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: COMMENT ON PESCOTT & JITLAL 2020: FAILURE TO ACCOUNT FOR MEASUREMENT ERROR UNDERMINES THEIR CONCLUSION OF A WEAK IMPACT OF NITROGEN DEPOSITION ON PLANT SPECIES RICHNESS
Review round: 3
Reviewer: 1

Output:
Basic reporting: Great - my comments and suggestions have been addressed
Experimental design: No comment
Validity of the findings: No comment
Additional comments: No additional comments


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

Input:
Title: NEW DISTRIBUTIONAL RECORDS OF THE SAMANA LEAST GECKO (SPHAERODACTYLUS SAMANENSIS, COCHRAN, 1932) WITH COMMENTS ON ITS MORPHOLOGICAL VARIATION AND CONSERVATION STATUS
Review round: 1
Reviewer: 1

Output:
Basic reporting: This is a very simple and straightforward paper about new records from an endemic gecko from the Dominican Republic. The data reported on variation is important, but one of the photographs of the gular area can probably improved. The new records are important, especially to assess its conservation status. I guess since the new records are from protected areas, this is great news for the species. The figures are good, but see my comment on the gular scales. Can you do a close up of the postmental scales? These seem to be more numerous in the eastern populations.
Experimental design: This section is adequate for the type of research. Perhaps doing some box plots might help showing better the differences among populations.
Validity of the findings: Sphaerodactylus geckos are species very hard to find a collect. This paper is important for the conservation status of a poorly know species.  I think the geological discussion is relevant to understand orogenic process in the area, but the age of this group is newer, so I don't think it has a lot of relevance for its distributional range. In this regard, maybe some niche models might me more relevant, or to determine what environmental factors are important, temperature, precipitation, etc.
Additional comments: This is a very simple paper, I like the general paper, but I would like to see more ecological data related to the habitat before this paper is published. The botanical assessment of the area is very vague, and basically would apply to many areas in the Caribbean. I recommend including additional microhabitat information about this species, which would be probably very useful (e.g. species of trees on the area, is the leaf litter moist or dry, is the leaf litter dense or sparce, etc) . You mention these geckos were found under rocks, would you classify the as rupiculous, or a combination of leaf litter dwellers using occasionally karst outcrops?


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 DISTRIBUTIONAL RECORDS OF THE SAMANA LEAST GECKO (SPHAERODACTYLUS SAMANENSIS, COCHRAN, 1932) WITH COMMENTS ON ITS MORPHOLOGICAL VARIATION AND CONSERVATION STATUS
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: see below
Additional comments: Reviewer Comments for "New distributional records of the Samana least gecko (Sphaerodactylus samanensis, Cochran, 1932) with comments on its morphological variation and conservation status"

The main focus of the manuscript is the discovery of previously undocumented S. samanensis localities, which expands the known distribution of the species and presumably warrants a re-evaluation of its conservation status as Critically Endangered. While this seems like a straightforward conclusion there are several things that make me question this line of thinking. First, a recent molecular phylogeny, that included many Hispaniolan species of Sphaerodactylus (Daza et al. 2019. The sprightly little sphaerodactyl: Systematics and biogeography of the Puerto Rican dwarf geckos Sphaerodactylus (Sphaerodactylidae, Gekkota). Zootaxa 4712:151–201) found S. ladae, S. difficilis, & S. darlingtoni were actually species complexes composed of more than one valid taxon. Thus, multiple widespread species of Hispaniolan Sphaerodactylus are in need of taxonomic revision and that could also include the now widespread S. samanensis. Analysis of molecular data would be helpful in this case to determine whether the eastern and western S. samanensis populations are reproductively isolated. Second, the authors detail significant phenotypic variation between the eastern and western S. samanensis populations. These morphological differences, when considered together, are sufficient to diagnose each population and perhaps even call them separate species. Third, the authors describe a biogeographic scenario (lines 174-190) that that could have resulted in allopatric speciation between eastern and western S. samanensis populations. Thus, while S. samanensis clearly has a larger distribution than previously known, it may not be as big as stated in this manuscript and the authors in all likelihood have discovered an undescribed species, closely related to S. samanensis sensu stricto. Thus, reconsidering the conservation status of S. samanensis, while clearly warranted, must be contingent upon a thorough taxonomic evaluation first.

Below are some additional comments that I hope the authors find useful.

Lines 52-54 - Should also mention that 21 Sphaerodactylus species are known only from the type locality (Meiri et al. 2017. Extinct, obscure or imaginary: The lizard species with the smallest ranges. Diversity & Distributions 24:262-273).

Lines 109-110 - Perhaps cite (Poe. 2012. 1986 Redux: New genera of anoles (Squamata: Dactyloidae) are unwarranted. Zootaxa 3626:295–299) as justification for using Anolis.

Line 158 - Should be Dodd Jr. & Ortiz (see also line 225).

Line 168 - "(intruders)" - not sure what this refers to.


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 DISTRIBUTIONAL RECORDS OF THE SAMANA LEAST GECKO (SPHAERODACTYLUS SAMANENSIS, COCHRAN, 1932) WITH COMMENTS ON ITS MORPHOLOGICAL VARIATION AND CONSERVATION STATUS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The manuscript has improved considerably compared with the previous version, However there are a few things that still are unclear and need to be addressed. See comments to authors section.
Experimental design: NA
Validity of the findings: The content of this manuscript is important and the data is valid.
Additional comments: Thanks for sending a revised version.

I am sorry to insist but dominant needs to be specified, is this in terms of biomass population density, or biodiversity? Certainly, Anoles are probably more diverse than sphaeros.

In the part where you write the species of trees, do not writte the abbreviation of species italics, that is incorrect.

I understand you want to wait to publish your phylogenetic analysis on a separate analysis, but the comment from the other reviewer is still valid. By the way, I could not find the tree mentioned in the rebuttal or a reference to it as supplementary material on the text. What I suggest you can do is to write a brief statement like the one below and make reference to the genetic distance of these populations:

An undergoing phylogenetic analysis of Sphaerodactylus from ______________ using molecular data has gound that population differentiation between the sampled localities is less than XX%.


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: EVALUATION OF RESISTANCE TO POWDERY MILDEW AND IDENTIFICATION OF RESISTANCE GENES IN WHEAT CULTIVARS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The introduction is self-explanatory.

Objectives, Material and methods, and Discussion are also clear.

Literature Review was sufficient.

The tables are clear but there is a typo error of a figure that didn't make sense.

From a breeding point of view, the introduction explained key points that any plant breeding wants to work on to develop a resistant cultivar.
Experimental design: They use the phenotyping and genotypic approach.
Both are very valid/strong approaches to identify R-genes
Validity of the findings: Relevant results.
It provides key information for breeding programs worldwide.
As long as they add a supporting figure/evidence of the Pm16 identification and correct the “typo error” of Fig 4-2, I would recommend this article for publication.
Additional comments: a)	Line 148: Molecular identification of Pm13 Is there Fig 4-2? I was not able to find it in the article. Is it missing?
b)	Line 153 – 158: Where is the figure/evidence for the identification of Pm16? Do you have a figure?
c)	The introduction is self-explanatory. From a breeding point of view, the introduction explained key points that any plant breeding wants to work on to develop a resistant cultivar. Objectives are also clear.
d)	Well written discussion. I enjoy the idea of pyramiding genes in elite wheat lines.
e)	Very nice review of the literature.


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

Input:
Title: EVALUATION OF RESISTANCE TO POWDERY MILDEW AND IDENTIFICATION OF RESISTANCE GENES IN WHEAT CULTIVARS
Review round: 1
Reviewer: 2

Output:
Basic reporting: This manuscript evaluate seedling resistance of 69 main wheat cultivars from Yunnan of China, and also conformed the Pm genes in these cultivars. The information can be used in future wheat disease breeding. However, several revisions should be carried out based on the suggestions listed blew. After revisions, the manuscript can be accepted for pubilcation in PeerJ
Experimental design: No comment
Validity of the findings: No comment
Additional comments: 1.Up to now, 61 loci (Pm1-Pm66) have been characterized to provide powdery mildew resistance, but not 58, please update the progress.
2.The documented Pm stocks in Table 2 were not near-isogenic lines, but in different genetic backgrounds.
3.Please describe the detail method to avoid cross-infection of different isolates after inoculation.
4.Different IT scores have detailed indicators, please re-describe them.
5.All the identification of the Pm genes were based on the amplification of parents but not populations of resistance and susceptible parents, so you can not confirm one cultivar certainly has one related Pm gene only by the special band comparison between the tested cultivars and Pm gene stocks. You need use “is most likely” to explain one cultivar is most likely have one special Pm gene based on the special band.
6.Yunnan is not the main wheat growing regions in China.
Why do you selected Pm3, Pm13 and Pm16? These three genes rarely exist in Chinese cultivars, especially there are no commercial varieties to be reported to have Pm13 and Pm16. Chinese cultivars mainly have Pm2, Pm4, Pm8 and Pm21.


