbers throughout my analysis.

I would recommend the addition of either a flow-chart which explicitly walks through the filtering process (including precise numbers of entries at each point) and how the provided datafile was generated and its relationship to the code. Clearly I took a slightly different process, and broadly speaking the numbers I obtain in my replication from the provided data are very similar, but not quite identical. A fully replicable process would enhance the confidence in the conclusions.

Analysis by funder

I have some concerns or misunderstandings about the analysis by funder. In my own analysis of the provided dataset I noted a lot of variant names for funders, some of which appear as if they are variants of the names used (see replication report). For instance Health Research Council, Health Research Council of New Zealand, and HRC all appear, as well as variants of the Ministry of Business Innovation and Employment and the Royal Society of NZ.

In my analysis the numbers for the precise string noted in Table 9 was usually the closest but this suggests a significant under counting of funded research. Interestingly there seem to be significant variations in the proportion of OA under different names, which might provide some help in estimating error margins?

It may also be of value to analyse some of the non-New Zealand funders that appear and potentially differences within funders for the case where there is a non-New Zealand corresponding author. It would be interesting, for instance if the levels of OA for the same funders were higher where the corresponding author is not from New Zealand.

Confirmation via conceptual replication using alternate datasources

In the provided replication report I have also conducted a conceptual replication for the overall numbers and levels of OA and for funders. I conduct this by querying a local copy of Microsoft Academic Graph for the GRID IDs of the eight New Zealand Universities, identifying a little over 9,000 outputs for 2017. Overall I find similar percentages for open access and for average citation counts based on MAG data which offers solid support for the main findings here.

I did not have ready access to SHERPA ROMEO data or to APC data, nor did I have corresponding author data so did not attempt a conceptual replication of those analyses.
Validity of the findings: The overall conclusions are supported by the evidence and both the direct replication and the conceptual replication (where conducted) support the overall findings.

As noted above I recommend a more detailed description of the data handling, filtering and precise details of the production of the provided dataset. I also recommend checking over the funder data.
Additional comments: Thank you for this work. As noted above my two main comments relate to:

1. The details of the methodology and production and filtering of the data.
2. The analysis by funder and possibility that some funders may have been missed in the analysis

On the content itself I wonder whether it is worth considering the degree to which repository mediated or green oa is contributing overall. In my conceptual replication I note that 29% of the content is available via green OA which is close to the 37% of total OA from my data. This to my mind strengthens the argument for green as the most direct route to increasing overall levels of open access.

This is not obvious from the current text because a proportion of this is labeled as gold. Given that those authors who are engaging with OA are clearly using repositories of one type or another for both closed content but also hybrid and gold, this supports the case that if more authors can be engaged then the green route is the most cost effective forward.

Further, I would argue for a change in terminology to make this explicit, noting that 'gold' here is "in an a pure oa journal" to distinguish between uses that combine this with hybrid, and using "green only" or some other term to make explicit that this applies to repository-mediated OA for outputs that are also published OA. It might help to actually add those two separate categories of green to the tables.


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: ONLY TWO OUT OF FIVE ARTICLES BY NEW ZEALAND RESEARCHERS ARE FREE-TO-ACCESS: A MULTIPLE API STUDY OF ACCESS, CITATIONS, COST OF ARTICLE PROCESSING CHARGES (APC), AND THE POTENTIAL TO INCREASE THE PROPORTION OF OPEN ACCESS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The article is generally well written in professional language. The structure of the article adheres to the PeerJ guidelines. The background acknowledges recent approaches to analyse open access in terms of productivity and citation advantage. Tables presents summary statistics; no figures are provided. The supplement contains article-level data. The authors share the source code of a tool developed for this study via GitHub, which is great!

I suggest to improve introduction and background by clarifying some open access terminology.

Most importantly, Peter Suber's seminal work on open access (OA) (MIT Press, 2012) is not acknowledged according to which Gold OA is defined as "OA through journals, regardless of the journal’s business model." In the article, however, Gold OA refers to fully open access under an APC business model (lines 63-64). Please discuss why you restrict the term "Gold OA" to journals with a specific OA business model, or revise the terminology. Similarly, "Green open access" is introduced as open access through institutional repositories (lines 67-68), but there are also other relevant types of repositories like subject-specific repositories. Is the focus of this work just on institutional repositories?

