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: CHARACTERISTIC CHANGES IN MALT, WORT, AND BEER PRODUCED FROM DIFFERENT NIGERIAN RICE VARIETIES AS INFLUENCED BY VARYING MALTING CONDITIONS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The English of the current manuscript is vey hard to understand sometimes.
the author has not cited most relevant references and so the manuscript is lack of novelty. The knowledge that the author want to display is not new for the brewing industry.
There was also no hypotheses within the current manuscript.

Line 76-77: why suddenly jumped from steeping to mashing? how about germination?
Line 78: how about other sugar?
Line 82-84, 85-87, what did the author mean here?
Line 83-84, why the author say that protein degradation is responsible for the cell wall degradation?
Line 88-89, why? in what aspects?
Line 94-95, any references?
Line 97-98, this is what you have done, not the research goal of the current study.
Experimental design: The experiment was not well-designed. For example, the author has not mentioned  why rice is a promising substitute for barley, as rice is the staple foods for human? in Line 80, this is not the reason that why the content of starch in rice is higher than that of barley. There are must be other reasons.
The mashing procedure was really really complicated and made no sense. This is definitely not the proper mashing procedure for the brewing industry.

Line 117, what alpha-amylase and beta-amylase? from which company? how about the enzyme activity?
Line 118, again, what protease?
Line 145, what is enzyme activity?
Line 153, why monitor starch?
Line157-158, the English is really bad and the author has no cited any relevant references.
Line 329-330, any references?
Line 334-335, why did not measure the starch content?
Validity of the findings: the findings of the current study was not new at all and there are already many many studies concerning the subject.
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: CHARACTERISTIC CHANGES IN MALT, WORT, AND BEER PRODUCED FROM DIFFERENT NIGERIAN RICE VARIETIES AS INFLUENCED BY VARYING MALTING CONDITIONS
Review round: 1
Reviewer: 3

Output:
Basic reporting: The authors conducted a extensive research about rice malt modifications in different malting conditions and its influence on wort and beer. The data  set is extensive and intesting to scientific audience dealing with brewing and malting. I agree that the article meets the standards of the journal.
Experimental design: The experiment is well designed and extensive. Perhaps the authors could reduce the number of pages by combining  tables, and reducing the M&M section.
Validity of the findings: The finding are valid and all explenations have been provided in the R&D part.
Additional comments: The paper is too long and should be significantly reduced prior acceptance (examples of how to reduce it are stated in the text before).


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: CHARACTERISTIC CHANGES IN MALT, WORT, AND BEER PRODUCED FROM DIFFERENT NIGERIAN RICE VARIETIES AS INFLUENCED BY VARYING MALTING CONDITIONS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The topic of the paper is interesting and it fits well within the framework of the journal PeerJ and conclusions that are of interest to the scientific community. In terms of the conception of the paper and the results presented, the paper can be considered good. The revisions that the authors have made to the manuscript are very effective.
Experimental design: The revisions that the authors have made to the manuscript are very effective. The research question is well defined, relevant and meaningful. The methods are described in sufficient detail with notes on replication.
Validity of the findings: The authors made many corrections that improved the quality of the paper. The results presented in the manuscript are interesting. The conclusions are well stated, linked to the original research question, and limited to supporting findings. The conclusions presented in the paper correlate with the results found.
Additional comments: The authors have incorporated all reviewer comments into the revised version of the manuscript.  I am satisfied with their responses and corrections. Therefore, I recommend the publication of the manuscript as it is.


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: CHARACTERISTIC CHANGES IN MALT, WORT, AND BEER PRODUCED FROM DIFFERENT NIGERIAN RICE VARIETIES AS INFLUENCED BY VARYING MALTING CONDITIONS
Review round: 2
Reviewer: 2

Output:
Basic reporting: The paper is extensive and I feel that the authors should reduce the number of pages or divide it to two papers.However, wheather this should be reduced or not, I would leave the decision to the Editor.  Also, please add rice into the title, since the title does not give a clear insight into the whole subject matter.
Experimental design: The experimental design OK.
Validity of the findings: OK.
Additional comments: You did an extensive research and obtained results that could be useful for future research and to scholars as guidlines for rice malting.


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

Input:
Title: A FRESH LOOK AT AN OLD CONCEPT: HOME-RANGE ESTIMATION IN A TIDY WORLD
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript describes new computational tools that enable researchers to apply a range of space-use estimators to animal tracking data. The ultimate goal of the work appears to be to provide researchers with a single platform for estimating these quantities. The ability to apply a range of different home range estimators in R, using a clean and straightforward workflow is of benefit to the broader community. Unifying multiple analytical frameworks is not an easy task, so I applaud the authors in their effort. The authors use clear and unambiguous language, have referenced the appropriate literature, have shared the raw data in an accessible way, and the work is self-contained.
Experimental design: The authors have designed a workflow that allows researchers to straightforwardly estimate quantities related to animal space use from animal tracking data. Experimental design, in its traditional meaning, is not relevant to the present study. Nonetheless, I do have reservations about the way several of the tools function.

First, the methods for estimating AKDE home range areas appear to require users chose an autocorrelation model as an option of the function (P. 11). Identifying the correct autocorrelation model for a specific dataset is one of the most important steps of AKDE home range estimation, and is a step that can be incredibly challenging in practice (note: there are currently dozens of unique models that can be selected from). This is especially true if users do not have access to any diagnostic tools to check that models’ assumptions are not being violated and that the fit is reasonable for the data. Given that the goal of the present work is to provide users with a streamlined workflow for estimating home range areas, I would recommend that the authors update the workflow of their package to be based on ctmm.select() by default instead of ctmm.fit(). The former performs automatic AICc based model selection, removing the subjectivity of the model selection step. I am not opposed to providing users the option of comparing AKDE estimates from different autocorrelation models as an additional option, but I would not make manual model selection the default.

Second, the authors briefly describe how users can estimate home range overlap, but no information is provided on what overlap estimator is implemented. For instance, the methods available to estimate overlap for probabilistic home range estimates are different from those that can be applied to geometric estimates, which can result in varying degrees of accuracy; Fieberg & Kochanny 2005). While the authors recognize this, it is unclear to me how the hr_overlap() function in amt accounts for this. The text on page 8 of the manuscript appears to say that it is a measure of intersection for geometric estimators, but that it can be based on one of “several other home-range overlap indices” for probabilistic home range estimates. Reading the helpfile on the hr_overlap() function from the version of the amt package currently available on CRAN, I can see no option for selecting which overlap measure is applied. Some more information on how this function works is needed if users are to know what the overlap numbers represent.

Third, all of the AKDE based home range estimates include confidence intervals on the area estimates. Confidence intervals on point estimates are particularly useful when applying an inferential framework, such as the male-female comparison presented in Fig. 3. Nonetheless, these are not depicted in the figure, and appear not to have been propagated into the comparison of male vs. female home range sizes. As a minimum, I would recommend that the authors include the CIs in Fig. 3. However, I would also encourage the authors to employ meta-analysis methods for these estimates using either the metafor R package, or the meta() function in the ctmm package.
Validity of the findings: All underlying data has been provided, the code is publicly available and well commented, and the findings based on sound science.
Additional comments: On P. 19, the authors mention briefly how the currently framework makes it easier to compare home range estimates via sensitivity analyses. This can be an incredibly challenging step in practice for many ecologists. While I recognize that these types of sensitivity analyses were beyond the scope of the present work, some guidance on how to approach this would likely benefit readers. For instance, what specific tools might research apply (e.g., cross-validation, bootstrapping, etc.), are there any functions in the amt package for doing this?


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

Input:
Title: A FRESH LOOK AT AN OLD CONCEPT: HOME-RANGE ESTIMATION IN A TIDY WORLD
Review round: 1
Reviewer: 2

Output:
Basic reporting: The authors demonstrate a convenient software package to perform home-range analysis across multiple individuals and multiple estimators, which the authors also promote, as a kind of sensitivity analysis. The topic is timely and the software offers clean solutions.
Experimental design: * With Scott LaPoint's fisher data, there is a complication that worries me in that the location errors are pretty big (10-100 meters) compared to how far the fishers travel during the sampling period (~10 minutes). There is error information in that data (e-obs inaccuracy estimate in meters) and the calibration was calculated in https://www.biorxiv.org/content/10.1101/2020.06.12.130195v1 S4.3.1 to be roughly 1.67. So when working this these data in ctmm, I would normally do something like

	uere(FISHER) <- 1.67
	GUESS <- ctmm.guess(FISHER,CTMM=ctmm(error=TRUE))
	FIT <- ctmm.select(FISHER,GUESS)

to fit with calibrated error in the model, which will return a less tortuous model with smaller home range. The occurrence distribution will also become less tortuous and less constrained to the location point estimates. I would check to make sure that this doesn't have a large impact on the results, though I recognize that this data complication is a big tangent to the purpose of this paper.