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: SMARTAR: AN R PACKAGE FOR DESIGNING AND ANALYZING SEQUENTIAL MULTIPLE ASSIGNMENT RANDOMIZED TRIALS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Since this is an article that presents a new R package, I have general comments about the structuring of the manuscript. Hence the review is below in the general comments section.
Experimental design: Since this is an article that represents a new R package, I have general comments about the structuring of the manuscript. Hence the review is below in the general comments section.
Validity of the findings: Since this is an article that represents a new R package, I have general comments about the structuring of the manuscript. Hence the review is below in the general comments section.
Additional comments: Authors present here an R package that they have developed for Sequential Multiple Assignment Randomized Trials (SMART). The overall manuscript is well developed, easy to read and the data is nicely represented. During this review, I went through the rOpenSci’s guidelines for reviewing a package too and checked if the manuscript fits in the review template provided by the rOpenSci community.
Here are my detailed comments. Within the introduction, the authors explain in the SMART trial approach. They illustrate how the approach is essential in developing trails for adaptive treatments with a couple of simple figures. Following that authors describe the current milieu of R packages that can be used for analyses related to SMART and briefly describe how the new package is advantageous over the others.
The authors describe in detail all functions presented in the package and that are required for the analytical pipeline of SMART. Besides tiny missing details, authors have described the functioning of the package very well. I would say that one important aspect of the case study is missing though that is the interpretation of results and outputs. To address those, I would recommend authors to first start with the Sample Size function (as the first step of SMART planning) followed by data visualization and statistical comparison. At each step, authors can explain the functioning of the package with code and data. This should then include discussion regards to the results.
For example: in lines, 307-311 authors say “In this case, conducting a global test among the 8 strategies 308 using the CODIACS trial data of 108 patients, it returned a test statistics Q = 36.03. Under 309 the null hypothesis, Q follows a centralized chi-squared distribution with degrees of freedom 310 ν = 5. As a result, we obtained a P-value less than 0.05 and claimed over statistical significant 311 difference among the eight depression management strategies in the CODIACS trial.”  This should be followed by discussing the significance in terms of ATSs and their outcomes. I would recommend seeing Elith et al. 2008 A working guide to boosted regression trees. To follow the idea of explaining the biological interpretations of a package developed.
Another comment I would include is to develop extensive documentation. Including various options for arguments.  Authors provide some explanations in the text, but centralized documentation is also essential as eventually users and developers would mostly refer to the package documentation. Similarly, help(SMARTAR) did not yield any documentation within my IDE while testing the package. There are additional small comments that I have noted while reviewing the package and they are attached below with the review.

Please see attached pdf document with a detailed review of the package based on rOpenSci’s guidelines.


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: SMARTAR: AN R PACKAGE FOR DESIGNING AND ANALYZING SEQUENTIAL MULTIPLE ASSIGNMENT RANDOMIZED TRIALS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The basic reporting is detailed enough and adequate to follow along easily.
Experimental design: ## Package Design

This package is on par with the design of its contemporaries, but it could use
some improvement that will improve the quality and make it stand out from other
packages. I will highlight a few things that struck me:

### Output

The output of the summary functions rounds the statistics before returning to
the user. Despite the fact that the "digits" option is available, I would
suggest to avoid this because it can be frustrating for users who do not want
their data rounded in the first place.

The output of smartest presents the $Strategy as a matrix instead of a data
frame. It's a small point, but it is more consistent to present this as a data
frame since each column represents variables of different modes.

### Tests

There are no formal tests in this package. As I mentioned before, this is on-par
with its contemporaries, but tests make sure a package provides both
reproducible and accurate results. Moreover, tests allow authors to refactor
code confidently without fear that it will break. I would recommend including a
test suite such as testthat or RUnit and writing tests that test known values
from published data sets and simplified scenarios. A very good resource on
writing tests is <https://r-pkgs.org/tests.html>

I would also strongly suggest the authors keep the package under version control
in a public repository (such as GitLab or GitHub) so that they can access
continuous integration tools.

### Documentation

The documentation provided in the package is enough to understand what it does,
but could use improvement. One suggestion is for the authors to write a vignette
describing a typical workflow. These are documents that live with a package that
are meant to describe how all the moving parts of the package work together.
 A resource on those can be found here: <https://r-pkgs.org/vignettes.html>

The examples in the package should use the CODIACS data set or another real
data set. Users should not have to run ~30 lines of code before they run the
example function. Having examples of different scenarios would also allow the
authors to demonstrate the flexibility of the package.

### ChangeLog

One frustration I had with the package is that the version on CRAN was different
from the version provided here and there was no record of changes that I could
easily inspect. One suggestion I have is to include a NEWS or ChangeLog file in
the package that describes changes that the user should be aware of that include
different plotting options, new parameters, outputs, or documentation.

### Code style

I would recommend using the package "goodpractice" on your code to highlight
potential issues. I will highlight some important ones here:

In several of the functions there are conditional statements
(if statments) that use `&` instead of `&&`. All conditional statments should
use `&&` (note: this does not include `which()`). The reason for this is that
`&` will return a vector of any length, but `&&` will always return a vector of
length 1.

The authors should use `seq(ns)` instead of `1:ns` in their code.
The reason for this is because if ns is 0, then `1:ns` will be the vector
c(1, 0) instead of just 0.

The authors should use `TRUE` and `FALSE` instead of `T` and `F` as the latter
can be overwritten with user variables and may cause problems.
Validity of the findings: I cannot comment on the validity of the statistical methods used as I do not
have expertise on clinical design. The data and code provided replicate the results observed.
Additional comments: SMARTAR is a new R package that supports SMART analysis. While there are several
packages available for SMART analysis, SMARTAR differs in the following aspects:

 - Supports Exploratory Data Analysis (L87)
    - Provides graphical tools at the treatment sequence and strategy level
    - Can generate SMART design diagram
 - Can construct (Bonferroni-corrected) simultaneous confidence intervals for
   ATS comparisons.
 - Provides sample size calculation for both pairwise and global comparisons.

The authors identify that there are five other packages that perform some facet
of SMART analysis, but stress that SMARTAR provides functionalities beyond that
of the previous packages. The authors then take care to describe the input data
in detail and provide a use-case based on a real data set.

The package itself works as presented when installed via the supplementary
materials, but does not work from the version on CRAN, despite identical version
numbers. There are things that could be improved that would make this package
stand out from its contemporaries in the field of SMART design, including
testing, documentation, and de-duplication.

### Line changes

 - L21--22: strike out "by far"
 - L70: "DTReg" -> "DTRreg"
 - L79: "limits" -> "limitations"
 - L85: "is appealing to" -> "appeals to"
 - L89--90: The sentence that starts with "It is by far..." is not needed.


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: SMARTAR: AN R PACKAGE FOR DESIGNING AND ANALYZING SEQUENTIAL MULTIPLE ASSIGNMENT RANDOMIZED TRIALS
Review round: 1
Reviewer: 3

Output:
Basic reporting: General notes:

The authors have created a useful R library that appears to address a need in randomized clinical trial design. The manuscript has been divided into the typical parts (Introduction, Materials & Methods, etc) though this is difficult to do efficiently with articles like this where the "study" being described is a piece of developed software. Additionally, the language is frequently awkward with incorrect and words and improperly used phrases. Some specifics are listed below, but there are too many examples in the entire paper to enumerate.

The "Real data example" appears around line 132, but there appears to still be more general functional explanation (lines 148, 171, 286, and equations 4-6). Especially with only 4 or 5 exported functions in the library, I believe it would be more clear to explain the library functions first, then provide examples of them in use later. This will allow clear sections laying out functions prior to involving real data.

Since the results of the CODIACS study is not the focus of the paper, it may be more appropriate to either explain how to use the results of the code in the discussion section. It would also be useful to compare how the package could have improved sample size selection, and how the the results produced by the the SMARTAR package compare to the results in Cheung et al. (2015)

The term "trialists" is an odd choice and in my experience not frequently used. In the discussion, the authors use the phrase "clinical investigators and data analysts" which is much more common parlance. Consider changing "trialist" to "investigators" or even simply "users" through out the document.

Other notes:

Introduction, first paragraph: Consider breaking this paragraph into several. The first paragraph should introduce the method, its use(s), its advantages, and perhaps begin the discussion about the analytic challenges of it presents. In the second paragraph, discuss more details and refer to the example. It seems odd to figure references in the first paragraph.

Line 61: "More details of ATS and SMART see [citation]" I believe this is should be "*For* more details see ..." This appears in several places, and it may be more appropriate to simply cite the respective articles in the preceding sentence.

Line(s) 109: The set notation here is somewhat confusing. For example A_1i  = { CBT, MED } , but the i subscript indicates which treatment the ith individual received, not the set of possible . It appears this should be A_1 =  { CBT, MED }, or A_1i is a subset of  { CBT, MED }, or A_1i = CBT.

Line 149: "We first lunch the SMARTAR, retrieve the example data..." This should be "launch" instead of "lunch." A better term is "attach" the library, but the code is so basic that it doesn't really bear mention in the manuscript; same with following line.