Because the APC business model plays a central role in the article, I furthermore suggest to acknowledge recent discussions about transitioning (library) spending for journal subscriptions to open access. In particular, the influential White Paper from the Max Planck Society from 2015 (https://doi.org/10.17617/1.3) is relevant as well as related work from the US-American Pay it Forward (https://www.library.ucdavis.edu/icis/uc-pay-it-forward-project/) project. I also suggest to review the work from Gumpenberger et al. (2018, https://doi.org/10.3389/frma.2018.00001) where the authors discuss bibliometric issues using corresponding authorships in the context of open access funding.

In addition to the Leiden Ranking, I suggest to consider the recent preprint "Evaluating institutional open access performance: Methodology, challenges and assessment" from Huang et al. (2020, https://doi.org/10.1101/2020.03.19.998336). In this article, the authors examined and discussed evidence sources to measure the OA performance of universities.
Experimental design: The methods could be described in greater detail.  Particularly, it is not clear to me when and how the underlying article DOIs were gathered. Did they originate from searching the Web of Science and Scopus? If so, what search queries were used? What was the overlap between these two sources relative to the New Zealand research output? And what was the proportion of articles without DOIs?

Research questions are descriptive, seeking to quantify the open access performance of New Zealand's university. I think the supplemented data is very comprehensive and would allow to ask for variations by subject or publisher. It would be also great to learn more about whether and why the OA uptake varies across the universities.
Validity of the findings: The authors supplemented article-level data. Unfortunately, I did not succeed replicating the presented results using the spreadsheet, but obtained slightly different numbers. Please find my R Markdown notebook attached to this review. Please also provide the underlying code used to generate the tables.

Besides, I am missing important context to assess the validity of the findings. Articles with more than 20 authors were excluded because they had a "tenuous connections" to the studied universities. Why did you choose the threshold of 20 authors? And how did you validate the "tenuous connection"? Did you consider alternative counting methods like fractional author counting?

I also miss background about how the accuracy of the retrieved article-level data was assessed in terms of recall and precision. How did you assure the quality of the tool and the open access evidence sources?

Drawing the conclusion that open access has a citation advantage based on the presented methods and results is very problematic. I feel that the citation window between the publication year of 2017 and mid 2019, the date of gathering the citation data, is too narrow to draw any meaningful conclusion. Furthermore, only mean citation rates were reported. The citation rates were also not normalized for differences in citation patterns between research fields, which is a standard practise in bibliometrics.
Additional comments: I would suggest that you narrow the focus of the study. The data you collected so far are great and provide more analytical opportunity than what was presented. One promising focus is the debate around transformative agreements and the economic consequences for library budgets and national library consortia in the future. Another promising approach is to highlight variations between universities and the exploration of potential factors affecting the institutional OA uptake. If the focus is on the tooling, then the paper should be revised to reflect the technical focus.


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: ONLY TWO OUT OF FIVE ARTICLES BY NEW ZEALAND RESEARCHERS ARE FREE-TO-ACCESS: A MULTIPLE API STUDY OF ACCESS, CITATIONS, COST OF ARTICLE PROCESSING CHARGES (APC), AND THE POTENTIAL TO INCREASE THE PROPORTION OF OPEN ACCESS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have addressed all of the raised issues with regard to clarity and some issues of confusion. I will note for the record that I disagree with some of the terminological choices but given there is no widely accepted standard for defining types of open access I do not think this is worth changing. The definitions are given clearly enough in the article.

Minor issue: Our preprint, referenced in the article at line 714 is now published at eLife - https://doi.org/10.7554/eLife.57067
Experimental design: The results are well defined and the process is now much clearer.

Minor issues: I can completely replicate the results given from the provided data. However there is one slight issues. There are 21 DOIs for which no authors appear to have been recovered and these are interpreted as having less than 21 authors. If this is correct then it would be valuable to make that clear in Figure 1.
Validity of the findings: I find the overall findings robust and clear. I note that I have conducted an outline conceptual replication of some aspects of the paper using an independent dataset and confirm the general findings.

https://github.com/cameronneylon/review_white_2020

There are variations in both the set of DOIs detected in the replication and the details of the results, particularly that the citation counts have subsequently changed following the data capture for the paper, but overall the conclusions are consistent with those drawn in the article.
Additional comments: Thank you for this article and the work done on it. I look forward to seeing the response to it.