There is a second complication with the fisher data, specific to movement model fitting, in that some of the individuals have accelerometer triggered sampling, so there is only sampling when the movement is more tortuous (which biases the fitted movement model). But I don't have any solutions for that.
Validity of the findings: > For convenience, and as is common in the literature, we will refer to the suite of methods used in this context as home-range estimators, even though these methods may have different statistical estimation targets (Horne et al. 2020).

I know this is the tradition, but it is also the tradition that there's no difference where there is, and when one class of estimators does a much better job at estimating Burt's notion of the "home range", don't you think it's confusing to researchers if both classes of estimators are referred to as "home-range estimators". Is there a better term that could be given to this, like "home-range and travel-log distributions" or something?

* I think there is a danger in using the occurrence distributions like this in Figure 5, as the confidence areas are quantifying a combination of animal movement and sampling quality. If the location fixes start to fail or become more erroneous, then the OD's area will change, even if the movement behavior is not changing.

* The IID OD in Figure 5 (I'm assuming no error model) is just the empirical distribution and so its area calculated here is probably just an artifact of the pixel resolution. Because the trends are all so similar, that then makes me wonder if the dominant effect here isn't the number of successful fixes. Can you check that?

> For example, a range distribution will not be appropriate for studying temporarily varying space-use patterns.

If p(x,y|t) is a function of time (non-stationary), then it can make sense to estimate p(x,y|t) as approximately constant over different windows of time as an alternative to modeling the time dependence explicitly, like performing a moving average versus a fitting a trend model. In either case, p(x,y|t) is something to be estimated and should not to be conflated with a time-average of p(x,y|t,data), which would be an occurrence distribution.

I think the danger of estimating range distributions over narrow windows of time is that the effective sample size can be too small to reliably support that: https://besjournals.onlinelibrary.wiley.com/doi/10.1111/2041-210X.13270

* If it was up to me, I would change the x-axis of Figure 4 to be the proportion of forest within the occurrence distribution, even though the y-axis uses the range distribution. In my mind, such a figure would encapsulate the most appropriate usage of both distributions, as using the range distribution on the x-axis can give false positives, while using the occurrence distribution on the y-axis would give negative bias.

Then, having an example with the occurrence distribution, Figure 5 could be dropped. But if keeping Figure 5, I would switch from occurrence to range distributions, while making sure that the time window can support that. For fishers, that might limit you to bi-weekly or weekly home-range estimates. I would also include the CIs where possible.
Additional comments: * Is there any chance the confidence intervals could be included on the AKDE point esitmates in Figures 3-4? I know the other estimators don't produce confidence intervals on their point estimates, but ideally they would/should.

* Can you explain the t-based confidence intervals in Figure 3? The mean area parameter estimate sampling distributions are given t-distributions? Where do the 3 parameters of that distribution come from?


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

Input:
Title: A FRESH LOOK AT AN OLD CONCEPT: HOME-RANGE ESTIMATION IN A TIDY WORLD
Review round: 1
Reviewer: 3

Output:
Basic reporting: Some minor problems with references.

Background could benefit from reorganizing.

Raw data is shared.

See below for more comments.
Experimental design: The gap is clearly defined in this manuscript: a common and reproducible workflow for estimating animal home ranges.

The code examples for a single individual and for multiple individuals show how this can be performed using amt.

See below for more comments.
Validity of the findings: no comment
Additional comments: In general, this paper provides a thorough set of examples for estimating animal home ranges with the amt package. The figures included (Fig 3, 4, 5) compare the results from different home range estimation methods available in amt. I appreciate the authors’ recommendation of sensitivity analyses to determine the influence of home range estimation method on results. However, I believe the authors’ have not sufficiently accompanied the amt’s functions with introductory information around the dichotomies of home range estimation methods mentioned (eg. “old” and “new”, geometric and probabilistic, and range distribution and occurrence distribution). In addition, the meaning and repeated emphasis on the authors’ implementation being “tidy” and “coherent” is lost for the reader, especially to one without a knowledge of the various packages and communities of the R programming language. I recommend that authors bolster the introductory information with clear comparisons of different home range estimation methods, potentially including those not currently available in the amt package, to assist the reader in selection and evaluation of home range estimators. In addition, a simplified description of the proposed home range estimation framework, using amt and other package dependencies, will help communicate the methods to a broader audience. Please see specific comments below.

General comments

I think this paper has the opportunity to clearly describe and categorize home range estimators. Since the goal of this paper is to present users with a standard workflow and to help users evaluate the influence of home range estimator selection, it should be clear how the available methods are fundamentally different. Here are some of the descriptors used throughout the paper:

L29: “statistical and modelling” - Are these different? Which of the different approaches presented fit into these categories?

L33-34: “range distribution” and “occurrence distribution”

L39: “old estimators”. These are introduced after the
mention of advances/new estimators. Consider reorganizing (see below).

L72: This is another dichotomy: geometry and probabilistic, but it wasn’t mentioned in the introduction.

Though this information is not clearly presented at the moment, I believe it is mostly contained within the paper and simply needs reorganizing to better present it. The introduction could more linearly track the historical progression from early uses (eg Darwin L25) to “old” estimators (L39) and advances “addressing issues related to autocorrelation” (L37), finally describing the niche amt is filling (L39:L52, L62:L70). This could also provide an opportunity to expand on the autocorrelated nature of GPS data and how that is being addressed by new methods.

In the current version, the “conceptual framework” section begins with a contrast between two types of home range estimators, geometric and probabilistic estimators, before moving quickly into the structure of the amt package’s functions for estimating home ranges. This structure is arguably no longer a “conceptual framework” and rather an introduction to the functions and structure of amt. Perhaps this section could be reorganized to include a comparison of estimation methods using the descriptors mentioned throughout the paper, before moving onto a description of amt.

Following this, I believe the reader would greatly benefit from a more detailed, sequential description of the home range estimation methods available in amt. What methods are available in amt and what are their parameters/arguments/methods? How are these methods characterized using the aforementioned descriptors? What are the key references for each method? Each function could have its own distinct paragraph followed by global methods for all types.

For example, in the current version the description of the local convex hull method and the expectation to provide the square root of the number of observations is mixed in with a description of the map function (L255:L257).

The following comments pertain specifically to the precision of the sections regarding amt’s functions, package dependencies and the language used to describe outputs and variables. I believe it is critical when writing about programming to use precise and simple language to avoid confusing the reader. In addition, all examples should be presented at a minimum reproducible level, for example there is no need to discuss approaches that are tangential, or include tools that are not necessary.

L105-107: Great! This is a really clear example of how amt makes it easy to work with different home range estimators.

L108: Replace “a tibble is very similar to a data.frame” with “a tibble is an extension of a data.frame” and please describe specifically how it is different. “improved properties” does not explain to a user why it is being employed over the base R data.frame. According to this (https://r4ds.had.co.nz/tibbles.html#tibbles-vs.-data.frame), the differences between tibbles and data.frames have only to do with subsetting, printing, and some default options when creating a tibble or data.frame. If I am not missing something, please remove “A tibble makes it is easy to work with list-columns” because this is not something that distinguishes tibbles from data.frames. To clarify: I hope that the authors’ will emphasize why they are using, for example, list columns rather than emphasizing a specific package’s function which is not different than base R.

L110, L156: The function hr_isopleth does not return a tibble. It returns an object which is of class sf and data.frame. A reader might benefit from a few clarifications: what is an sf object/what is the sf package? What is the meaning of the column sfc_POLYGON?

L119-120: The Supplement 1 points to a Zenodo repository (https://zenodo.org/record/3991482). In the repository the “all-code.R” file does not contain any preparation steps for the dataset “fisher_preprocessed.rds” that are not presented in the manuscript.

L122-124: Is it necessary to load the tidyverse package? I believe that it is clearer for the reader if you only use specifically the packages that you need, as opposed to metapackages such as the tidyverse. In this section “One individual or sampling instance”, you do not present any figures so there is no need to mention ggplot2. In addition, there is no manipulation of dates so there is no need to mention lubridate. On the other hand, where does the “filter” function come from? Where does the “CRS” function come from?

L131: I would try and use language consistent with R help manuals here. Perhaps “Results from any estimator in amt are stored in a named list with three elements: crs, data, levels.” Then describe each in the subsequent sentences, dropping the semicolon.

L190: Instead of framing this in terms of what amt lacks, it would be clearer for the reader if you simply stated that to estimate home ranges of multiple animals you recommend using list columns. On this note though, list columns are not unique to tibbles. They are simply a column type that can be used with data.frames, data.tables, etc. This could be simplified by specifically stating why list columns are used and how, when combined with the map function, they can be used to estimate home ranges for multiple animals.

L377-384 (also L1, L19): What is specifically “tidy” about the proposed workflow? I understand that there are a subset of R packages released under the “tidyverse”. Does “tidy” therefore mean that the workflow is compatible or perhaps dependent on these tidyverse packages? It is not clear to me why the authors use this word often, other than it being a trending term in a subset of the R community that does not seem to carry an obvious meaning in this case.