Line 188: "We input a SMART data..." The word 'data' plural; this should read "we input SMART data ..." or "We input a SMART data object ..."
Experimental design: Based on the text and limited review of the included code, the design of the SMARTAR package appears to be sound. The example code runs in R 3.6.2 and returns results consistent with the manuscript text.

The 'seqmeans' function reports mean and variance to [digits] places. I would recommend reporting mean and std. deviation to the same number for significant figures, but providing more decimal places for the variance.
Validity of the findings: Again, the results reported by package appear to be consistent with the manuscript inputs and supplemental materials. However, it is somewhat difficult to assess the validity of the results without access to the 'unbuilt' code (the body of the library's functions are accessible via the body function, but it's much easier to read raw code). I would suggest the authors create a public-facing GitHub page to allow review and modification of the code for transparency. Also, an open-source license should be declared.

In the introduction, the authors mention several other packages designed to assist investigators in designing and analyzing SMART trials. The authors should compare and contrast the relevant output of the of their package to the results of the established packages.
Additional comments: Overall, the package developed by the authors appears useful and is consistent in quality with CRAN standards, but the language issues make manuscript very challenging to review. Additionally, given there are software packages that already exist to assist with these calculations, the performance of the current package should be compared to the results of applying the same data to other packages.

I would suggest having the paper reviewed and re-written with the assistance of a native English speaker to resolve these issues prior to the next round of review.

Other coding notes:
In the 'astmeans', the family and method arguments should be limited to a set of possible values. For example, the family argument could be defined using: astmean <- function(..., family = c("gaussian", "binomial") ). Arguments declared in this way will default to the first value in the concatenated set.

The authors may want to consider using informal S3 classes for their output. This allows the use of pre-existing generics like 'summary', 'print', and 'plot', while still allowing the constituent elements of the returned list objects. See Hadley Wickham's advanced R page, specifically http://adv-r.had.co.nz/S3.html


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: SMARTAR: AN R PACKAGE FOR DESIGNING AND ANALYZING SEQUENTIAL MULTIPLE ASSIGNMENT RANDOMIZED TRIALS
Review round: 2
Reviewer: 1

Output:
Basic reporting: Authors have meticulously worked on the comments, improving the structure of function outputs, and improving version control. Vignettes provided online will be useful for package users improves the usability of the package. I like the way authors have explained functions with additional interpretation. The overall flow of the manuscript is also improved.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The authors have satisfactorily addressed all my comments and have improved the reproducibility of the package.


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: SMARTAR: AN R PACKAGE FOR DESIGNING AND ANALYZING SEQUENTIAL MULTIPLE ASSIGNMENT RANDOMIZED TRIALS
Review round: 2
Reviewer: 2

Output:
Basic reporting: The reporting is detailed and easy to follow.

Line 123: it is not necessary to state that the article ends with a discussion and conclusion.
Line 625: the "Strategy" output in list 6 still needs to be updated to the current output format.
Experimental design: I would like to thank the authors for taking the effort to add NEWS and implement tests for their package.

I believe the tests could stand for a bit more improvement. At the moment, the 64 tests take 400 seconds to run on my macOS and upon further inspection, none of the tests actually check that the output is correct, but instead rely on testing that there are no warnings generated by the code, of which there are three places that could generate such a warning.

I would strongly suggest to the authors to test that the outputs are equal to known quantities or at least within expected ranges.
Validity of the findings: No comment
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: SMARTAR: AN R PACKAGE FOR DESIGNING AND ANALYZING SEQUENTIAL MULTIPLE ASSIGNMENT RANDOMIZED TRIALS
Review round: 3
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: I believe the authors had misunderstood my comments from the previous review. When I looked at the testing code for the package, no tests to check validity of output were added to the test suite. The added simulation study is a nice exercise, but it is not what was requested and not a substitute for adding tests for validity to the suite.

The simulation study is performed only once and tests the software as it is at a single piont in time, while the test suite is run every few days on CRAN and will make sure the package continues to produce correct results as the source code and the software it depends on changes.

I would point the authors to read Section 12.3 in "R Packages" by Wickham and Bryan for more information about the strategy for writing tests: https://r-pkgs.org/tests.html#test-tests and request once again that they put validation tests in their test suite.
Validity of the findings: no comment
Additional comments: The effort you have put in for testing and design of this package outpaces the efforts of the competing packages and I think you should be proud of that. Adding the verification tests to the suite will make your package even better and make it easier for you to refactor or update your code in the future.


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: ASSOCIATIONS BETWEEN SELF-REPORTED AND OBJECTIVE FACE RECOGNITION ABILITIES ARE ONLY EVIDENT IN ABOVE- AND BELOW-AVERAGE RECOGNISERS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript is written in clear, professional language and provides sufficient context for the study. The research question is clearly stated. There are minor typos throughout the manuscript (e.g., Line 119, second sentence should begin with Observers’), but these do not detract from readability. I have highlighted these typos in the attached copy of the manuscript. The figures are also very clear and are referenced appropriately in-text. I included this comment in the manuscript, but I think a reference figure for the CFMT-Chinese would be helpful to the reader, as the procedure described is at times confusing.

All raw data can be accessed. The only piece of the data file that is not immediately clear is the column labeled ‘Three Groups.’ This information is not referenced in-text, nor is it identified in the data file. Does this refer to the PI-20 score?

Identifying information is also included. I highlighted these in the document (e.g., Lines 116 – 117, Lines 123 – 124).
Experimental design: The research question addressed in the article is well-defined and easy to identify. The introduction nicely sets the stage for the method described and contains sufficient information to convey how this research contributes to knowledge about the association between self-report and objective measures of face recognition abilities. The Method of the study includes several areas of confusing phrasing or points that would benefit from clarification, noted in the attached document. These areas begin at approximately Line 136 (the CFMT-Chinese description). For example, in Line 138, it is not clear if the target orientations appear 6 times each over the course of the 18 trials, and if the distractor identities also appear in the same orientation or can vary.
Validity of the findings: The data appear to be robust and statistically sound, and the results are laid out clearly. In the attached manuscript, I highlighted Line 160 in the results with the following suggestion. It would be helpful to the reader to include some summary statistics (e.g., average scores for the CFMT and PI-20; how many participants fell into each quartile; how quartiles were divided, as they are not equal intervals) before describing the associations. I also think it would be relevant to highlight low and high scores identified by previous work (e.g., what qualifies as prosopagnosia, according to these tests; what qualifies as a super-recognizer; is there an “average” or “normal” score across either of these two tests?). These comparisons would help when assessing the current study. I highlight these points again in the conclusions because a major focus of the introduction is on prosopagnosia. I also wonder how the data in the current study compare to the re-analyzed data from Gray et al., in broad strokes (means and ranges, for example).

I find the speculation about the explanation for why only above- and below-average recognizers seem to have insight into their abilities very interesting. I think the manuscript would benefit from additional speculation about why these patterns emerge, and the implications it might have for real-world face identification.

As a final note: I wonder how the results of the PI-20 and the CFMT compare with other measures of face recognition ability – for example, those using famous faces, or the Glasgow Face Matching Test. I don’t know how prosopagnosia is typically diagnosed (aside from the noted aim of the PI-20 to help diagnosis of face recognition disorders), but I would be interested to see if other subjective or self-report measures of face recognition show similar associations with objective abilities. To that end, I think it would be worth including if there are any other phenomenological measures of face identification beyond the PI-20, if only as an example for the reader.
Additional comments: Summary
This manuscript represents a study that investigated the association between self-report measures of face recognition abilities, the 20-Item Prosopagnosia Items (PI-20) and objective measures of face recognition abilities, the Cambridge Face Memory Test Chinese (CFMT-Chinese). To examine whether previously observed associations between these tests are driven by low- and high-scoring participants, 280 students completed both tests. Using correlational analyses, the authors show negative associations between PI-20 and CFMT-Chinese performance primarily for individuals who score lower or higher than average.

I have attached an annotated copy of the manuscript. Some of the comments in the PDF will be redundant with what is listed above, but I highlighted areas in which I had questions, comments, or suggestions about the content.


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: ASSOCIATIONS BETWEEN SELF-REPORTED AND OBJECTIVE FACE RECOGNITION ABILITIES ARE ONLY EVIDENT IN ABOVE- AND BELOW-AVERAGE RECOGNISERS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The English is not clear throughout, and there are some problems with references not being included in the reference section. I've listed some examples:

Line 25: ‘typical average’ – not clear; only one of these words is needed.
Line 48: These citations could be more clearly linked to the two statements. Which provide evidence that acquired prosopagnosia is low prevalence, and which provide evidence that developmental prosopagnosia is up to 3%?.
Line 55. ‘This task can be completed in approximately…’
Line 74: It’s unclear what you mean by the sentence “one clear advantage..”. It suggests that the advantage of using self-report is because it doesn’t need adapting for different populations, but that’s not the case, as it needs to be translated.
Line 77: Consider changing ‘famous’ to ‘widely used’ – more scientific.
Line 103: ‘objective’
Line 156: no references are given for Estudillo, 2020, or Estudillo et al., 2020.
Line 153: ‘five-point’
Line 154: ‘reverse scored’
Line 160: ‘we first explored observers’ insights’
Line 161: It’s unnecessary to have the R2 here and throughout when you’ve already said how much of the variation is explained, the R2 is superfluous/repetitive.
Line 174: ‘closer inspection of Figure 1B’
Line 201: ‘on the PI20’
Line 226: ‘someone not seen in years’
Line 244: ‘association’
Line 251: ‘objective’
Figure 2 – it should be the CFMT, not CFMT-Chinese.
Experimental design: The research question is well defined, relevant and meaningful. There are some details missing from the method:

Line 117: How fast was ‘abnormally fast’?
Line 130: How was the pI20 translated into Mandarin? Were there any checks on the validity of the translation?
Line 148: the reliability of the PI20 has been previously given as 0.96, which is considerably higher than the current sample.