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: ONLY TWO OUT OF FIVE ARTICLES BY NEW ZEALAND RESEARCHERS ARE FREE-TO-ACCESS: A MULTIPLE API STUDY OF ACCESS, CITATIONS, COST OF ARTICLE PROCESSING CHARGES (APC), AND THE POTENTIAL TO INCREASE THE PROPORTION OF OPEN ACCESS
Review round: 2
Reviewer: 2

Output:
Basic reporting: No comments
Experimental design: No comments
Validity of the findings: The revision did not sufficiently addressed my concern regarding the citation analysis. By just comparing mean citation counts, neither a citation advantage for open access nor for  OA types can be derived. To illustrate my concerns, I used the provided spreadsheet and compared the OA distributions (for replication, my R Markdown notebook output is attached using the "annotated PDF file" functionality"). My exploratory analysis suggests that the citation distributions closed vs OA as well as by OA type are quite similar with a few outliers. The exemption is diamond OA, but this OA types accounts for smallest number of publications in the sample. Moreover, at least two highly cited and highly collaborative OA publications were miscoded in the spreadsheet as having 20 or less authors, and should have been excluded according to the study design.

Again, I strongly suggest to revise the citation analysis and provide a statistically valid proof for an OA citation advantage. Data errors need to be fixed.
Additional comments: Thank you for the revision and the nice job addressing most of my comments. However, I think that the citation analysis cannot prove a citation advantage, and should be thus substantially revised or omitted.


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: INTEGRATIVE SPECIES DELIMITATION BASED ON COI, ITS, AND MORPHOLOGICAL EVIDENCE ILLUSTRATES A UNIQUE EVOLUTIONARY HISTORY OF THE GENUS PARACERCION (ODONATA: COENAGRIONIDAE)
Review round: 1
Reviewer: 1

Output:
Basic reporting: 1. The color labels in Figure 2 and Figure 3 for presenting different species may make readers puzzled. The colors for different species and for different analysis methods have some overlaps. Besides, it is hard to distinguish the colors for P. hieroglyphicum and P. sieboldii.

2. It was declared that "species delimitation based on a single gene may be questionable", however, species delimitation based on two genes/markers may still be questionable.

3. Only one mitochondrial marker and one nuclear marker were used in this work, but the conclusion was made as "The discordance in mtDNA and nDNA data ...".

4. For the conclusion "The discordance in mtDNA and nDNA data also indicates the value of using multilocus datasets in species delimitation studies", can two markers be viewed as multiloci?

5. The photo of a key species, P. barbatum was absent in Figure 8.

6. For the hybrid individuals shown in Figure 9, they should be indicated correspondingly in Figure 2, Figure 3, Figure 4, and Figure 5.
Experimental design: From Figure 1, it seems that the sampling for P. melanotum and P. v-nigrum were not enough according to the geological distribution. To show the robustness of sampling, the analyses of ecological niche reconstruction should be made.
Validity of the findings: 1. It was concluded that "Our results indicated that the genus Paracercion includes nine confirmed species." But in lines 82-83, it was demonstrated that "We now think that P. pendulum and P. malayanum should both be junior synonyms of P. melanotum". Viewpoint can not be used as evidence.

2. All of the so called "introgression, hybridization, recent speciation and incomplete lineage sorting" are speculations. They should not be taken for sure as "The existence".
Additional comments: 1. The study should focus on the relationships between P. v-nigrum, P. barbatum, and P. melanotum rather than on the whole genus, as some species were not included.

2. It is not that necessary to make too many speculations of introgression, hybridization, recent speciation, and incomplete lineage sorting and thus make the study appear meaningful in broad sense. In strict sense, each of the above speculations needs more data and analyses to serve as evidence.


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: INTEGRATIVE SPECIES DELIMITATION BASED ON COI, ITS, AND MORPHOLOGICAL EVIDENCE ILLUSTRATES A UNIQUE EVOLUTIONARY HISTORY OF THE GENUS PARACERCION (ODONATA: COENAGRIONIDAE)
Review round: 1
Reviewer: 2

Output:
Basic reporting: The abstract needs more specific details. How many species were analyzed (out of how many in the genus)? What, specifically, were the “multiple methods” used for species delimitation, both in terms of data and in terms of analysis?

The sentence on line 83 needs support: either provide evidence or a citation. The sentence on line 87 does not follow from what came before. If the authors “now think” think that P. pendulum and P. malayanum are junior synonyms of P. melanotum, why not actually establish that fact in the present manuscript?

***The material and methods section needs to specify how the specimens were identified, who identified them, what resources were used to identify them, and the collection depository of the specimens. Ideally, the specimen data would be digitized and made available on-line. I am not an expert on Odonata, but it might be useful to describe how easy or hard it is to identify the specimens to species. Without this information, how is the reader to know that the results weren’t just based on incorrect specimen determinations?

The sentence on line 103 should specify that these 16 and 8 sequences were in addition to those acquired in the lab for this present study.