L377-384: Similarly, I’m not sure anyone but a reader or user can decide if this is a “coherent” workflow. Perhaps a different word choice? I believe this is an impressive “standardized” workflow, that allows users to easily estimate home ranges by different methods.

Figure 1: This figure and its caption uses confusing language: hr_locoh has “type” and “n” - these are parameters for the function. In contract, hr has “levels”, “crs” and “data” - these are attributes of the resulting object. In addition, why show the empty boxes when there are no specific methods or attributes?

Minor comments

L13: Please clarify or consider rewording “computational point of view”.

L32-33: Please replace “whether they estimate one of two estimation targets:” with “whether they estimate either”.

L37: Could you expand on the autocorrelated nature of GPS data and, briefly, how these new methods address this issue?

L38: Please remove “modern day”.

L62: Please use more confident language: “we propose a general framework that can accommodate most home-range estimators”.

L69: Please review the citation style for the reference “Wickham and others 2014”.

L69: Why is the R Core Team cited in reference to the amt package? Maybe it could be clarified with something like this: “using the package amt (Signer 2019) for the R programming language (R Core Team 2020) and a previously published…”

L70: Please include species name for “fishers”

L97: Please replace “and/or” with “and”.

L98: Please remove “for example”.

L99: This paper describes amt. Please remove “here and in amt” and simply refer to the name of the property in amt.

L100: Please remove “should also”.

L117: Consider citing a reference for Movebank. According to their website (https://www.movebank.org/cms/movebank-content/about-movebank#citing_movebank) you may cite either or both listed in the references below.

L126: What is the meaning of the “init:epsg:5070” string?

L128: What is the meaning of the “default reference bandwidth”?

L130: Please remove “note just the ones illustrated here”.

L196: Please change “to demonstrate list columns” to “to demonstrate estimating home ranges for multiple animals” or something of the sort.

L203: Please use “absolute home range size” as in Fig 4 caption.

L203, L369: These two sentences repeat the same information. This is an example of where information presenting differences in home range estimation methods is not clearly organized for the reader.

L227: Please consider replacing vague and assuming “in the regular way” with a description of subsetting with square brackets.

L242: Please explicitly state which package the mutate function comes from and possibly what it does.

L244, L260: Why bother mention lapply if you don’t use it? This could be confusing for a reader.

L375: Please add “the” - “how choice of estimator”.

L377-380: Please consider simplifying this sentence, its meaning is unclear.

L377-384: I think this paragraph would be better off restating how the amt package and the proposed sensitivity analyses allow a user to use and compare different home range estimator methods using a common tool. Does the amt package provide or intend to provide all “new methods” that are being developed to maintain this common interface for users? That could be mentioned here if so. I would also argue that with papers such as Tucker et al. 2018, wildlife biology is definitely already in the era of big data and your proposed home range estimator functions are a welcome tool for engaging with it.

L397: Please correct this citation from “Beest, Floris M van” to “van Beest, Floris M”.

Figure 3, Figure 4, Figure 5: Colors selected are not colorblind safe or greyscale friendly. Please consider the viridis color palette or other colorblind safe palettes.

Figure 5: Please correct “ggplot” to “ggplot2”. The function geom_smooth uses a smoothing method, which one is selected for this plot? Please mention it in the caption. Please replace “year day” with “Day of Year” or “Julian Day”. Consider changing the break points in the M5 panel because 82.5 is an impossible value for integer type Day of Year. Consider mentioning the different x-axis scales, presenting from 4 days to >30 days.

Comments on Supplemental material

The following comments pertain to the “all-code.R” file included in the Zenodo archive (https://zenodo.org/record/3991482/files/all-code.R).

Please remove knitr chunk labels (presumably returned by knitr::purl) and replace with informative comments and perhaps sections matching the paper’s section names. As described above, hr_area returns a tibble, but hr_isopleth does not.

In addition, when I ran the all-code.R file, I encountered a number of messages, warnings:

 • messages: “ff is invalid” many times and “Minimum sampling interval of X minutes in unknown” many times
 • warnings: >50 warnings mentioning the CRS object and 25 warnings mentioning the geom_smooth function

References

Kranstauber B, Cameron A, Weinzierl R, Fountain T, Tilak S, Wikelski M, Kays R (2011) The Movebank data model for animal tracking. Environmental Modelling & Software 26(6): 834–835. https://doi.org/10.1016/j.envsoft.2010.12.005

tibbles vs data.frames in R for Data Science. https://r4ds.had.co.nz/tibbles.html#tibbles-vs.-data.frame

Tucker et al. 2018 https://science.sciencemag.org/content/359/6374/466.abstract

Wikelski M, Davidson SC, Kays R [year]. Movebank: archive, analysis and sharing of animal movement data. Hosted by the Max Planck Institute of Animal Behavior. www.movebank.org, accessed on [date].


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

Input:
Title: A FRESH LOOK AT AN OLD CONCEPT: HOME-RANGE ESTIMATION IN A TIDY WORLD
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: After carefully reading the revised manuscript, I have found that the authors have adequately addressed all of my initial concerns in their revisions. I only have one minor comment that I leave up to the authors' discretion. In Figure 3, panels A and B contain similar information, but A is on a linear scale, and B on a log. I would recommend putting these on the same scale as this would allow for these to be more readily compared.


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

Input:
Title: A FRESH LOOK AT AN OLD CONCEPT: HOME-RANGE ESTIMATION IN A TIDY WORLD
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: I appreciate all of the work that the authors have put into the revisions, even though some of my recommendations turned out to be unnecessary worrying. I still think "For example, a range distribution will not be appropriate for studying temporarily varying space-use patterns." is too strong of a statement, and now seems in conflict with Fig. 5 as I understand it, but the authors are allowed to have their own opinions. My only additional comment is that the last two figures might be easier to decipher with some color.


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

Input:
Title: A FRESH LOOK AT AN OLD CONCEPT: HOME-RANGE ESTIMATION IN A TIDY WORLD
Review round: 2
Reviewer: 3

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: This manuscript provides a clear set of examples for using the amt package
in calculating and comparing home range estimates. The authors have
made significant improvements through the previous round of revisions. My
comments here are few and minor.

Throughout the manuscript, "home-range" and "home range" are both used. Please consider selecting one and using it throughout.

Lines 114-124: This paragraph, describing the output formats of the hr_area and hr_isopleth functions, serves an important role for describing the outputs of these functions.
Two object types are presented: tibbles and sf objects.

"the function hr area() in the package amt, will always return a tibble"
"Similarly, the function hr isopleth() in amt will always return a data.frame with a list column where the isopleths are saved as simple features as implemented in the sf package"

I am not sure what is similar in these two: hr_area returns a tibble, and hr_isopleth returns a "data.frame with a list column...". Please remove "similarly". In addition, I am unsure if "will always" is necessary in these cases, unless the authors of the `amt` package are planning on introducing backwards-incompatible changes.

I appreciate the clarification presented here on why tibbles are used instead of
data.frames. I think, however, the description of sf objects could be improved (Lines 120-124, 178-180).
While sf objects are in fact a data.frame, I think "return a data.frame with a list column where the isopleths are saved as simple features as implemented in the sf package" is overly wordy and could be rephrased simply as "return an sf object". This is immediately
clear to any user that is familiar with sf objects. Then the following sentence
could be used to clarify further for those unfamiliar. For example, taken from the sf package's README: an sf object "represents simple features as records in a data.frame or tibble with a geometry list-column".

R: class(hr_isopleths(hr_mcp(deer)))
[1] "sf"         "data.frame"

Note as well, much of the information presented in this paragraph is repeated
throughout the rest of the manuscript. While I appreciate this repetition and
reinforcement helps a user understand how to use the `amt` package for home-range
analysis, some more justification type repetition could be pared down ("makes it easy to perform further GIS-type work").

hr_area: Lines 114-120 & Lines 164-167
hr_isopleth: Lines 123-124 & Lines 178-180

Lines 142-145: Comments concerning the definition of coordinate reference systems.
I appreciate the clarification of setting the CRS using EPSG codes. However,
given recent updates to GDAL and PROJ, R spatial has changed their expectations
with regard to setting coordinate reference systems. This is the relevant
page describing this: https://www.r-spatial.org/r/2020/03/17/wkt.html.
This presents itself in two places relevant to this manuscript.

Firstly, running the lines:

R: library(amt)
R: data(deer)
R: hr_isopleths(hr_mcp(deer))

Returns the following warning:

Warning message:
In CPL_crs_from_input(x) :
  GDAL Message 1: +init=epsg:XXXX syntax is deprecated. It might return a CRS with a non-EPSG compliant axis order.
Calls: hr_isopleths ... st_crs<- -> st_crs<-.sfc -> make_crs -> CPL_crs_from_input

Highlighting internal code in `amt` that should be updated to use the new syntax.
In addition, in the manuscript (Lines 142-146) the CRS is set using the following:

CRS("+init=epsg:5070"))

My interpretation of the above link is that the recommended way to set a CRS
in R for use with the `sf` package is

st_crs(5070)

Finally, I think that writing a manuscript describing code, analytical details
and trying to reach an audience of mixed programming skill can be challenging.
There are innumerable ways of coding any one task, and describing each of these
approaches can be overwhelming. In the case of this manuscript, there are
a few instances where the authors present their method then
in parenthesis or as an aside, mention an alternative. I believe that
for a reader, especially a novice, presenting only your approach can
yield a more succinct and clear manuscript. This might help avoid reader
confusion (eg. map is used to iterate but in base R, lapply could work too).
This is simply a comment, not meant for any substantial changes. Something to
consider when parts of the manuscript feel bogged down in details or confusing.


