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: PHYLOGENY OF PALEOZOIC LIMBED VERTEBRATES REASSESSED THROUGH REVISION AND EXPANSION OF THE LARGEST PUBLISHED RELEVANT DATA MATRIX
Review round: 5
Reviewer: 2

Output:
Basic reporting: Paper is long and meandering.

Digressions and casual discussions need to be eliminated.

Paper still lacks a clear rationale that supports the experiments outlined.

Citations and responses to works in progress by other research groups need to be removed.

General paper structure appears to promise a methodological investigation of how reproducible matrix construction is. Instead, paper claims to deliver a phylogeny of tetrapod relationships and a resolution to the lissamphibian origins problem. In actuality, it is very uncertain what the paper actually delivers at all.
Experimental design: Experimental design is not clear. Many experiments are reported but how these support the rationale is unclear. Basic summary statistics on character recoding are not reported, making it difficult to assess whether any patterns in supposed errors exist. Sensitivity analyses are not conducted or reported, making it impossible to tell why the results differ. Statistics of tree similarity are not reported, making it difficult to tell whether differences between trees are actually meaningful.

This is not the first paper the authors have written with this methodology, and it seems that no matter the source dataset, these authors get the same general set of results. It is unclear whether everyone else's dataset is bad, or whether the authors are introducing biases in morphological interpretation or character treatment. One way to solve this problem is to bring in outside collaborators from other research groups and statistically compare the resulting matrices. This is obviously not done here either.
Validity of the findings: The actual findings of the authors are difficult to interpret because their phylogenetic experiments are not appropriately controlled and do not employ appropriate sensitivity analyses.

The findings that the authors appear to want the reader to accept (overall phylogenetic patterns and resolution of the lissamphibian origins problem) are not valid for the reason that they are not actually tested here. The experiments here test whether it is possible to break the underlying dataset of Ruta & Coates 2007, but does not test how much change is necessary to do so (this would require sensitivity analysis) nor does it test how much change is actually reasonable to impose here (that would require replicate efforts by several independent researchers/groups).

The specific conclusions of the authors are difficult to determine, but much of the discussion is only tentatively linked to the experiments reported and is not supported even by the data reported here.
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: SPICULOUS SKELETON FORMATION IN THE FRESHWATER SPONGE EPHYDATIA FLUVIATILIS UNDER HYPERGRAVITY CONDITIONS
Review round: 1
Reviewer: 1

Output:
Basic reporting: This manuscript has two major components: 1) freshwater sponges are grown from gemmules under hypergravity conditions, 2) freshwater sponges are fed 13C and 15N as a measure of their ability to take up DOM. These two parts of the manuscript are linked by the hypothesis that hypergravity conditions may deplete energy reserves, causing developmental abnormalities, and that nutrient uptake could compensate for this effect.

The manuscript is clearly written, simply designed and appropriately controlled.
Experimental design: The experimental design seems fine. I have only three comments:

1. 13C and 15N enriched medium was added to some sponges, the solution removed and replaced with the regular growth medium. Was there a wash step, or only replacement of the nutrient enriched medium? And if the latter, could the isotopic signatures reflect carry-over of residual nutrient-enriched medium that was not taken up by the sponges? To control for incomplete washing it may have been prudent to add the nutrient-enriched medium to control sponges at the end of the experiment, then immediately remove and replace with regular medium, just to ensure that residual enriched medium did not carry- over as a liquid volume. Also, how were 13C and 15N treatment levels determined? It could be very convincing to titrate this enriched medium to show graduated changes in uptake.

2. Gemmules were pretreated with H202 to minimize fungal and bacterial contamination, but freshwater sponges always have algal and bacterial associates, even after such treatment. How can nutrient uptake be firmly attributed to the sponge and not associated microorganisms?

3. How do g forces relate to ambient pressure. Sponges routinely grow at great depth. Should the expectations for hypergravity could affect sponges be calibrated to account for the fact that sponges have a hydrostatic skeleton? In other words, is 20x g as extreme for a sponge as for an astronaut, or any terrestrial organism? Would it be worth testing a broader range of g force until dramatic effects are detected?
Validity of the findings: No additional comments.
Additional comments: My only real objection to this manuscript is that the two topics don't seem conceptually connected. It seems that the authors have an interest in DOM uptake in sponges, and that they had access to a large diameter centrifuge. Conceptually, the two parts of the study seem largely unrelated and I would probably choose to publish as two discrete manuscripts. Albeit, both may require further development in that case.