***GenBank accession numbers need to be included for the DNA sequences, probably in Table S1.

The authors report on the number of variable and informative sites for CO1, but do not say anything about ITS (line 184). Also, were ITS sequences acquired for all nine species? Line 182 does not specify if it refers to one or both loci.

Split the sentence beginning line 223 into two. The first sentence reporting on CO1 (include the following sentence describing the 3% threshold, the second sentence reporting on ITS.

The “Morphological checking” paragraph (lines 285-290) should probably be included in the introduction and/or material and methods. Then, the molecular methods can be set up as a test of the morphological concepts.

In the figures, the colors for P. hieroglyphicum and P. sieboldii need to contrast better: I can’t tell them apart.
Experimental design: The experimental design is appropriate.
Validity of the findings: The findings are valid.
Additional comments: The manuscript is a valuable contribution to the field. The research is presented clearly and succinctly and highlights the challenges of molecular species delimitation.

The conclusions are somewhat self-contradictory, however. The authors state that we should recognize species based on a congruence of methods (line 356), but they favor splitting the putative taxa over lumping them: that is, they recognize species delimited by CO1 data that are not delimited by ITS data, and they recognize species delimited with ITS that are not delimited with CO1. If they suggest that congruence is important, shouldn’t they only recognize species that are delimited with both datasets? In the end, in recognizing nine species (line 365), they validate the congruence of the morphological data with either, but not necessarily both, of the molecular datasets. Personally, I think this approach is appropriate, but the authors should clarify their discussion accordingly (especially the “Species delimitation methods” section and the “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: INTEGRATIVE SPECIES DELIMITATION BASED ON COI, ITS, AND MORPHOLOGICAL EVIDENCE ILLUSTRATES A UNIQUE EVOLUTIONARY HISTORY OF THE GENUS PARACERCION (ODONATA: COENAGRIONIDAE)
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The authors have made a clear thing seem complex and puzzling.
In the conclusion, they declared that "our results indicated that the genus Paracercion includes nine confirmed species". However, 6 or 7 bars were indicated by the results based on COI in Figure 2, while 7, 10, or 12 bars were indicated by the results based on ITS in Figure 3. That means neither COI nor ITS well supported the conclusion of 9 species, which was in fact based on morphological evidence. Certainly, compared with the results based COI, those based on ITS are more close to the value 9, as this value was covered by the range from 7 to 12. Therefore, it is necessary for the authors to really make major revisions to their whole manuscript from the title to the conclusion part. As for the title, "Species delimitation based on a single gene may be questionable" is nothing new and too story-telling. It is better to have a more serious title such as "Improved species delimitation based on COI, ITS, and morphological evidence: ...".


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: INTEGRATIVE SPECIES DELIMITATION BASED ON COI, ITS, AND MORPHOLOGICAL EVIDENCE ILLUSTRATES A UNIQUE EVOLUTIONARY HISTORY OF THE GENUS PARACERCION (ODONATA: COENAGRIONIDAE)
Review round: 2
Reviewer: 2

Output:
Basic reporting: Abstract:“For ITS, Paracercion barbatum and Paracercion melanotum can be combined into one putative species, which suggests a hybridization event between them.” It seems this sentence is missing information. Is it the fact that CO1 did separate the two nominal species whereas ITS did not that suggests a hybridization event?

There is some confusion as to the reporting of the two new synonyms and new combination.
The introduction should present the fact that all 12 nominal species were examined but that P. luzonicum was eliminated after preliminary analysis. Therefore, 11 nominal species were analyzed with the molecular data. If they want, the authors can mention the fact that two of those species are understudied and questionable, and even state their validity as among the hypotheses to be tested in the present study.
The materials and methods should then reassert the fact that 11 nominal species were examined.
The results, in turn, state that P. pendulum and P. malayanum were genetically indistinguishable from P. melanotum. Specimens putatively identified as these nominal species should be indicated in the various figures so the authors can see how they are clearly embedded within P. melanotum.
Finally, the discussion should propose the formal synonymy of these three nominal species (two junior synonyms and one senior).
The abstract should include mention of the removal of P. luzonicum from the genus and the establishment of the two new synonymies.

The species color codes should match across all figures. For example, P. barbatum is purple in Fig 1, orange in Figs 2 and 3, and red in Figs 4 and 5: make them all the same color.
Experimental design: The experimental design is appropriate.
Validity of the findings: The findings are valid and the discussion improved from the previous version.
Additional comments: Although the text is understandable, the English could use some editing and the syntax and message could be improved to make the article more readable and much more cited. “Decimation” (line 317) means something completely different than “delimitation”.