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: DNA METABARCODING OF ZOOPLANKTON COMMUNITIES: SPECIES DIVERSITY AND SEASONAL VARIATION REVEALED BY 18S RRNA AND COI
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript would be improved by careful editing for English throughout.
Experimental design: The main purposes of the paper as stated at the end of the Introduction are to explore the composition of the Sanmenxia zooplankton, the seasonal dynamics and the environmental factors driving the seasonal changes. Based on this, the only people who will want to read this paper are those interested in the Sanmenxia Reservoir zooplankton, a very narrow audience! The paper could be re-framed to provide advice on how future zooplankton metabarcoding studies should be carried out. For example, the abstract mentions this study highlighted limitations and advantages of using two metabarcoding markers for zooplankton diversity. Currently this is not covered in the Discussion, but would be a useful addition to the paper. For example, was the 18S marker better at detecting protozoa, or did COI do a good job too? Would you recommend using both markers for future studies, given the two markers detected essentially the same patterns? If not, which marker would you choose? This could be based on the level of taxonomic detail each marker provided (but see comment on taxonomic accuracy below).
Alternatively, are there broader management implications for these results? The authors state several taxa indicative of eutrophication are present (L376-379), should efforts be made to reduce organic pollutants in the Sanmenxia Reservoir? Large sections of the Discussion currently repeat (or belongs in) the Results (e.g., L386-412), the Discussion could be better used to place the results in context and discuss the implications for future metabarcoding studies.
The taxonomic assignment of OTU sequences needs to be repeated with a more rigorous approach. Was any effort made to confirm the taxonomy was accurate? A ‘top-hit’ approach is prone to errors. Programs such as MEGAN are useful to ensure taxonomic assignments reflect uncertainty, particularly important for markers like 18S with good taxonomic breadth but poor taxonomic resolution. This is particularly relevant for the comparison with historical data based on morphology (L342-345).

The methods section does not provide enough detail to replicate the results. For example, how long was the plankton net dragged for? What volume of water was sampled for the laboratory analyses (chemical oxygen demand etc.)? What MiSeq kit was used, and with how many base pairs? How was the bioinformatic quality control done, e.g., what were the settings used? The first data processing step is converting the FASTQ files to FASTA, suggesting it was not based on the sequencing quality scores. Line 162 suggests replicate samples were collected, this should be detailed in the section on zooplankton collection.
Validity of the findings: No comment
Additional comments: The authors present a study on seasonal changes in zooplankton diversity in the Sanmenxia Reservoir based on metabarcoding data from 18S rRNA and mitochondrial COI. Winter and spring zooplankton communities were similar, but summer and autumn communities were distinct. These changes were related to water temperature and other environmental variables, with both markers showing similar trends. The number of samples collected across many parts of the reservoir suggest the results observed are robust.
Much of the paper assumes that differences in read abundance for each marker reflect the abundance of the zooplankton OTUs. This assumption may not be accurate (in contrast to the statement on reliably detecting relative abundance on L103) and needs to be acknowledged. The authors should also clarify whether they used the binary Jaccard distance (as opposed to ‘Jaccard’.
The reason some of the statistical tests were chosen is unclear. For example, a parametric test (ANOVA) is used for the zooplankton group richness, but a non-parametric test for overall zooplankton diversity (Kruskal-Wallis). Welch’s t-test is used to look at changes over the four seasons, but is designed for testing the difference between two samples (in this case seasons). A different test would be more appropriate.
The manuscript would be improved by careful editing for English throughout.
Minor comments:
L69-70: This sentence is not connected to the rest of the paragraph. Rather, it sounds like you need a new method to look at zooplankton diversity.
L114: Many studies have used more than one marker to look at zooplankton diversity (whether fresh or marine), including Berry et al. (2019, cited above) and the ‘Tree of Life’ approach developed by Stat et al. (2017).
L116: I suggest the main advantage of using more than one marker is the increase in taxonomic breadth, which is especially important for diverse groups like zooplankton. For example, the COI marker used in this study is designed for metazoans but not protozoa, whereas the 18S rRNA marker should detect most protozoan groups.
L128: No hypothesis is stated.
L153: Why were samples ‘filtered through’ 0.22 um filter paper? Was this to remove water?
L216-217: It would be useful to provide the range of sequencing depths separately for both markers.
L220: The singletons should be removed prior to creating rarefaction curves.
 L226 and 234: Only 714 of ~1600 OTUs were zooplankton, and only a small percentage were unclassified, what were the remaining >800 OTUs? Similar for the 10-15% of sequences that were not classified as 18S or COI.
L263 and throughout: The division of zooplankton groups used is odd, as Protozoa are not a clade, rotifers are a phylum and cladocera and copepod are classes. However, if you state this is the division you are using in the methods it can be accepted.
L295: If this is the main figure for this result, it should be in the main text.
L344: References should be provided for at least the 1985 study.
L376: Suggest changing Brachiosaurus to Brachionus.
L427-428: Relative abundance always sums to 100%, so if everything else goes down, rotifers would have to increase.
L452: This statement needs references.
L457: Do you mean OTU-level, given that is what is shown in Figure 8?
Figure 1: Change to bar charts.
Figure 3: Define A and B in legend.
Figure 5: These could be combined into two panels instead of 6. Does the x-axis shopw the mean relative abundance per sample? If so, the standard deviation or standard error should also be shown.
Figure 6: Are the ellipses 75% confidence intervals? This should be included in the legend.
Figure 8: I’m not sure panel A is informative. It is not referred to in the text.
Figure 9 is not referred to in the Results (but is in the Discussion). I suggest reducing the number of taxa shown to 10-15 at most, as it’s difficult to discern the differences between many shades of the same colour. Also, taxa could be colour-coded by zooplankton group, e.g. rotifer genera are all shades of grey, copepods are shades of blue etc.


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: DNA METABARCODING OF ZOOPLANKTON COMMUNITIES: SPECIES DIVERSITY AND SEASONAL VARIATION REVEALED BY 18S RRNA AND COI
Review round: 1
Reviewer: 2

Output:
Basic reporting: Article structure can be improved. For example, not all figures are explained in the Result Section. Labels in some figures are too small and not clear, and readers cannot understand results authors present. Raw data of sequence reads are available; however, each data is not fully explained. In the current version of the data deposited, it is very difficult to be used secondary in the future. I also recommend to use database in NCBI BioProject, in order to promote secondary uses of authors’ data. I’m not native speaker, and I cannot evaluate English in the manuscript.
Experimental design: There are significant flaws in the method section.

Methods are poorly explained, and there is insufficient information to be reproducible by another investigator. In particular, detailed information of bioinformatic analysis are not provided, and I couldn’t fully evaluate accuracy of results in the manuscript. Authors should explain details of quality filtering steps. Also, they should clarify how they annotated OTUs into taxonomic groups (species, genus or other taxonomic level). There are also no information if authors have DNA barcoding data for species in the study area.

Primers for COI gene are mainly constructed for Metazoa. In my impression, these primers couldn’t evaluate protozoa accurately. In addition, 18S is relatively conserved for species-level. Authors present data with combination of two markers (18S + COI). However, because of these biases, these markers should be analyzed separately. I also doubt that OTUs derived from a same species present in analyses of 18S + COI. Biases between makers are clearly visualized in Figure 1.

I found OTUs classified into marine taxa. I think taxonomic classification is not properly performed in the manuscript, or there are insufficient database for 18S or COI. Anyway, I couldn’t judge cause of mis-identification, because of lacks of information in Methods.

Authors removed a singleton read of OTUs. So diversity cannot be evaluated by Chao 1 index.
Validity of the findings: Although results are descriptive, data obtained in this study is valuable. The metabardcoding data can be compared in the future under different environmental conditions. However, as described above, accuracy of data couldn’t be fully evaluated because of insufficient explanations in the method section.
Additional comments: No additional comments


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

Input:
Title: DNA METABARCODING OF ZOOPLANKTON COMMUNITIES: SPECIES DIVERSITY AND SEASONAL VARIATION REVEALED BY 18S RRNA AND COI
Review round: 1
Reviewer: 3

Output:
Basic reporting: - English could be improved in some parts of the manuscript.

- General lack of references that would allow comparing the results to previous studies on freshwater zooplankton
Experimental design: - Sampling, laboratory workflow and bioinformatic processing should be described in more detail. Replication would not be possible with the information provided at the moment.
Validity of the findings: - Results should e compared to previous findings. It is not clear if the work is highly novel or if similar studied have been conducted on other reservoirs.
Additional comments: The authors conducted an interesting study on freshwater zooplankton. I am sure that the results merit publication, but several aspects of the manuscript suffer from a lack of references, and the methods used should be described in more detail to allow other researchers to reproduce results/adopt protocols. I therefore recommend major revisions.