Gray et al. actually performed two studies; the data used in the current paper collapses across these two samples. Can you give a justification for doing this?
Validity of the findings: I have some major problems with the approach taken in the analysis. Specifically, the selection of quartiles is arbritary. When looking at the scatter plots, it does not look as though there is a non-linear relationship between the variables. I understand that investigating sections is theoretically driven (in that both good and poor performers could have better insight), but using quartiles doesn’t match up to our definitions of good and poor performers. If one took a random slices of any correlation, there may well be differences between the strength of the relationship. Have the authors thought of fitting non-linear models to the data? Or using a more theoretically driven approach to the partitioning of the data?

Given this, it is particularly problematic that the authors do find a significant correlation in the third quartile of Gray et al.’s paper. The existence of a correlation in this quartile (where participants are neither good nor bad at face processing) directly refutes the conclusions of the paper. Therefore, the conclusions are not supported.

There also seems to be some misunderstanding on how correlations work – reducing the range will always limit the strength of a correlation. In the discussion, the authors say that “only good and bad recognisers have (limited) insights”, and discuss this in the final paragraph of the discussion, suggesting they are over-interpreting the drop in R2 which is driven instead by the reduction of score range.
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: ASSOCIATIONS BETWEEN SELF-REPORTED AND OBJECTIVE FACE RECOGNITION ABILITIES ARE ONLY EVIDENT IN ABOVE- AND BELOW-AVERAGE RECOGNISERS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The manuscript in much improved, I found few grammatical mistakes in this version:
Line 75: "have"
Line 200: "observers' insights"
Line 201: "observers' scores"
Line 255: "of the PI-20"
Experimental design: The added information makes the paper much clearer.
Validity of the findings: I continue to have reservations about the calculation of the correlations/quartiles, which I will attempt to explain.

There are uneven numbers of participants in the groups allocated in both experiments’ quartile split, this is because in the middle of the distribution many of participants share the same score. Can the authors describe and justify their approach to allocating scores into the quartiles? In the current experiment, if participants are randomly split evenly between the nearby groups (such that each group is made of 63/64 participants), the correlations change considerably, suggesting the current findings are not robust to the cut-offs used.

The variability in both scales are important when assessing a correlation - the PI20 also does not have an even range across the quartiles. Can the authors also provide the range of PI20 scores for each quartile in Table 1?

One way to help the authors and readers interpret the correlations would be to include 95% confidence intervals on them.
Additional comments: I appreciate the authors’ rebuttal, and their thoughtful response. I apologise for my misunderstanding of the direction of the effect in Q3 in Gray and colleague’s data.

In the title, the authors are suggesting that the CFMT represents ‘actual’ face recognition ability. However, the CFMT is a match-to-sample test with a reasonably high probability of guessing the correct answers by chance (33% on most trials). The CFMT is an objective face test, but is not a wholly accurate test of face recognition ability ‘in the wild’. The authors should change their framing of this in the title and throughout the manuscript. The fact that the PI20 accounts for a relatively small proportion of the variance of CFMT scores could be due to noise within the CFMT rather than issues with the PI20.


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: SILK DERIVED FORMULATIONS FOR ACCELERATED WOUND HEALING IN DIABETIC MICE
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript by Tariq et al entitled “Silk derived formulations for accelerated wound healing in diabetic mice” examines the potential of doping silk fibroin and sericin hydrogles with Aloe vera to generate a potential wound treatment product. The manuscript contains a range of data sets but fails to reach the minimum scientific rigour required by this journal. I have got the following specific comments that may help to further improve the manuscript:

Selection (many more problems):
(1)	The in vivo study is flawed. Rodent wound healing is by contraction. In the absence of the splint model the data are misleading. Please see: Adv Wound Care (New Rochelle). 2013 May; 2(4): 142–148.
doi: 10.1089/wound.2012.0424
(2)	Results: Figure 2. I am not convinced this is silk. This looks like salt crystals. Authors also mix up scales. SEM does not appear to be in the nanometer range as described in the body of the manuscript.
(3)	Results: Figure 3. Duplication of images (left and right hand side). Top left figure panel: 102 nm is incorrect.
(4)	Results: Table 3 needs to be converted into a bar chart.
(5)	Discussion Line 256 following: Based on Table 3 not obvious how day 11 and 15 were selected and seems arbitrary.
(6)	Mice: The wound area was not shaved properly. What was the sex of the animals?
(7)	The vast majority of references are outdated. For example, the introduction cites the 2003 Altman review despite numerous timely and up-to-date reviews published over the past 5 years.
(8)	Remove Figure 1 because it does not have any content.
(9)	Figure scale bars are absent or too small.
(10)	Figure 4. Overall appears sloppy: Too small, the annotations are not clear, scale bars are missing etc.
(11)	Results: All figure legends need to be expanded to include more key information.
(12)	The manuscript would benefit from English language editing (e.g. abstract “finest biocompatibility”; “wounds in control group (polyfax) were healed” etc.
Experimental design: Please see Basic Reporting
Validity of the findings: Please see Basic Reporting
Additional comments: Please see Basic Reporting


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: SILK DERIVED FORMULATIONS FOR ACCELERATED WOUND HEALING IN DIABETIC MICE
Review round: 1
Reviewer: 2

Output:
Basic reporting: In general, language used is clear and professional English is used.
Sufficient background information is provided with proper citations except for some parts under methodology (shown in the manuscript as track changes).
Proper article structure is used with figures, tables and raw data.

Following comments are for further improvement
Keep a space between a numerical value and units
Additional reference is requested under methodology
Some figures need revisions (font size of labels, additional labels in Figure 4, comment on Figure 5 T4 day 11 panel)
Experimental design: Research question is well addressed
Ethical standards were maintained.
Experiments conducted have used accepted standards.

Following comments are for further improvement
Originality could be more emphasized- focusing on to wound healing in diabetes
One objective may be removed (shown in manuscript with track changes)
Comments-on extraction of sericin and measurement of wound contraction need to be addressed
Validity of the findings: In general results are supported with relevant figures and tables
Conclusions match with research question

Comment on figure 5 T4 panel need to be addressed (shown in manuscript with track changes)

Usefulness of tables 1 and 2 are not well documented
Additional comments: Comments are covered by 3 areas above


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

Input:
Title: SILK DERIVED FORMULATIONS FOR ACCELERATED WOUND HEALING IN DIABETIC MICE
Review round: 2
Reviewer: 1

Output:
Basic reporting: Addressing point 1 is beyond the scope of a revision. The model system is not robust and thus not of the required standard for Peer J.
Experimental design: See above.
Validity of the findings: n/a
Additional comments: Addressing point 1 is beyond the scope of a revision. The model system is not robust and thus not of the required standard for Peer J.


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

Input:
Title: SILK DERIVED FORMULATIONS FOR ACCELERATED WOUND HEALING IN DIABETIC MICE
Review round: 2
Reviewer: 2

Output:
Basic reporting: The article entitled "Silk derived formulations for accelerated wound healing in
diabetic mice” by Tariq et al is written in clear English. The language needs some improvement, but the formulated hypothesis and scientific method to address research question are clear and well structured.
Experimental design: In vivo study design is fairly solid with five treatment groups and a negative control. Moreover, given the ethical constrains, six animals per treatment group are convincing numbers "N" to conclude the rigor and strength of the findings based on statistical grounds. Method has sufficient detail and where aver possible well supported by relevant reference.
Validity of the findings: Although the silk proteins are known to induce beneficial wound healing effect however combination with medicinal plant extract in this current study has significantly improved wound healing in diabetic rodent model. Findings are strong and may eventually seek applications in pharma industry and medical sciences.
Additional comments: I reviewed the article entitled “Silk derived formulations for accelerated wound healing in
diabetic mice” by Tariq et al. In this manuscript, the authors have developed a new wound healing formulation by combining silk derived proteins, fibroin and sericin and aloe vera plant extract (gel). They then examined wound healing potential of this formulation in vivo on the chemically induced diabetic mouse model. The findings suggested that the combination of fibroin and Aloe vera gel provides beneficial wound healing effects as opposed to fibroin alone or a combination of sericin and aloe vera gel.