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: SPICULOUS SKELETON FORMATION IN THE FRESHWATER SPONGE EPHYDATIA FLUVIATILIS UNDER HYPERGRAVITY CONDITIONS
Review round: 1
Reviewer: 2

Output:
Basic reporting: 1. The entire manuscript should be re-written to fit the general style of a scientific manuscript.

(i)	The Introduction section should include sufficient background information to set the work in context. The aims and importance of the work should be clearly stated.

(ii)	Results should be presented in a logical sequence in the text, tables and figures. The current description of the results is not detailed enough for readers to understand what is the aim of this experiment, how the experiment was performed and what is each result. Even though the experimental procedures are written in the section of Materials and Methods, the Results section should be written to make it possible for the readers to understand the experiment without reading the Materials and Methods section.

(iii)	Figures should be cited in more detail in the text. For example, the current citation is as “(Figure 3, 4)”, but it should be cited as “Figure B upper panel”, or “blue arrows in Figure 3B upper panel”, etc.

(iv)	The Discussion section should consider the results in relation to any hypotheses advanced in the Introduction and place the study in the context of other work. To discuss the results in this study, each data should be cited in detail. Currently any data was cited.

2. The English language should be improved and should accurately use correct technical terms to ensure that international readers can clearly understand this manuscript. Some examples are:

(i)	hatched gemmules (should be “juvenile sponges hatched from a gemmule”).

(ii)	Line 67, degenerative sponge bodies (gemmules), (this is incorrect. Only stem cells are inside the gemmule, and the gemmule is not a degenerative sponge body.

(iii)	Line 104 Does “internally-stored energy” mean vitelline platelets that are contained in totipotent stem cells inside the gemmule coat?

(iv)	The description “spicules are erected” during the process of skeleton formation is fine. But “sponges erect spicules” or “sponges transport their spicules (line 122)” are scientifically inappropriate descriptions. Since the authors described previous studies of Nakayama et al. as “transport cells” transport spicules and possibly erect spicules, the subjects of these sentences should be “cells” not whole animals (sponges).

(v)	So far as I know, “spiculous skeleton” is used instead of “spicule skeleton”.

(vi)	Line 129 “relative surface area” needs more explanation; otherwise, it might mislead readers, since the authors seemed to use “surface area” as the dimension of basal epithelia (basopinacoderm) and not as the dimension of the total external surface of the sponge body. Furthermore, what is the meaning of “relative”? what are compared is not clear in this sentence.

3.	Previous studies and knowledge should be accurately described. Some examples of the sentences in which this seems to need to be corrected are:

(i)	Line 73-77, totipotent stem cells do not differentiate inside the gemmule coat. Stem cells differentiate after they migrate outward from the gemmule coat. What is the difference between “germination” and “gemmule hatching”? During gemmule hatching (development of the juvenile sponge body), stem cells not only attach to the substrate but also differentiate into various types of cells. “cells attach sponge to its substrate” is not an appropriate description. “Cells firstly migrate out from the gemmule coat and differentiate to basal epithelial cells that are attached to the substrate” might be a more accurate description.

(ii)	Line 84, So far as I know, it has not been clearly shown that filter systems of juvenile sponges are only developed after spicules are held up. Funayama et al. 2005 showed that choanocyte chambers started to form from developmental stage II. Thus, which phenomenon (spicule holding up or starting the filter system development) is earlier seemed to be difficult to determine at present.

(iii)	Line 88, what are the “different locations”? different from what?

(iv)	Line 90, what is the “third type of specialized cells?” What are the first and second types of specialized cells? Furthermore, Nakayama et al. clearly reported that basopinacocytes (basal epithelial cells) around the held-up spicules secrete short type collagen to embed the basal end of held-up spicules. Thus, the “third type of specialized cells” in the text should be basopinacocytes around the held-up spicules.

(v)	Line 122, Nakayama et al. described that transport cells carry spicule stochastically. Thus, it does not seem likely that transport cells have a certain correct orientation to transport spicules.