Abstract:
18 - 21: You might want to split this sentence in two.

22: Please mention that COI is a mitochondrial gene

31: It is not clear what 'biological interaction' means in this context.
Please specify

Introduction:

93: There are several publications on bias introduced by morphological
identification of invertebrates, e.g. Haase et al. 2006. Please cite at least one or two.

103: The statement that metabarcoding can reliably detect abundances is debatable and not necessarily true for all environments and all taxonomic groups, and also depends on the genetic marker and library preparation method used. Please tone down this statement or clarify which taxonomic groups and ecosystems you mean.

113: Please cite papers which show the limitations of single marker approaches regarding the detection of certain taxonomic groups

119-125: You explain what you expect, but your expectations are probably based on previous observations or patterns known from other freshwater ecosystems. Please provide some references.

132: Please provide a reference for metabarcoding using the 18S v4 region, as readers without a background in molecular biologiy do not necessarily know what the v4 region is.

133: Please mention the full name of COI and that it is a mitochondrial gene

General remark: Is there any data on the taxonomic diversity of the Sanmenxia zooplankton or is your study the first to look into this? Is there any data on zooplankton communities from similar dams in the area? If so, please cite previous work. If not, please explicitly mention this.

Material and methods

141: When was the dam completed?

145: Which geographical features were used to decide on sampling locations?

150: Was the sampling standardised by sampling identical volumes? If not, did oyu correct for this?

153: Why did you filter the samples?

158: Did you collect additional water sampled for these measurements?

162: Why did you decide to pool different samples?

168: Please elaborate on the workflow and mention all library preparation steps or cite the protocol that you followed.

173: Did you use the PEAR default settings?

174: Trimmomatic: Which settings were used?

177: Which 'additional quality filtering' steps were conducted using USEARCH?

182/183: How did you proceed if an OTU could not be assigned to a species name? Did you assign the OTU on higher taxonomic level or did you exclude it from downstream analyses?

General remarks:
Did you  normalise your data by subsampling?
Did you remove samples with extremely low read numbers (if there were any)?
Did you process biological or technical replicates?
Did you run negative controls to check for contamination?
Did you  take into account that OTU clustering both 18S and COI with 97% similarity could lead to a bias,as these markers do not have the same evolutionary history and mutation rates?

Results

Please provide some basic information on the number of obtained reads and if any samples had to be excluded because of low read count or low quality.

If I understood correctly, you pooled OTUs obtained through 18S and COI metabarcoding. However, this means that some species were present with two or more OTUs (one in 18S, one in COI). Some species might have een amplified using the 18S primers, but not the COI primers or vice versa. Did you somehow account for that? If not, this could greatly bias your data. If you did account for that or used a different approach, please explain this in more detail to make it clear.

252: Please specify which group was the most abundant. Also keep in mind that this result could be a result of primer and/or amplification bias. Please discuss this carefully. Maybe you can cite literature showing that that copepods and/or rotifers are commonly the most abundant taxa in reservoirs?

320-330: Did you apply a Bonferroni correction to account for multiple testing?

329: Please elaborate on the different results you found for the two markers

Discussion

344: Please provide the references for the surveys conducted in 1985 and 2005

350-351: Do you mean 'Therefore, studies on zooplankton diversity can only be conducted by using the same method and sampling location, which allows obtaining comprehensive and reliable data and comparing results'?

356: Please provide a references for the 1986 study

359: Do you mean'the Protozoa were dominant'?

360: 'disadvantage' is probably not the right word here. Please rephrase.

361: Can you be sure that predation was the main factor driving protozoan abundance? It could also be a change in biomass ratio caused by other factors, which can lead to different sequencing results. Please discuss potential amplification and primer bias.

362: I assume you want to say that they are filter feeders? 'fed filters' is not correct.

367: Are there studies showing how incomplete the reference databases for Protozoa are?

371: Isn't a reservoir a relatively stable, non-dynamic environment?

377: Diversity in terms of OTU number?

381-382: This sentence should be rephrased

385: Other freshwater reservoirs, or what kind of location?

387-388. Did you expect this? If so, why? Also, please discuss why you found differences between 18S and COI datasets.

397-412: Many of these numbers could go to the Results. Also, please discuss them and compare your findings to previous studies. More references would help understand what your results actually mean.

415-417: Were any of these factors autocorrelated? Can you say that all were relevant, or could it be that only one or few factors were the actual drivers?

422: Please provide references for the statement that different groups have different temperature optima.

450-452: Many statements without references.

462-467: Please rephrase and split into two or three sentences.


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: DNA METABARCODING OF ZOOPLANKTON COMMUNITIES: SPECIES DIVERSITY AND SEASONAL VARIATION REVEALED BY 18S RRNA AND COI
Review round: 2
Reviewer: 1

Output:
Basic reporting: While the text has improved, the authors might want to ask a native English speaker to proofread the manuscript.
Experimental design: No comment
Validity of the findings: No comment
Additional comments: The authors addressed all concerns raised during the first round of review and rewrote large parts of the manuscript. The results are now presented more carefully, and limitations of the dataset are described and discussed. I feel that the manuscript and the reported datasets are interesting and should be published. However, the authors might want to ask a native English speaker to proofread their manuscript before publication, as some parts still read rather awkwardly.


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: NETWORK BASED MULTIFACTORIAL MODELLING OF MIRNA-TARGET INTERACTIONS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Based on ceRNA, this article comprehensively considered the influencing factors of the
interaction between miRNA and mRNA, and builds a model to discover perturbing genes
and miRNAs, which helps to understand the interaction between miRNA-mRNA.

In the preparation process of the article, there are the following suggestions.
1. In the results and discussion part, the content covered in this article should be in the past tense or past perfect tense, rather than the present perfect tense which the author uses more. The content described in the cited literature is the general present tense.
2. The use of the cited documents in the article is not accurate, please refer to the writing of other articles.
3. Part of the expression was difficult to understand.
    Line 243-244
Experimental design: The research ideas of the article are clear.
But the author can still consider drawing a flowchart. For bioinformatics analysis research reports, the flowchart can help readers quickly and accurately understand the research ideas of the article. At the same time, it can also be used as a supplement to Table 1, so that readers can effectively identify the difference between Sample/Sample+ and Real/Real+.
Validity of the findings: 1. For Figure 11S, it would be better to label the significant difference in gene expression levels between normal tissues and cancer tissues.
2.The article elaborated on the research results that he considered important, but failed to display the relevant data results completely and clearly.
   Line 274-285, Line 219-226.
Additional comments: Authors can refer to similar research articles to improve the writing skills of research papers.


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: NETWORK BASED MULTIFACTORIAL MODELLING OF MIRNA-TARGET INTERACTIONS
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Selcen Ari Yuka and Alper Yilmaz present a useful bioinformatics tool to run ceRNA analysis. The introduction provides a good, generalized background of the topic that quickly gives the reader an appreciation of the wide range of applications for this tool. The online R document is neat and easy to follow.
 I have a few comments as below:
1, since one miRNA might turn off thousands of coding genes, the ceRNA network is very huge. What is the cutoff to control the size of the constructed ceRNA network.
2, Your example analysis is just focusing on one TCGA case. I am wondering how to conduct multiple samples-based ceRNA network construction? Shall we just choosed concordant regulatory motifs? It will be better to discuss this.
3, The network visualization in R might not the best choice. So, I am wondering if the network file could be imported in Cytoscape for further analysis.
4, it is not very clear about what is the “perturbation efficiency”, and how this could be applied to the ceRNA network construction.


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: NETWORK BASED MULTIFACTORIAL MODELLING OF MIRNA-TARGET INTERACTIONS
Review round: 1
Reviewer: 3

Output:
Basic reporting: - This manuscript has many small grammer issues that should be fixed prior to publication.
Experimental design: - Runtime analysis: The authors comment on this but do not make any concrete statements, e.g. "perturbation efficiency calculation is still longer than anticipated" or "Our tool can successfully simulate perturbations in large network" which do not give an impression of the limitations of the tool.
	- In Equation (1),  I still don't understand why i is outside the sum since it is also defined as a running variable. I presume this is the relative expression of one gene i vs all other genes (say gene j). Correct me if I'm wrong, but this would just be the read count normalized by sequencing depth of the RNA-seq experiment, i.e. counts per million or CPM.
	- The methods section does not explain how many iterations of the method are run and if there is an automated stopping criterion for determining the steady state.
Validity of the findings: - In the discussion, the authors higlight that a dynamic simulation considering degradation rates etc. would be impractical but what's missing is a statement why the results of ceRNAnetsim are still of importance, e.g. the results do not capture these important factors but nevertheless help to understand the network dynamics in ceRNA regulation and pinpoint critical players that may be worth studying in a follow-up.
Additional comments: The competing endogenous RNA effect is an important concept for understanding systemic effects of microRNA regulation in which we consider miRNAs as resource various transcripts with binding sites compete over. ceRNA regulation can be studied on a global (genome-wide) or on a local scale using partial correlation or conditional mutual information based measures. However, we lack a tool allowing us to study the effect of expression changes on the ceRNA network. The authors close this gap and offer an R package that simulates miRNA target repression in a ceRNA networks over a large number of iterations. This allows studying indirect effects and to assess the potential of single genes or miRNAs to disturb this complex networks. The simulations that can be created here are likely of limited practical use as important mechanisms such as degradation are not considered. Nevertheless, I find the user-friendly, openly accessible and well documented R package provided here useful for the community for gaining a better understanding of the ceRNA theoretical aspects.