Although, as authors indicated, the wound healing effect conferred by the cocoon’s derived silk proteins is well known, however, the idea of combining silk protein with the extract of naturally existing medicinal plants and subsequently, testing the formulation on diabetic mouse model is very promising, which may eventually contribute to the fields of pharma industry and medical sciences.

As such the SEM images (fig 1 and fig 2) look suboptimal. I would suggest combining both figures as one with the same scale bar. If possible another confirmatory test could be done to show that the extracted proteins are indeed silk fibers. Since both proteins are available commercially, maybe a head to head western blot comparison or an in vitro cell based motility assay (doi: 10.1371/journal.pone.0042271) could be sufficient to show authenticity of the extracted proteins.

Authors should indicate statistical significance (p value) on the bars of figure 4 showing comparison of at least control versus silk protein and aloe vera formulations treated groups.

Labels within the H&E stained images are not readable, the authors should increase font size to make it visible.


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: SILK DERIVED FORMULATIONS FOR ACCELERATED WOUND HEALING IN DIABETIC MICE
Review round: 2
Reviewer: 3

Output:
Basic reporting: The authors have successfully addressed all the queries raised by the other reviewers about the manuscript "Silk derived formulations for accelerated wound healing in diabetic mice". I have no further queries.
Experimental design: The experimental designs are looking good.
Validity of the findings: The conclusions are well stated and relevant to the current scenario of the field.
Additional comments: No further comments.


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

Input:
Title: SILK DERIVED FORMULATIONS FOR ACCELERATED WOUND HEALING IN DIABETIC MICE
Review round: 2
Reviewer: 4

Output:
Basic reporting: Interesting question, how different silk components act together with Aloe extracts in diabetic wound healing.
The manuscript is a revised version, which was clearly improved based on the reviewer’s comments.
Still the references are in inconsistent formats and partially incomplete eg.

Bouzghaya, S., Amri, M. and Homblé, F., 2020. Improvement of Diabetes Symptoms and Complications by an Aqueous Extract of Linum usitatissimum (L.) Seeds in Alloxan-Induced Diabetic Mice. Journal of Medicinal Food .
REV:Missing details
Cassinelli, C., Cascardo, G., Morra, M., Draghi, L., Motta, A. &Catapano, G., 2006. Physical-chemical and biological characterization of silk fibroin-coated porous membranes for medical applications.Int J Artif Organs., 29(9):881 .
REV:Only first page
Actabiomater ., 48:157-174.
REV: Unclear Journal name
Experimental design: Alloxan and streptozotocin are the most popular diabetogenic agents used for models, it is not mentioned why alloxan was used in this model and which type of diabetes does this represent.
A non-splinted excision model was used in mice. It should be mentioned what Polyfax is (antibiotic ointment)
Validity of the findings: I cannot find all the underlying data, only single examples of in vivo and histology figures are given.
Although the figures might have been improved, they are still of low quality. Fig 1 and 2 do not give a clear information, which comes also from the very limited figure legends. Quality of histology is limited.
It is unclear for which purpose the tables with MS results are shown, because there was no research question associated and also no discussion presented.
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: CONSTRUCTION AND VALIDATION OF A NOVEL PROGNOSTIC SIGNATURE OF MICRORNAS IN LUNG ADENOCARCINOMA
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comments
Experimental design: no comments
Validity of the findings: no comments
Additional comments: The authors performed an analysis using TCGA data to identify a miRNA signature with prognostic value in lung adenocarcinoma patients. They found a signature of 6 miRNAs associated with prognosis.
A series of points should be clarified:
- The end point of the statistical analysis should be better clarified and specified
- Did the statistical analysis consider the adjuvant or neoadjuvant treament variable? This should be specified
- Figure 4 is related to "gastric cancer" patients. May be there is a mistake
- The english languages should be revised


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: CONSTRUCTION AND VALIDATION OF A NOVEL PROGNOSTIC SIGNATURE OF MICRORNAS IN LUNG ADENOCARCINOMA
Review round: 1
Reviewer: 2

Output:
Basic reporting: There are multiple grammatical and stylistic errors over the text. The English should, therefore, be thoroughly revised by a Native speaker (ideally professional proofreader).
Lines 112-113. The phrase “... significant correlation with lung adenocarcinoma” is not clear. Please indicate the exact parameter with which correlation of eight miRNA expression was observed (e.g. stage of the disease, the aggressiveness of the tumor, etc.).
Lines 111-123. From the sentence “The Cox univariate regression analysis was 112 employed for the preliminary screening of potential miRNAs” it remains unclear what is meant after “potential miRNA”. Please clarify.
Figure legends 4, 5 and 6 should include more information (description) of the corresponding plot.
Experimental design: The MMs and results sections are too short and should contain more details. Specifically, the calculations and data analysis procedure should be described in more detail (ideally with exact formulas used) and include references to the corresponding methodology literature or reports where such calculations are described. Next, the format in which the miRNA expression data were collected as well as the method used to measure miRNAs expression should be indicated. Ideally, the raw miRNA data downloaded from the TCGA database should be included in the supplementary material. Finally, it is not described how exactly (methodology, software, etc.) Gene Ontology and KEGG analysis were performed.
Validity of the findings: The reference list must be rechecked and corrected. For instance, reference 5 is incomplete (lacking the authors' names). The reference 26 seems to lack the page numbers. Reference 29 lacks the journal name. References 39 and 43 have no publication year and journal volume/pages indicated. Some references contain the indicated months and the others are not.
Additional comments: In their work entitled “Construction and validation of a novel prognostic signature of microRNAs in lung adenocarcinoma” the authors analyzed miRNA expression data in a large number (499 in total) tumor specimen obtained from Cancer Genome Atlas (TCGA) database. Furthermore, preliminary miRNA screening using Cox univariate regression analysis resulted in a selection of eight miRNAs which demonstrated a statistically significant correlation with disease status; next, using LASSO cox regression analysis the authors established a prognostic signature for overall survival which included miR-1468, miR-299, miR-4709, miR-5571, miR-582 and miR-3653. Finally, the authors showed that indicated target genes of the above-mentioned miRNAs were related to Wnt and mTOR signaling pathways, which are strongly associated with carcinogenesis. The presented miRNA signature has the potential to be used for personalized therapy strategies for lung cancer patients.
All statistical calculations seem to be performed adequately; however, a more detailed description is necessary.  The data are convincingly presented in the figures. The article can be reconsidered for a publication only after several points mentioned above are 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: CONSTRUCTION AND VALIDATION OF A NOVEL PROGNOSTIC SIGNATURE OF MICRORNAS IN LUNG ADENOCARCINOMA
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript by Li et al. provides a 6-miRNA signature with prognostic value for overall survival y patients with lung adenocarcinoma. Background/context has been adequately provided and the references are appropiated. The following caveats need to be solved before publication:

1.-English used throught the text needs to be extensively revised. It lacks clarity and is often confusing. Some examples:

Abstract:"Lung cancer (LC) is highly implicated in biological process" (makes no sense)
p. 3, l. 41: "...which is the most common pathological type of LC" (all cancers are pathological)
p.3, l. 46: "The limited survival benefits indicate the deficiency of the current staging system and the presence of more unknown tumor characteristics" (needs proofing and rephrasing)
p. 3, l. 57: "With consistency" change to "Consistently"
p. 4, l. 71: "And it is of promising value..." (no sense) change to "It is promising..."
p. 4, l. 72: "Herein, in our study..." is redundant, just leave "Herein"
p. 4, l. 76: "Material and Method" change to "Material and Methods"
p. 5, l. 86: "...resample model inclusion proportion (RMIP)" change to "...resample model inclusion probability (RMIP)"
Throught the text OS is defined as "Over Survival" instead of "Overall Survival"

These are jus a few correlative examples but it goes on throught the entire manuscript.

2.-Raw data for the Cox univariate regression analysis has not been provided. This is an important flaw. The authors only provide final Kaplan-Meyer graphs for their "selected 8 miRNAs".

3.-The identification of potential target genes remains speculative without proper assessment  by immunohistochemistry in tumor samples.
Experimental design: The work presented in this manuscript is original and withing the aims and scope of the jounal. The experimental design suffer from the following deficits:

1.-As already mentioned, this study heavily relays on the statistical analysis of tumor expression data from the TCGA database, however, the raw data obtained after the analysis has not been provided.

2.-Fig. 1A: the Y-axis is labeled as "frecuency", however the scale consist of absolute numbers (samples)

3.-p. 5, l. 121: a risk score formula is provided without any further explanation nor is the methodology to get such formula explained.

4.- p. 5, l. 126: the authors define high risk and low risk groups of patients, however it is not clear which group corresponds to high or low levels of the 6-miRNAs.

5.-p. 6, l. 150: the authors use different well-stablished algorithms to obtain a miRNA-mRNA regulatory network and predict ontology terms and putative targets. However, none of the identified targets (their expression levels) has been assessed in a tumor samples.