4.	Only previous studies or knowledge that is directly related to the present study should be described or clearly explained in relation to the present study. An example of a sentence in which the relationship is not clear is:

(i)	Line 289, Chitin in the gemmule (coat?) of E. fluviatilis. This does not seem to be related to the skeleton formation.
Experimental design: 5.	To avoid possibly misleading readers into thinking that Figure 1B shows the results of this study, Figure 1B should be entitled “Our working hypothetical model of the effect of hypergravity (and nutrient uptake) on skeleton construction of juvenile sponge hatched from a gemmule”. Adding a question mark in the illustrated model is commonly used to describe the working hypothesis. Since the current title of Figure 1 is “ Developmental stages of germination in the freshwater sponge, Ephydatia fluviatilis”, Figure 1B could easily be misunderstood as showing the results of this study.

6.	The main question in this study should be clearly stated, especially in the Abstract and Introduction. Since the nutrient uptake is described in earlier parts of both sections, it is not clear whether the authors focused on the skeleton construction under hypergravity, or on the effect of nutrient uptake on skeleton construction.

7.	Do the data shown in Figure 3 show non-fed juvenile sponges under several different strengths of gravity? If so, it should be clearly described both in the Results section and Figure legend. Photographs of fed-juvenile sponges should be shown, especially since the authors argue that development of the oscula is different between fed- and non-fed juvenile sponges under hypergravity.

8.	Does the legend of Table 1 mean that the authors determine the developmental stage of juvenile sponges as “sponges with held-up spicules but without a developed osculum” as Stage III sponges and “sponges with a developed osculum” as stage IV sponges?
Validity of the findings: 9.	In the Discussion section, several arguments or conclusions of authors seemed to be overstatements, or I could not follow the authors’ scientific logic. This might be partially because of the inappropriate wording. For example:

(i)	Line 343 (the first sentence of the conclusion). Since this study did not examine any of “the process of skeleton formation”, current statement is overstatement. Not the process but whether the skeleton is formed (spicules are held up) is examined in this study.

(ii)	Line 277-278, why does the result that juvenile sponge could be developed under the hypergravity forces show that a pivotal step in the seasonal life-cycle of E. fluviatilis is highly “resilient”?  If “a pivotal step in the seasonal life-cycle” means development of a juvenile sponge from a gemmule, I would like to suggest that the authors cite the literature that describes that gemmule hatching is seasonal in this species. So far as I know, Ephydatia fluviatilis generally can survive winter as adult sponges, and gemmule hatching is not really included their life-cycle. In contrast, the sister species Ephydatia muelleri generally survives winter as gemmules (and thus gemmule hatching is included in its seasonal life-cycle).

(iii)	Line 282. the authors described that sponges showed a significant “decrease” in surface area under increased g-force exposure But in Figure 4, all specimens showed increase (>100%). It is confusing and could not follow what the authors would like to describe.

(iv)	Line 303- Since only juvenile sponges in the process of developing the body, including the filter-feeding system, were used in this study, it is difficult to distinguish whether the results suggest that the process of skeleton construction relies mainly on intrinsic energy or whether they suggest that juvenile sponges simply could not take up sufficient nutrients to affect skeleton construction in these developmental stages.

(v)	Line 306- it is interesting that development of an oscule was significantly less frequent in fed juvenile sponges, especially under hypergravity force. but the data did not show or describe this effect (numbers, etc.). So, it is difficult for readers to evaluate how “significant” the effect was.
Additional comments: 1.	One of the major problems is that the authors did not show data concerning whether  there is any difference of body growth between non-fed and fed juvenile sponges in the general culture condition (gravity 1g).  I suppose that juvenile sponges grow similarly in non-fed or fed conditions, since in the working hypothesis in Figure 1B, in 2.5g the illustrations of the morphology of juvenile sponges show that they are the same.
          If so, this suggests that in juvenile sponges from developmental stage II to IV (stage IV is the stage when the oscule forms and the filter-feeding system becomes fully functional) there is not much uptake of nutrients from the culture medium. The fact that the filter-feeding system is not completed (without an osculum to expel water) in the developmental stages the authors used in this study also supports this possibility. Thus, it is difficult to follow the logic of the authors’ working hypothesis to expect difference between non-fed and fed conditions.
            Since the experiments themselves using hyper gravity are interesting, I suggest that the authors re-consider the stream and logic of the manuscript.