I've previously reviewed this manuscript and find many of my previous comments successfully addressed by the authors. Most importantly, the authors conducted additional analyses I requested that leverage information from more than one patient and use ceRNA network inference methodology to refine the network.


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: MOLECULAR BARCODE AND MORPHOLOGICAL ANALYSIS OF SMILAX PURHAMPUY RUIZ, ECUADOR
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript athough clear and unambiguous, could do with professional English proofreading throughout. The paper is technically correct text. The article conform to professional standards of courtesy and expression.

The article includes sufficient introduction and background to demonstrate how the work fits into the broader field of knowledge. Relevant prior literature have been appropriately referenced.

The structure of the article conform to an acceptable format of ‘standard sections’.

Figures are relevant to the content of the article, of sufficient resolution, and are appropriately described and labeled.

All appropriate raw data have been made available in accordance with our Data Sharing policy.
Experimental design: No research question is defined. However, it is stated how the research will fill an identified knowledge gap.

The submission clearly define the research problem, which is relevant and meaningful. The knowledge gap being investigated was identified, and statements should be made as to how the study contributes to filling that gap.

The investigation have been conducted rigorously.

Some parts of the methodology is incomplete- thus preventing repeatability/replication.
Validity of the findings: All underlying data have been provided; they are robust, statistically sound, & controlled.
The data on which the conclusions are based must be provided or made available in an acceptable discipline-specific repository.

Conclusions are well stated, linked to original research question & limited to supporting results.
Additional comments: TITLE
1.  Suggested modification: Molecular barcode and morphological analysis of Smilax purhampuy Ruiz,   Ecuador

ABSTRACT
2.       The rhizomes was also analyzed. For what?
3.       Results showed that … First report on micromorphological characteristics of the leaves, 2nd report on rhizome analysis, 3rd report on molecular barcode characterization – as per sequence of method reported above

IINTRODUCTION
4.	The Introduction cannot consist of just one long paragraph with a number of different topics – each topic = separate paragraph. This reviewer attempted to create a number of self-contained paragraph, each with their own focal area.
5.	The Introduction section provide NO information on Smilax purhampuy in Ecuador, which is the core species of this paper. Is it indigenous to Ecuador? What medicinal value does it have? Is it taxonomic position uncertain? etc etc.– remedy this by inserting information on S. purhampuy /or/ replace some of the general information of the genus with more specific information related to S. purhampuy.

MATERIALS AND METHODS
6.    Collection of plant material - Indicate season of collection; indicate is leaves were sun-exposed leaves or shade-exposed leaves?
7.    Micromorphological analysis - Incomplete: Refer to example provided in annotated manuscript

RESULTS
8.   Morphological analysis  - Incomplete: Refer to example provided in annotated manuscript
9.  "...while other genera (indicate names of genera) were in other clades (Fig. S1)."
10. "On the other hand, the ITS2 phylogenetic tree revealed that the two Smilax samples (name them) are in a different clade (bootstrap 100).."

DISCUSSION
Microscopic analysis
11. The cross section of S. purhampuy leaf indicated that the abaxial and adaxial epidermis are uniseriate, with the existence of an easily perceptible cuticle (how thick was the cuticle?) – not mentioned in results section.
12. :In S. purampuy, a dorsiventral ....Martins and Appezzato-da-Glória (2006) to S. polyantha and Martin et al. (2013) for (which species?).
13.  A peculiarity of the species studied was the fact of findingpresence of six vascular bundles of variable size at the level of the central nerve vein of the leaf (was this indicated as such in the results section?), which is in discrepancy with other Smilax species (indicate which species) that present three vascular bundles (Martin et al., 2013). Cannot have a 1 sentence paragraph
14. In other species (indicate which species) of the Smilax genus from Cuba and Guatemala, the ..."

Molecular barcode sequence
15.  The genotypic characterization of plant species is important since most plants, although they belong to the same genus and species, could show considerable variation (in what?).
16. "...011, CIBE-012) were more similar between them than other Smilax species (name them)..."
17. "Furthermore, the ITS2 revealed low percentage (be specific) ..."

REFERENCES
18. Some references not found in text - check all


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: MOLECULAR BARCODE AND MORPHOLOGICAL ANALYSIS OF SMILAX PURHAMPUY RUIZ, ECUADOR
Review round: 1
Reviewer: 2

Output:
Basic reporting: In the Introduction section, the authors focused on the medicinal value of the genus Smilax, but did not introduce the reason why DNA barcodes are needed for its identification. Perhaps it is difficult to separate it from its closely related species based on the morphological characteristics? In addition, what methods have been used for the identification of this genus? The author can elaborate on the above issues in more detail.
Experimental design: - The Smilax genus consists of 310 species, but the authors only analyzed one of them (Smilax purhampuy). What is special about this species?
- Very limited number of specimens were analyzed in this study.
- Where does the “powder drug of the rhizomes” (Line 112) come from?
- When constructing a phylogenetic tree, bootstrap replicates is generally set to 1000 to make the results more reliable.
Validity of the findings: - For the same species, it is strange that the lengths of the amplified sequences from the same marker vary greatly (Data_S1). Before the blast analysis and alignment, it is necessary to cut the sequences from the same marker with a uniform standard to avoid false positives. The sequence length of different barcodes should be mentioned in Results section. Correspondingly, the results of the phylogenetic trees also need to be updated.
- “Smilax samples” and “samples of Smilax” in Results section should clearly refer to Smilax purhampuy. Please revise thoroughly.
- After comparing all the barcodes in this study, the author should conclude which barcode is the most effective for identifying Smilax purhampuy. The discussion of candidate barcode selection for Smilax purhampuy or Smilax genus should be enriched.
Additional comments: -This work reported the micromorphological characteristics and the molecular barcodes of Smilax purhampuy collected in Ecuador. The most prominent problem lies in sample size and pre-processing of data, which need to be improved and modified.
-The English language should be further improved.


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

Input:
Title: MOLECULAR BARCODE AND MORPHOLOGICAL ANALYSIS OF SMILAX PURHAMPUY RUIZ, ECUADOR
Review round: 1
Reviewer: 3

Output:
Basic reporting: I believe that both the literature review on molecular methods, the description of results and the discussion of part concerning barcode analysis require improvement and extending.

1. I would advise to enrich the Introduction with an outline of the phylogenetic and taxonomic knowledge of the genus Smilax. Has any phylogenetic research been conducted on this genus? If so, it is also worth referring to them in the Discussion.

2. I think it is worth to mention that presently in GenBank database there are only few complete genomes of Smilax published, namely: four genomes representing three species. The methodology applied in the study would be also more transparent if the Authors state it clearly, that plastomes of other Smilax species which appear in The Results as blastn hits are not published as complete genomes but only chosen genes are sequenced and deposited in GB. It should be explained to the reader that the results of the blastn analysis showing similarity to a given species are based on the incomplete database.

3. The Authors should also explain why it was not possible to conduct a barcoding-gap analysis, or to use automated species identification such as Spider, SpeciesIdentifier or FASTACHAR, routinely used in studies concerning barcodes. What I mean is the fact that for these kind of analyses more than one representative for each species is needed. In this study S. purphampuy is represented by three samples collected for this study but the Genbank database unfortunately does not provide multiple representatives within species taken into comparison.

4. In my opinion, the results would definitely become more readable if one or several of the best-developed trees were presented in the main article body and not in a supplement. Instead, table 1 could be moved to supplementary information.

5. The best blastn hits described in the Results in lines 170-178 could be successfully presented in Table 2 by bolding the highest results. I think In the text The Authors should emphasize the most important results, e.g.: that the rpoC1 gene didn’t discriminate species within Smilax genus at all. I would also mention that the use of matK as a barcode didn’t discriminate S. purphampuy from S. fluminensis. The variability of rbcL sequence is also worth discussing. The identity of rbcL sequences in this study was always over 99%, however it is not known whether this was due to interspecies variation, or also largely to variation within species. The latter possibility would be indicated by the result of the ML analysis, which does not group Smilax species into species-specific clades.
Experimental design: No comment
Validity of the findings: 1. In lines: 31 and 81 the Authors stated that morphological and molecular barcode analysis S. purphampuy are lacking in Ecuador. Have they been made in any other country? The reader may doubt whether the research is innovative in a given area or for the species at all.