The methos section could be improved by extending the description of the different methodologies ( (particularly, the statistics) and being more specific in the details.
Validity of the findings: -The lack of some raw data (as mentioned), together with the above-mentioned flaws in the experimental design and the too-speculative nature (not supported by experimental data) of some predictions regarding genes potentialy regulated by the 6-miRNA signature limit the scientific soundness of the manuscript.
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: CONSTRUCTION AND VALIDATION OF A NOVEL PROGNOSTIC SIGNATURE OF MICRORNAS IN LUNG ADENOCARCINOMA
Review round: 3
Reviewer: 1

Output:
Basic reporting: The authors adequately responded to the concern raised in the first round of revisions, however, I do have two more issues:
1. The authors provide AUC values both on training and validation groups (section Validation and evaluation of the miRNAs signature). However, they do not provide values of sensitivity and specificity. It is really important to do this since AUC only measures "classification potential" of the classifier ignoring actual accuracy (AUC can be really high with very low accuracy). As we can see, these values actually are presented at the bottom of ROC curves in Figure 2, however, they should be clearly stated in the text.
2. In the Data and sources, section authors state that the LUAD patients were randomly assigned to the training and validation groups. Can the authors repeat this step multiple times to assess what will happen with AUC, sensitivity and specificity? Obviously, other partitions to training and validation groups can both increase and decrease the reliability of the classifier. Thus, it will be helpful to see means/variations/confidence intervals.
Experimental design: The authors provide the original research within aims and scope of the journal.
Validity of the findings: Please see the second issue upper.
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: SYNERGISM BETWEEN CALCIUM NITRATE APPLICATIONS AND FUNGAL ENDOPHYTES TO INCREASE SUGAR CONCENTRATION IN FESTUCA SINENSIS UNDER COLD STRESS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The article was written in a clear and technically correct English, and a sufficient number of references is supporting how the works fit into the field of study. However, Table 4 is not too clear and potentially will causes confusing in the readers to interpret it. I strong suggest that they reorganize this table into two different ones.  All appropriate raw data have been made available, but, due to inadequate statistical description, the same model used by the authors can't be reproduced easily. Please, include  the statistical model (with equations).
Experimental design: i) Lines 100-108: They are inducing the reader to understand that they performed a completely randomized design, which does not apply to most of  the experiments conducted on the field. More information detailing the experimental structure should be necessary to support the understanding about their experimental design. If the authors had structured  in the field a completely randomized design, what were the reasons to apply this approach?

ii) Line 101: Authors have to be more clear if they considered 36 plants by line or a total of 36 plant considering both lines. Just on the line 107 the reader can indirectly understand that a total of 36 plants was used on. Please, make this clear before.

iii) Lines 100-108: The treatment looks a factorial structure, as they combine levels of  seeds (infected or not) with levels of Calcium (0, 25, 50), however there is no information about treatment structure. Please, provide this details in the manuscript.

iv) Lines 108-110: As the author described that 12 plants under each level of Ca for either E+ or E- were harvested independently over three time periods. As those plants were established into same plot, there is a spatial relationship between them, consequently, this cannot be viewed as a random sample. How the authors control confounding between plots and treatment on plants?

v) Lines 108-110: As the authors are using a subsample on the plots (12 plants), how they included the effect of plots (sample) and the effect of plant (subsamples) on the statistical analysis? This description will be fundamental to guarantee that their analysis is correctly developed, and, consequently, make their finds more reliable. Probably the effect of plots and subplots should be added in the analysis of variance.
Validity of the findings: The paper can potentially has a high impact into to improve sugar concentration in Festuca sinensis. However, as the authors did not describe their experimental conductions nor statistical model sufficiently, some doubts arise about the validity of the results obtained. I suggest that the authors re-write the Statistical Analysis including details about the approach used and justifying it.  Furthermore, all comments described about the experimental design should be clarified in order to prove the validity of their results.
Additional comments: i) Lines 170-176: There is no statistical model provided over the paper. Please, include it as the model can help to understand how the experimental structure was modelled.

ii) Lines 110-111: As the authors measured the temperature over the experiment during day and night. How they included this information in the statistical model?

iii) Lines 113: Could be interesting the inclusion of the most important response variable, categorized by treatment, in the Figure 1, as the interaction between treatment  and temperature has an direct effect on the response.

iv) Lines 180-181: The authors could provide, as supplementary material, a plot of the residuals versus fitted values to check error independence assumptions, Q-Q plot of residuals versus theoretical residuals to check the error normality assumptions. In case of completely randomized design, the Shapiro-Wilk test for normality and Levene test to check homoscedasticity could also be provided in order to guarantee the validity of their finds.

v) Lines 188-189: What was the reasons  that E- plants under 25 Ca had higher root activity than E+25?

vi) Line 206, 222: Authors should not assume that there were substantial time series effect as they don't have enough information over the time. I encourage the authors to focus in describe the effects considering the time as a factor rather than as continuous.

vii) Lines 257-259: Authors described that exogenous application of calcium in combination with endophyte actively effects soluble sugar accumulation. I can see an possible significant marginal effect of the time on soluble sugar accumulation, but the effect of exogenous calcium on E+ and E- plants are not enough evident based on means and standard errors, and without model assumptions checked. As the authors have a factorial design, should be appropriate that they included, in the supplementary material, the the effects of each factor estimated at each levels of the other factors, to show that they are yielding conclusions that are valid over their experimental conditions.

viii)  Lines 312-313: Authors said that "Interestingly, the synergistic effects of endophytic fungi and exogenous calcium on root activity occurred early after cold treatment in natural field conditions (Fig. 2)".  What  they means about "Natural Condition"? This results could also shows that there is a possible confounding  between natural field conditions and treatment effects. How the authors could explain it?

ix)  I disagree with the authors about they infer that calcium treatment enhanced the water content of leaves. I would like to see the parameters estimations of the three-way ANOVA, as the Figure 3 suggests the opposite, that means, there are more water content of leaves without calcium treatment.


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: SYNERGISM BETWEEN CALCIUM NITRATE APPLICATIONS AND FUNGAL ENDOPHYTES TO INCREASE SUGAR CONCENTRATION IN FESTUCA SINENSIS UNDER COLD STRESS
Review round: 1
Reviewer: 2

Output:
Basic reporting: Generally good, but see comments in last box below
English language would benefit from additional checking
Experimental design: The research question could be better linked to the cited references.
There is a concern that effects attributed to Ca might be a response to N since Ca(NO3)2 was the key treatment.
A map of the spatial arrangements of the treatments with the raw data would be desirable.
Comments below expand on these comments
Validity of the findings: The possible influence of applied nitrate on the results needs to be addressed.
The citation of other work is sophisticated, but the linkage of the authors' work to the cited work is actually quite weak, as indicated by very generalized conclusions in section 5.
Additional comments: PeerJ MS 45909; Zhou et al. Synergism between calcium applications and fungal endophytes to improve sugar concentrations in Festuca sinensis under cold stress
General comments
1.	Overall the MS is well prepared. An interesting plant-endophyte symbiotic system was studied and a pleasing range of physiological parameters measured. It is a pleasant change to have the raw data for inspection, and for the most part I was able to replicate the ANOVA Tables presented in Table 1. (However, I think the F-value of treatment time for root activity should be 1454, not 145.4, according to my ANOVA). The English grammar shows clear signs of checking by a native speaker, though some words and sentences requiring attention have been missed, especially in the results section and Figure and Table captions (or adjusted by the Chinese authors after the checked MS was received). The data on root activity differences between E+ and E- plants is novel and interesting. Despite these good points, I do have some significant concerns about the MS as outlined below.