2.	Although the title of this manuscript is “Spicule skeleton formation in the freshwater sponge Ephydatia fluviatilis under hypergravity conditions”, it was not clear the main question of this study is whether the authors focused on the skeleton construction under hypergravity, or on the effect of nutrient uptake on skeleton construction. As there are quite a lot of description about nutrient uptake in Abstract, Introduction and Discussion, it is very confusing and difficult to follow the main stream of this manuscript.

3.      The entire manuscript should be re-written more accurately. There are many inappropriate descriptions of previous knowledge and works in Introduction section. Furthermore, many parts of Discussion are overstated, especially conclusion. Conclusion should be based on the results of this work.


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: SPICULOUS SKELETON FORMATION IN THE FRESHWATER SPONGE EPHYDATIA FLUVIATILIS UNDER HYPERGRAVITY CONDITIONS
Review round: 1
Reviewer: 3

Output:
Basic reporting: The English writing could be improved
Experimental design: OK - no comment
Validity of the findings: no comment
Additional comments: PeerJ 		#30193

Evaluation of the manuscript “Spicule skeleton formation in the freshwater sponge Ephydatia fluviatilis under hypergravity conditions” by Bart et al.

This manuscript describes a work, which fits publication in PeerJ. The authors examined growth and formation of a 3D skeleton in freshwater sponge under hypergravity conditions. They assumed that under such conditions is energetically more costly than under normal g force, and hence with increasing g-force the growth will be slowed down. They examined this assumption using growth post germination of freshwater sponge gemmules. Furthermore, in order to evaluate if lack of energy is driving such hypothesized phenomenon, they supplied half of the gemmules with DOM (dissolved organic matter) in the form of (C and N) marked amino acids. I liked the experiment using the Large Diameter Centrifuge designed to examine behavior/physiology under various gravity forces.

Some minor comments are given below – but I consider the manuscript suitable for publication.

There are comments I do not write which I assume are the result of converting the text to a pdf format (many places were space is required between words).
The number of specimens treated (n) should be given in all places relevant (including figures 4 & 5).

The other remarks are written in order of appearance in the text.

L. 73 - Ilan et al., 1996 does not appear in the references
L. 93 - Nakayama (2015), should be Nakayama et al. (2015),
L. 277 - E. Fluviatilis should be E. fluviatilis
L. 278 - ” under extreme levels of mechanical stress” What do you consider to be “extreme levels”?
L. 285 - sponging should be spongin
L. 285 - Regarding chitin in adult E. fluviatilis - I am not aware of chitin reported from mature Ephydatia. See: PloS one 13.5 (2018): e0195803.
L. 287 - “ invertebrates and marine demosponges” implies that sponges are not invertebrates. I would change to “invertebrates including marine demosponges”
L. 298-301 – What about adult E. fluviatilis microbiota?
L. 308 - prologue should be prolonged.
L. 316 - McMurray 2016 should be McMurray et al., 2016
L. 355 - DOM uptake was shown here in a developing sponge - not in an adult.


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: TEMPERATURE-MEDIATED FEEDING BETWEEN SPRING-ASSOCIATED AND RIVERINE-ASSOCIATED CONGENERS, WITH IMPLICATIONS FOR COMMUNITY SEGREGATION
Review round: 1
Reviewer: 1

Output:
Basic reporting: The authors use clear and unambiguous English but there are few minor errors throughout the text. For example: Line 23: “trails” should be “trials”. Line 39: delete the s from Temperatures. Line 81: “Purpose of this study…” should read “The purpose of this study…”

The introduction of the paper gives sufficient background to show the context of the work. It is clear how the authors are building on previous research.

The literature is well referenced and shows the relevance of the study. However, there are many errors in the text of the Literature Cited Section (see below). I recommend thoroughly checking all references.
Line 232. Behen: Reference incomplete.
Line 290. Poff, Brinson and Day: reference is not complete.
Line 295. Portner, Peck and Somero: Reference has a repeated information.
Line 306. De staso: reference is out of place and repeated here for a second time
Line 309. Stevens: Reference incomplete.
Line 324. Watson: Reference incomplete.

The manuscript structure conforms to PeerJ standards.

There are absolutely no figures in this paper, this is very strange. The authors should include: Fig. 1 - a map of the study location. This should include an inset of the relative location of each site. Fig. 2 – a picture panel of the two species or perhaps of the containers that they were placed in for observation. Fig. 3 - a basic graph of the feeding results.