2. Please emphasize the fact that there is little molecular data on Smilax available, what will emphasize the novelty of this work and at the same time dispel the reader's doubts about the correctness of the methodology used in the study.

3. The valuable information which I lack in Conclusion is an indication of which barcode or barcodes best identify species of the genus Smilax.
Additional comments: The manuscript entitled “Molecular barcode and morphological analysis of the medicinal plant Smilax purphampuy Ruiz collected in Ecuador” presents the results of the study on the representative of Smilaxaceae family. The studied species gained the attention of the Authors of the research because of its usage in folk medicine and because of the lack of morphological and barcode analysis for S. purphampuy in the literature.
The subject of the research is in line with the current research trends, and the problem undertaken in the research is absolutely justified. The manuscript describes the research conducted in two ways. Some of them concern morphological research, the other part molecular analysis. I do not feel like an expert in the field of morphological research, although both the methods used seem to me to be reasonably selected and presented, and the description of the obtained results is understandable, clear, consistent and exhaustive. This part gives the impression of being well thought out and thoroughly conducted. Regarding the description of research conducted with molecular methods, I have a few comments. The laboratory methods themselves are well selected and described and do not raise my concerns.
I hope that questions and detailed comments on specific chapters listed in my review will help to improve the manuscript.

Below minor comments to the manuscript are listed:
Please, standarize the nomenclature of genes – relates to the use of italics in gene names.
Line 51: „… therefore, the taxonomy classification of Smilax was confirmed” –this conclusion goes a little bit too far and is a bit of a oversimplification.
Line 133 – should be sequenced instead of sequence
Line 134 – Replace sequences with the word sequenced
Line 139 – Please, replace sequences with sequence
Line 141 – I don’t understand, what number did the Authors wanted to mention in brackets?


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: MOLECULAR BARCODE AND MORPHOLOGICAL ANALYSIS OF SMILAX PURHAMPUY RUIZ, ECUADOR
Review round: 2
Reviewer: 1

Output:
Basic reporting: Minor grammar issues persists.
Minor punctuation issues persists
Minor typographical issues persists
Check maximum number of words allowed for Abstract, according to journal guidelines
Some references (Costion) (Chang), amongst other, not found in text - please check all citations in Bibliography against the text
Experimental design: Anatomical preparation not adjusted - how were the leaves handled prior to dissection? - see previous version example attached by reviewer.
Validity of the findings: Uniseriate epidermis not mentioned in results
Additional comments: Much improved manuscript.


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

Input:
Title: MOLECULAR BARCODE AND MORPHOLOGICAL ANALYSIS OF SMILAX PURHAMPUY RUIZ, ECUADOR
Review round: 2
Reviewer: 2

Output:
Basic reporting: The text has been thoroughly corrected according to all the reviewers’ comments. Now it is comprehensive and understandable. English language is clear and professional. In my opinion in the present form, the manuscript meets the standards of PeerJ Journal. However, there are some minor, almost cosmetic changes that must be made before publication of the manuscript. After making pending corrections in the text, I recommend the manuscript to be accepted for publication.
The most pending is spelling of Latin names in italics. I realize nowadays there is a trend to write generic names without italics, although in the Shenzhen Code from 2018 International Code of Nomenclature for algae, fungi, and plants (iapt-taxon.org) All the generic names and species names are still written in italics. Please, check the spelling in Your manuscript, also in figure captions and table headings.
The text needs to be checked for double spaces and for spaces before commas.
In line 234 there is double “the” – one has to be deleted.
Experimental design: The paper presents original study. The methods applied in the study are correct, described with sufficient details, the results were correctly interpreted and discussed.
Validity of the findings: The strong point of this study is considering both morphological and molecular studies in one paper, which unfortunately, nowadays isn't very common practice in the studies concentrated on molecular methods. The second advantage of the study is an object of a research which has been poorly investigated so far, being at the same time broadly used in traditional medicine.
Additional comments: I encourage The Authors to continue their studies on Smilax to complete the barcode sequences database and broaden possibilities of data analysis.


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

Input:
Title: SUPPLEMENTARY FEEDING OF CATTLE-YAK IN THE COLD SEASON ALTERS RUMEN MICROBES, VOLATILE FATTY ACIDS, AND EXPRESSION OF SGLT1 IN THE RUMEN EPITHELIUM
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript describes changes in the microbial composition of rumen samples in two groups of cattle-yak receiving different diets. The study provides an interesting point of view to evaluate the benefits of supplementing feed with concentrates, in an effort to increase animal welfare and resistance to harsh conditions. This work poses some interesting basis for further research, given the hints to the socio-cultural background that would potentially benefit from such a study.
The work seems well organized, although improvements could be done throughout the whole manuscript.
The introduction section presents well the context and the general purpose of the study, but a clear hypothesis is missing. I understand the work is an exploratory study, but predictions and expectations could be formulated in this section, discussed and confirmed or rejected in the conclusion part. The conclusions section itself is more a repetition of results than a real conclusive statement. I would recommend rewriting this section and mentioning the limitations of the study.
I could not find a reference for the deposited raw sequences.
I noticed that many of the references cited are quite old (before 2010). I know many papers, especially regarding ruminant physiology, are quite dated, but I think there is a lot of recent material that could be consulted, for example, on microbial activity and pathways. I would suggest trying to update at least some of the references.
Tables’ headings could be expanded, adding descriptions that would help the reader to better understand the contents without the need to look for the references in the text. This could also be done for figures. Please include acronyms explanations in figures/tables descriptions.
Please check though all the manuscript: all bacterial taxa (phyla, genera, species, etc) must be formatted in italics.
Furthermore, I would suggest a native speaker to check the English language throughout the manuscript.
Please see the general comments section for specific comments per each line.
Experimental design: Methods description could be improved, as some sections seem lacking important details and the experimental planning could be clarified.
It would be crucial to add details about the animal husbandry. Can the authors provide information about the animals housing conditions? How were the groups kept separated? How did they receive the supplemental feeding?
Details regarding the sampling procedure are also missing. How was the rumen fluid collected and how much? Which region of the organ was sampled? Which instruments were employed and how was cross-contamination avoided? Additionally, may I ask to which purpose were the animals slaughtered? Was it only for the experiment?
Please add specific details regarding the kits used for DNA extraction and how were concentration and purity checked. How was the library prepared? Please provide more details on the sequencing process.
The authors did not specify why they choose to investigate only SGLT1. It seems odd to test the expression of only one gene: weren’t other genes investigated? Was β-actin the only housekeeping gene tested?
It is not clear from the text how were bioinformatical analyses performed. I would suggest to clearly state what software was used for each of the steps.
I would strongly recommend including plots showing beta-diversity. Why was beta-diversity not discussed at all?
Validity of the findings: The total number of reads and consequently of OTUs is very low. Do the authors have an explanation for that?
The authors detected several KEGG pathways that could be discussed. Nevertheless, the discussion focuses only on carbohydrate metabolism, with unexpected findings, I would say. The G+S group was receiving a carbohydrate supplement, therefore it would be expected to find higher activity associated with carbohydrate metabolism in this group. How do the authors explain the higher carbohydrate metabolism-related activity in the group that did not receive the supplement? I would recommend discussing also the other outcomes of the PICRUSt analysis.
Could the higher expression of SGLT1 in the G group be due to any other reasons, apart the ones discussed? Could the higher activity of microbes in G+S group ferment most of the glucose available and therefore cause a relative lower expression of the SGLT1 gene?
As stated previously, the conclusions seem a repetition of the results section. I would strongly recommend including in the discussion and in the conclusive statement the limitations of the study. I might suggest taking into account, for example, the limited number of samples and the possibility to induce ruminal acidosis with a high content of concentrate in the diet. Furthermore, include a confirmation/rejection of the hypothesis. Do the results confirm the hypothesis? Are the expectations on the outcome of the experiment met?
Additional comments: Please consider the following comments for specific sections of the manuscript:

Line 22: areas
Lines 25-26: use n=3 in brackets after each group description, instead of writing “three each”
Line 40: please rephrase “after grazing”
Line 41: please specify the geographical location
Line 45: delete “of ruminants”, as it sounds repetitive
Line 50: is there a reference for this statement?
Line 57: Aschenbach et al. 2000 is cited twice
Line 59: please consider rephrasing
Lines 62-63: could the authors specify more clearly how are the animals used for “hide and fuel”?
Lines 64-70: please add references for these statements
Line 73: supplementing feed in winter only provides positive effects?
Line 85: please consider adding the age of the animals
Line 86: please consider rewriting (see comment for lines 25-26)
Line 97: what is the name of the kit?
Line 98: how were concentration and quality assessed?
Line 132: how was the taxonomy assigned? Which version of the database was used?
Lines 139-140: please consider rephrasing
Lines 143-146: this part should probably be included in the statistical analyses part
Line 155: how were values equal to 0.05 considered?
Line 159: what to the authors mean with pairs of reads? Weren’t the reads merged as a first step of bioinformatical analyses?
Line 160: what do the authors mean with “clean” tags?
Lines 164-166: the rarefactions curves in the figure only show the number of OTUs. Please consider rephrasing.
Line 169: plural of phylum is phyla
Line 196: please specify “other VFAs”
Line 205: please consider rephrasing; for animals it would be more appropriate to use “feed” instead of “food”
Lines 235-238: if I understand correctly, the authors suggest that G+S group eat more fiber with their diet. How is that possible if they were receiving grain as supplementation?
Lines 252-255: Please add references.
Lines 263-264: what do the authors mean with “long-term intake of hay”? When did the animals receive hay?
Lines 275-281: How can the authors state that when VFAs decrease SLGT1 expression increases? Correlation indicates only the degree of relationship between two variables, please consider a regression analysis to strengthen this point, or add a reference to previous studies.
Line 284: please rephrase “rumen internal environment”
Lines 286-294: do not repeat all the results in the conclusion, but just focus on the most relevant.