Specific comments
1.	Confounding of Ca and N effects: A major concern I have about this piece of research is that the results are presented as a response to calcium, but the treatment was Ca(NO3)2, so plants have received a double stimulus of Ca and N. Inspection of the Methods section shows that the quantity of elemental N added with the Ca treatments was up to 42 mg per plant. I find no mention of plant total dry weight but if plants were of a smallish size this could potentially be in excess of 2% plant DW which would be a major physiological stimulus. I can not see how we can differentiate the Ca response from the N response under the current experiment design. It would seem that to satisfy the aim of measuring a calcium response, either a calcium salt with a plant-inert negative ion would need to be used, or the differential response calculated between the Ca(NO3)2 application and an equivalent amount of N applied without Ca in a form such as urea or NH4NO3. For publication, this issue must be satisfactorily addressed. This would include mention somewhere in the MS of typical plant root and shoot DW at harvest and a logical explanation of the separation of Ca and N effects in the results interpretation, since they are confounded in the treatment application.
2.	Quality of research hypothesis: For this reviewer the research hypothesis seems very general and without detail. In my observation, the traditional approach to research in China is to identify the system one wants to work with (in this case a Festuca-Epichloë symbiosis) and then introduce a perturbation (in this case Ca application) and record the response of the experimental system. The results are then published as response observations in juxtaposition with some nominally relevant results of other experiments and speculative or very general mechanistic interpretation by authors (as seen at lines 343 – 367, e.g, leading to a lack of specificity in the closing sentence at lines 387–389). This is different from the Western approach where there is much more focus on hypothesis development, identification of an experiment design to address the hypothesis, and interpretation of results according to physiological mechanisms at play. This reviewer’s impression is that the experiment might have been designed under the traditional Chinese approach  and then presented to seem to fit the Western research philosophy. Specifically with respect to the way the introduction builds to the  research hypothesis, there is a very general assertion (with copious cited references) that cold retards plant growth and Ca can be a stimulus, but when we come to the research aim at lines 83 – 84, there is as yet no detail at the physiological level of plant processes that may be affected by cold, with the cold response ameliorated by Ca. For example, at lines 72 – 73 we have “These observations have led to the hypothesis that agricultural strategies to assist plant adaptation to low temperature stress include exogenous chemical applications or symbiotic technology”. For this reviewer some detail from the cited references of the exogenous chemicals used in the cited references and their impact on the plant-endophyte interaction and relevance to the hypothesis at the physiology level would be highly desirable. This detail could be constructed so as to give insight into the reasons for the choice of measurement parameters for the experiment (root activity, chlorophyll levels, relative water content, soluble carbohydrates).
3.	Experiment field layout, randomisation and statistical analysis validity:  Lines 101–102 mention 2 rows of 36 E+ and 36 E– plants per replicate, so that is 216 plants in the experiment plot, but the data have only 54 rows. Therefore it is not clear how the plants for the various treatments were spatially arranged in the field. This is important because valid ANOVA requires randomisation to satisfy the assumption that each data point is independent of the others. Even if it is not published,  it would be good that the authors supply to the reviewers or for inclusion with the raw data in the word file, a map showing the special placement of each of the 54 sampled plants and their treatments (Endophyte, Ca and day). For example if Ca0, Ca25 and Ca50 treatments are grouped along a row and the rows have some stronger and weaker patches, then this can lead to the false identification of a treatment effect or interaction effect in the ANOVA. The MS as presented does not show there is a problem (though separation of E+ and E– into different rows is a possible concern and might be better treated as a split plot design, not a factorial ANOVA as used by the authors); but equally it does not confirm there is not a problem, and that would be solved if a map of the spatial organisation of the plants from each treatment is available. Regarding 216 plants and 54 data observations, maybe 4 plants were planted in May for every one needed at harvest in October/November? Meanwhile layout details at lines 107–108 seem to be different from those given in lines 101-102. It highlights the value of providing a map for clearer understanding. Did harvests at days 0, 14 and 28 use the same plants or different plants each time?
4.	Use of could in reporting and discussing results: In Chinese science writing use of ‘ke yi’ (可以) is common when reporting or discussing results but in English we like to report the result from the experiment as a fact, without saying ‘can’ or ‘could’. In English ‘could’ is a subjunctive verb form expressing doubt if something will happen. So at line 385, it might be better to choose another phrase instead of “it could be”. (Also line 317.)
5.	Low soluble CHO levels: The CHO levels in Table 4 seem low. Is it because the plants are growing in harsh conditions, or could there be an analysis calibration problem?


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: SYNERGISM BETWEEN CALCIUM NITRATE APPLICATIONS AND FUNGAL ENDOPHYTES TO INCREASE SUGAR CONCENTRATION IN FESTUCA SINENSIS UNDER COLD STRESS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The paper has a clear aim, and part of comments done on the first review were included in the new version. The proposal manuscript has merit to be published, however,  some questions still arising through methodology and results that should be clarified. All questions were described in details in the following sections.
Experimental design: Line 106 - Authors said that "the trials were arranged using a split-plot design with E+ and E– as the main plots, Ca(NO 3 ) 2 levels as the subplots, and sampling time as the sub-sub-plot". I agree that model could be viewed as described by the authors, however It should be interesting they describe how the repeated measurements on subject was captured by their model on the methodology, as I've had requested in the first review. For example, an way to model a design like that is through mixed-effects model. Furthermore, as the repeated measurements on subject are probably correlated, they also could explore the individual variability to describe how strongly subjects in the same group resemble each other, using the intraclass correlation coefficient. This will enhance the discussion of the paper as well.
Validity of the findings: 1) I requested to the authors, as supplementary material, a plot of the residuals versus fitted values to check error independence assumptions, Q-Q plot of residuals versus theoretical residuals to check the error normality assumptions, and Levene's test to check homoscedasticity in order to guarantee the validity of their finds, however they provide just Levene's test.

The Levene's test shows that most of response variables were not obtained based on random sampling from a population with equal variances (p-value < 0.05). Thus, authors should include a more complex structure in the variance-covariance matrix to correctly fit the model to their database, probably including different variances by treatment.  In this sense, I recommend the implementation of a new variance-covariance structure in order to get correct inference and p-values.

2) What do authors mean by "Univariate analysis of variance was conducted tests of between-subject effects according to the general linear model"? This phrase is not clear at all. Please provide the formulation of this general linear model.

3) Table 4 is showing no two-way nor three-way interaction effect for Soluble sugar concentration (root), Fructose concentration (shoot), and Glucose concentration (root and shoot) (see p-value > 0.05). In this sense, please do a stepwise selection procedure for each model in order to select a more parsimonious model for each response variable.
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: SYNERGISM BETWEEN CALCIUM NITRATE APPLICATIONS AND FUNGAL ENDOPHYTES TO INCREASE SUGAR CONCENTRATION IN FESTUCA SINENSIS UNDER COLD STRESS
Review round: 2
Reviewer: 2

Output:
Basic reporting: The first submission was generally good; the authors have tried hard to address issues raised by reviewers. The English grammar, especially in revised sections, needs checking by a native English speaker. There are a number of mistakes (e.g. 'more much' at line 343, many 'awkward' sentences, and frequent mismatching of singular and plural adjectives, nouns and verbs.
Experimental design: Both reviewers raised concerns on the first version. The site map now supplied by the authors confirms the reviewers' concerns were valid. The issues raised by the reviewers remain unadressed by the authors. Currently the authors state they used a split-split-plot design, but the present ANOVA Tables in their results are for a three-way factorial design. I have provided extended comment on this question in an attached file. Regardless of which design is chosen for reporting purposes, the authors at least need to present the ANOVA Tables for the design they state in the materials and methods that they used.
To assist the authors, an example ANOVA table for a split-split-plot design, is compared in the attached file with a three-way factorial ANOVA for the authors' root activity data.
Validity of the findings: A critical point in my first review was No.1, 'confounding of Ca and N treatment effects. Since the treatment was Ca(NO3)2, I do not see how we can exclude the possibility that the observed plant physiology responses are to N and not Ca, or to both elements working together? The authors' answer does not satisfy me. I have added further explanation of my concern in the attached file. Perhaps this is now a matter requiring the Editor's decision to settle it.
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: SYNERGISM BETWEEN CALCIUM NITRATE APPLICATIONS AND FUNGAL ENDOPHYTES TO INCREASE SUGAR CONCENTRATION IN FESTUCA SINENSIS UNDER COLD STRESS
Review round: 3
Reviewer: 1

Output:
Basic reporting: I recommend a carefully review about experimental design, statistical methodology and the inclusion of a paragraph to discuss the limitations of this study.
Experimental design: In a new revision of their map of the spatial arrangements of the treatments I found serious experimental design errors. It appear that they systematically distributed the levels of each factor on plots and subplots rather than using a randomization procedure.

One of fundamental principle of experimentation is the randomization, where the probability of a unit receive any treatment should be the same. Furthermore, randomization reduces confounding among observed and unobserved factors. This can puts all considerations and conclusions of this study as being unreliable. There is no way to distinguish if the effect of levels of Ca nor plants, nor interaction between them were different due to a "true" effect of them or possibly because there is a confounding between them and experimental error. In a split-plot design there are still more concerned about error and factor confounding as there are two source of errors, one from the plot and another from split-plot. Perhaps this is now a matter requiring the Editor's decision to settle it.
Validity of the findings: 1) My question about validity of their findings was not properly answered by the authors.

Although they are using split-split-plot design, all models have strong assumptions that should be verified before report any findings. Including diagnostic plots, and statistical tests about model assumptions for Reviewers or as supplementary material are fundamental to certify that significant effects, and hypothesis tested are validity. I would like to see a plot between fitted values vs. residual and a statistical test for homoscedasticity.

Reference: Montfomery, D. Design and Analysis of Experiments, John Wiley & Sons, 8 ed., 2013

2) In the last review I requested a model selection procedure because some interactions were not significant, however, they did not addressed this questions. Additionally, In the new Table they reported p-values higher than 1, how they found it as p-value takes value between 0 and 1?
Additional comments: I did not see any paragraph in their manuscript discussing about a possible confounding of Ca and N treatment effects (as mentioned by Reviewer 2), which could be relevant for further studies.


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

Input:
Title: SYNERGISM BETWEEN CALCIUM NITRATE APPLICATIONS AND FUNGAL ENDOPHYTES TO INCREASE SUGAR CONCENTRATION IN FESTUCA SINENSIS UNDER COLD STRESS
Review round: 3
Reviewer: 2

Output:
Basic reporting: Covered in previous review
Experimental design: With a map of the experiment layout now provided and the ANOVA now presented as a split-split plot design, this reviewer is now happy. We should also not that in many cases the statistical F values are very large so debate about which model to use is less relevant as any model will declare significance.
Validity of the findings: My number one concern was always that the experiment design does not allow any logical separation of the effects of the element calcium and the element nitrogen when adding calcium nitrate as a treatment. In this second revision, the authors continue to avoid any direct answer on this point. The reference cited for work on a tree species is not at all convincing. The purple-colored track changes comments added then removed again near line 93 ('We ignored .. cell.') are revealing. I note, however, that in the conclusions of the later versions, unlike the first submission, the authors now refer to the effect of 'calcium nitrate', instead of simply 'calcium'. This is much more scientifically correct. If the authors will now do the same in the title and refer to synergy with 'calcium nitrate' this reviewer will be satisfied.

In hindsight it occurs to me that the authors very likely still have residual leaf samples from their work. If so, analysis of leaf tissue for Ca and N concentration would likely be possible within days and would quickly resolve whether or not the nitrate component in calcium nitrate could have played a role in generating the results observed.

I consider all other comments and requests have been addressed well. There remain some minor grammar points. For example I think 'a' (indefinite article) inserted before split-split-plot at line 188 would be better. Maybe cosmetic adjustments like this can be carried out by the typesetting team.
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: SYNERGISM BETWEEN CALCIUM NITRATE APPLICATIONS AND FUNGAL ENDOPHYTES TO INCREASE SUGAR CONCENTRATION IN FESTUCA SINENSIS UNDER COLD STRESS
Review round: 4
Reviewer: 1

Output:
Basic reporting: Thanks to the authors for answering all my questions in this session.
Experimental design: Thanks to the authors for answering all my questions in this session. Their experimental design appears to be systematically randomized rather than give the same probability of a whole plot receive a specific dosage of Calcium nitrate and the same happened in subplot. I reccomend the authors consider apply all the principles of experimentation on their next experiments. When one of them are broken we involuntary can introduce biases that sometimes is really difficulte to evaluate in the data analysis process. The model proposed to analyse this experiment, e.g.,  is not accounting for the systematic process of randomization.

Example: If you observed the lines within plots, there are a systematic inclusion of E+ and E-. This shows that the authors introduced a new factor in the experimental design due to the randomness process adoped. Clearly, the impact of this new factor in the whole experiment is confunded with the treatment effect.

On the other hand, the inclusion of the experimental map help readers to undertand this limitation. If the authors can guarantee that all the phisics and enviromental factors were the same for each plot, thus the effect of this new factor tends to be small.
Validity of the findings: The authors presented the Levene's test for homoscedasticity, The Table S1 shows that for most of their response variables there is a no acceptation of the null hypothesis. This shows that the variance between treatment were not the same. There are some statistical solutions to account for heterocedasticy in the model. However, I initially suggest a variable transformation (log, square root, etc.). Generally, variable transformation affects variance function in order to obtain more similar variances for treatments, and sometimes also improve normality assumption. The correction of the variance issue in fundamental to get comparison between treatments more reliable. I know that the authors have been doing a lot of work in our suggestion, and they are doing a nice work here. However, I see myself obligate to strong recommend this correction in order to improve their results.

In addition, the log transformation of Root activity was not sufficient to stabilize the variance.

I recommend to the authors read the paper titled as 'An Analysis of Transformations' described by Box and Cox in 1964. It could be helpful in addresses this issues.
Additional comments: No more comments here


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

Input:
Title: SYNERGISM BETWEEN CALCIUM NITRATE APPLICATIONS AND FUNGAL ENDOPHYTES TO INCREASE SUGAR CONCENTRATION IN FESTUCA SINENSIS UNDER COLD STRESS
Review round: 4
Reviewer: 2

Output:
Basic reporting: The Map in FigS1 is incompatible with Tables 1 and 4 in the MS; see below.
Experimental design: I have to apologise that in my response to the second revision I did not notice that the experiment map was changed. Was it mentioned by the authors anywhere? If it was, I missed it, but when reviewing revision 2, I simply referred to the first map which I had downloaded and saved. Never in my previous experience have the researchers changed the map of their experiment during MS review.  For this reviewer, the new experiment map now presented as Figure S1 is incompatible with the MS. To explain, I will call the first map supplied with the first revision Map 1 and the new map now supplied with revision 3, Map 2.
In the original submission it was impossible to know the physical layout of plants in the experiment from the information in method and materials so I asked for a map. Figure S1 (Map 1) was supplied by the authors with Revision 1 and the authors called it ‘a map of the spatial arrangements of the treatments’. It showed 3 replicates, each with an E- and an E+ main plot. E- and E+ as reviewer 1 pointed out were not randomised but I was prepared to overlook that even though I would expect a high impact journal to reject a MS for this kind of problem. As reviewer 1 rightly points out, any soil property gradient across the field where the experiment was located would differentially affect E+ and E- in the same way in all three replicates so experimental error from the soil property gradient and treatment effects will be inseparably confounded in this scenario. It is not reasonable to say as the authors did that soil fertility gradients were believed to be controlled by cultivation; firstly because cultivation mixes soil vertically more than horizontally within quite a small area and not over a distance across a field, and secondly because in the trait for which I did a trial ANOVA (previously suppplied), the replicate effect (which the authors did not extract) had a very large sum of squares.
Map 1 was consistent with the statement by the authors that they had used a split-split plot design with Endophyte as the main plot effect and CaNO3 as the split plot effect. At the main plot or whole plot level there are 6 plots (E+/E- x 3 replicates) (though unrandomised). For CaNO3 there are 3 plots within each mainplot (0, 25, 50) and those are randomly allocated – their spatial position is different in each replicate. In this way there are 6 main/Split plot combinations with 3 replicates = 18 obvious plots in the experiment. Each of those 18 obvious plots has 12 plants.  We are not told exactly how the Time effects (0, 14, 28 days) were allocated to plants but that is perhaps a minor point.
It is believable that researchers inexperienced in statistics might first analyse Map 1 as a three-factor factorial design. The non randomisation of E+ and E- suggests it was not conceived as a split plot design but the three factors are obviously and clearly all present so a factorial design could seem logical. But if it was only a ‘diagram’ that did not accurately show the plot positions, why give a diagram when a map was asked for and why label it a ‘map of the spatial arrangement of the treatments’?

If we analyse Map 2 which I have become aware of only while reviewing revision 3, we see that the columns labelled ‘Whole plot’ (I, II, III) are actually replicates and that within these we see the main plot factor is actually CaNo3 (3 randomised main plots per replicate) and the split plot factor is E+/E- which is allocated differently within each CaNO3 plot. Each E+ or E- split plot is a strip of 6 plants that provides for 2 plants for each of three times (0, 14, 28 d) in each of three replicates. However there are 4 strips, not 2 strips in each split plot so the authors have not stated the reason for the extra plants. Was it to have spare plants to ensure healthy plants were always used for measurement, or was some other research done with the other plants for publication elsewhere? If other research it is good to be transparent and tell the readers about it.

If Map 2 is the true map of the experiment then in my opinion all the ANOVA tables have to be recalculated with CaNo3 as the main plot factor and E+/E- as the split plot factor. All the F values and P values will change! (This applies to Table 1 and Table 4 in the MS.)

It is not so easy for this reviewer to believe that if the researchers carefully laid out the experiment in the field as in Map 2 for a split-split-plot design, they would then present their MS with a simple factorial analysis, or that they would make a mistake now by wrongly identifying which was the main plot factor and which was the split plot factor in their Map2. That said, this reviewer is human, so maybe I am making the mistake! I wonder if the United States and New Zealand authors or their statistics colleagues have had any input to the statistical decisions in this data analysis and/or if they have inspected and approved this part of the work?
Validity of the findings: When the map of plot layout shows CaNO3 as the main plot factor and E+/E- as the split plot factor, it is necessary that the ANOVAs in the Tables follow the same.

In the trait I prepared an ANOVA for the replicate effect seems more important than the authors recognised and maybe should be extracted in the main plot stratum of the ANOVA.
Additional comments: Other points:
As reviewer 1 points out, a P value by definition can not be greater than 1. Even so the authors still keep two P values of 1.019 and 1.027 in their Table 4. It would be good to fix this problem.

I remain concerned that the total soluble sugar levels of shoots at 14 – 18 mg/g are lower than I would expect for grass shoots, unless there is a major proportion of dead material in the sample. I looked for another reference on soluble sugars in Festuca pratensis and I found a paper of Wang et al. (also from Lanzhou University) in Functional Plant Biology 2017 where the stated levels are 6 – 12 µg/g (less than the free proline content). At least I am certain those other authors would not be correct, but it does not help provide a benchmark to know if these values are reasonable. Since the authors already gave assurance on this point I will not pursue it any further.

I can’t help noting that in the first submission of this MS the authors were sure the Ca component of the CaNO3 treatment was the cause of the treatment effects. Now they allow that it could be a nitrogen effect and they carefully avoid any discussion about whether there is evidence in favour of one or the other. So it is a different paper now, with endophyte effect on root activities in Fig.2 now the most interesting part.