Table 2: could the authors explain the indexes in the description?
Table 3: please report the values also for each of the other VFAs measured.
Figure 1: may I suggest trying colorblind-friendly palettes for the bar charts?


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: SUPPLEMENTARY FEEDING OF CATTLE-YAK IN THE COLD SEASON ALTERS RUMEN MICROBES, VOLATILE FATTY ACIDS, AND EXPRESSION OF SGLT1 IN THE RUMEN EPITHELIUM
Review round: 1
Reviewer: 2

Output:
Basic reporting: In the submitted manuscript, Sha and co-workers investigated the effects of supplementary feeding in the cold season on the rumen microbes, VFAs, and expression of SGLT1 in the rumen epithelium of cattle-yak. Overall, the topic of manuscript is of interest. This study applied 16S rRNA sequencing and covered the functional analysis of KEGG pathways and COG analyses. While the reviewer has major concerns regarding the experimental design/discussion part.

The English sentence structure should be improved in some sentences.

The figure legends should be modified, adding the comparision of treatment groups and statistical analysis (P-value) under the tables.
Experimental design: This study provided changes in microbiota population and VFA, however, no details about animal host (gender) and the power of statistic calculation for the number of animals in each treatment group were presented since these factors can react to these changes and statistical analysis. Also, the experimental design should be crossover designed with including the washout period to reduce the variation of animals, which reduced the readability and robust of this manuscript. Does the author have any explanation for the experimental design without crossover animals?

For microbial profiles, the family level is missing in the results. The authors represented the phylum and genus level in the abstract and results. The phylum consisted of a variety of microbial community, which have diverse function; therefore, the phylum is not well representative of the microbial community. It showed only general trend, the family and genus level are more validity. Please explain why you used this phylum level.

Regarding to the functional analysis, the results of KEGG pathways and cog genes from the experiment can be used to explain the changes of microbial population. Why does the author decided to put in the supplementary materials? I recommned to put in the main manuscript. Also, few results presented in the result and discussion section. I would recommend explaining more details related the functional analysis, not only mentioned “carbohydrate metabolism”

For concerning to the gene expression, Actually, there are other genes involving in glucose transporter found in the mucosa. What bases were these particular genes selected? Why did you interest only the expression of SGLT1?
Validity of the findings: In general, the finding provided the sufficient results according to the method. However, the data presentation should be improved. There are several repetitions in the results and discussion and the conclusions are not supported. It must be strengthened. All over-interpret data must be removed. Please reconsider and rephrase. The manuscript would gain substantially by making better use of the wealth of literature data for interpretation of the experimental results.
Additional comments: Abstract part:
Please reconsider to report the results at the family and genus level.
Introduction part:
Line 55-56: The author stated “some glucose in the rumen is transported directly to the blood via sodium ion-dependent glucose transporter 1 (SGLT1), thereby saving energy for fermentation (Aschenbach et al., 2000; Aschenbach et al., 2000; Aschenbach et al., 2002)”. The authors should provide clearly understanding for your text how it saves the energy for fermentation.
Line 74-79: The hypothesis is missing.
Materials and Method:
Line 84-85: Ingredient and chemical composition of experimental diets should be declared.
no animal gender related data was provided. Does the author use crossover design?
Line 86-88: How special is the “supplementary feeding “?
Please reconsider to explain factors “soluble or digestible or…………..”
Line 105: The condition for processed the  library and sequenced on an Illumina MiSeq platform (Illumina, San Diego, CA, USA) should be provide.
Line 149: “The microbial communities in each group (identified by 16S rRNA sequencing), based on both the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Clusters of Orthologous Groups (COG) databases”. Does the author report the relative abundance at what level?
Result part:
Line 168:  Paragraph “Microbial community structure in the rumen”
Please reconsider to provide the result at the family level.
Does the author report the relative abundance at what level?
Line 200:  The sentence is hard to follow.
Figure
Discussion part:
Overall: Please remove all repeated results “were/was significantly” “higher or lower” throughout the paragraph.  Please provide more explanations and give more reason and scientific data from previous literatures or your correlating results from the experiments to support how the diet-related shift in microbiota and functional capability.

Line 210-215:  “Firmicutes possesses many genes encoding energy metabolism-related enzymes”. The sentence is over-representing data. The Firmicutes comprised of several microbial group and may shift their functions depending on the substrate availability. Therefore, presenting the specific microbial groups at family and genus level would gain substantially by making better use of the wealth of literature data for interpretation. Also, the microbial group should be consistent with the KEGG findings on carbohydrate metabolism.
Line 223-226:  Please add references
Line 240: “Therefore, we speculate that appropriate supplementary feeding after grazing in the cold season can promote the degradation of carbohydrates in the feed, which can allow the cattle-yak to more effectively cope with the harsh cold season”
The sentence is over-interpreting data. The authors need to explained how diet-related change in microbial groups and shifts in KEGG and COG pathways. Not only Carbohydrate metabolism, but also other pathways should be discussed.
Line 243-245: “In both the KEGG and COG analyses, the carbohydrate metabolism gene families”
Please provide the lists of gene families which affected by the diets.
Line 263-264: Please consider to interpret the data with the result of the KEGG and COG analyses
Line 274: Please add references

Conclusion part:
The conclusions are repeated from the result and not supported the finding. Provide more novel data further research.


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: SUPPLEMENTARY FEEDING OF CATTLE-YAK IN THE COLD SEASON ALTERS RUMEN MICROBES, VOLATILE FATTY ACIDS, AND EXPRESSION OF SGLT1 IN THE RUMEN EPITHELIUM
Review round: 2
Reviewer: 1

Output:
Basic reporting: In general, the revised version is much better but the reviewer has major concerns regarding the grammar error and conclusion part. The Figures and Tables legends should be expand and also include the treatment groups in the titles.
Experimental design: No comment
Validity of the findings: In general, the data presentation has improved from the previous version. The discussion and conclusion must be strengthened and removed all repetation.
Additional comments: Materials and Method:
Line 94: Typing error “…..”
Line 100 - 105: Grammar error and wrong sentence structures. The sentence in the Materials and Method part should be past tense. Also, these are not sentence “Take 50ml of the rumen contents for each cattle-yak and filter the rumen content with four layers of sterile gauze. Divide the filtered rumen fluid into 3 sterile cryo tubes, which were immediately placed in liquid nitrogen for preservation, and bring them back to the laboratory for further processing 16S rRNA analysis”
Please recheck the whole Materials and Method part.
Line 128: Please rewrite the sentence. “The detector temperature was “………” Better to combine with previous sentence.

Line 144: it should have verb after and
“and ……………………….chimera removal (UCHIME v4.2) in order to obtain optimized sequences (tags)”

Line 149: Please reconsider to change the sentence. “P<0.05 is significant difference, p<0.01
168 is extremely significant difference”. The extremely significant” is not scientific. Better to use “considered significant levels at ………..”
Result part:
Line 177: grammar error. It should be past tense.  “ indicate the species diversity and species richness”
Line 204-211: grammar error. It should be past tense.  Group G+S is significantly higher than group G. Among the 25 COG   gene families, transcription was the most frequent. In addition, the most abundant functional Carbohydrate transport and metabolism, Amino acid transport and metabolism, and Energy productions and conversion are significantly different. Among, Carbohydrate Transport and Amino acid transport and metabolism show as G group with significantly high G+S group. However, in terms of Energy Productions and Conversion, G group is significantly lower than G+S group, which is consistent with the KEGG function analysis above.

Line 246-249: grammar error. It should be past tense
Discussion part:
Overall: Please remove all repeated results “were/was significantly” “higher or lower” throughout the paragraph.  You can keep the same meaning but paraphrase the sentences.
Line 260: grammar error. It should be past tense
Line 305-310: grammar error. It should be past tens

Conclusion part:
The conclusions are repeated from the result and very weak. Please rewrite the whole section. Provide more novel data further research.
The structure of conclusion should be
             1	Restate your research topic.
	2	Restate the thesis.
	3	Summarize the main points.
	4	State the significance or results.
	5	Conclude your thoughts./ Provide next steps