All raw data on first and total feeding attempts has been supplied and is part of Table 1.
Experimental design: This paper contains original primary research on temperature-mediated species performance of two fish species, one spring and one river-associated. Dissimilar to previous research this manuscript focuses on two species that are both warm water forms with similar temperature tolerances, but that have different habitat preferences.

The research question is well defined. Past research has focused on temperature-mediated performance between warm and cold water forms (that change in distribution over altitudinal gradients). This study tested whether temperature-mediated performance might be a mechanism to segregate the habitat use of two warm water forms that have similar temperature tolerances. The authors state how their research fills an identified knowledge gap.

Additional details in the methods section are required to provide enough information for the study to be replicated. For example, were the different species collected from the different sites on the same days? How were the fish transported to the lab? How long in total were they in the lab before experiments were performed? How long did it take to drip acclimate them to 23 degrees C?
Validity of the findings: The feeding data are sufficient to address the question. However, data should be presented on the sizes of the fish to confirm that the visual matching of size and sex was sufficient to make the comparisons and that there was not a size bias by species.

The conclusion states that temperature mediates the distribution of these species. I think that it should be worded less strongly because other factors are also influencing the distribution of these species. Furthermore, this was just one feeding study at different temperatures and it didn’t include actual measurements of physiology, etc, so don’t conclude too much. The conclusions are linked to the original research question.
Additional comments: This manuscript is clear and concise. It flows logically, states a prediction, and presents the data set to support the prediction. A few things should be improved:
1.	Your most important issue is the lack of figures in the manuscript.
2.	Secondly the literature cited section needs to be revised to ensure completion of all references.
3.	Please add some more details in the methods section.
4.	Additional results on the sizes of the fish in the trials should be reported.


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: TEMPERATURE-MEDIATED FEEDING BETWEEN SPRING-ASSOCIATED AND RIVERINE-ASSOCIATED CONGENERS, WITH IMPLICATIONS FOR COMMUNITY SEGREGATION
Review round: 1
Reviewer: 2

Output:
Basic reporting: This article is clearly presented and the context is sufficient.  The structure is professional, the data are clearly presented and the hypotheses are clearly articulated.
Experimental design: The experimental design is sound in many ways.  It is not very original.  The only novel component is in the specific fish species that they used.  The basic design and motivation mimic other studies.  The overall research question is relevant and meaningful but the study addresses only a small component of the overarching goal.  The research is technically sound and meets a high ethical standard.  The methods would be easy to replicate.
Validity of the findings: The data are robust and statistically sound.  The conclusions are supported with respect to the specific hypothesis but because the experiment is limited in scope, the conclusions in light of the over all theme are overstated.  The data collected are very specific and do not adequately address the larger context articulated in the introduction.
Additional comments: This paper presents results from a laboratory study testing how temperature affects feeding performance in two small fish.  The conceptual framework is well developed and the predictions and hypotheses are well articulated.  The research itself is modest. The study is straightforward, testing if fish feeding performance differ for two species at different temperatures. The methods are sound all though they are not novel. The small data set is clearly presented in a Table with appropriate statistical analysis.  The results support the hypothesis that the fish species collected from springs performs better at the temperature of springs and the fish species collected from the river fish performs better at the temperature of the river.

In addition to the small scope of this paper I have concerns with the interpretation of the results, given the experimental design. Because these are paired trials - there is an inherent assumption of competition yet there is no data on performance of the two species in isolation.  This study would be stronger if it were coupled with individual feeding trials to test if there is an underlying physiological basis for the difference in performance of the two species with temperature.  As the title suggests the authors are ultimately looking to understand community segregation - inferring that the underlying mechanism for separation is different competitive abilities under different habitat regimes.

The experiment is somewhat contrived as there is only one food source, food is limiting and species are forced to interact in a small space.   The food was almost entirely consumed during some of the trials.  It is unclear if the mean number of food items consumed is driven more by food limitation then by the rather forced constraints of feeding.  The authors need to address how this study is relevant to field conditions.

On a more detailed note, the results are largely driven by one trial run where one of the species didn't feed at the high temperature.  Given that these results are so different from the other trials it makes me wonder if there was something else going on with this batch of fish.  Could they have been more stressed than the other fish?

This research would be stronger if the experiment were coupled with growth rate measurements, other behavior or physiology data to give a broader picture of fish performance under different temperature regimes. The authors develop predictions based on contrasting stenothermal vs. eurythermal environments but set up an experiment to test two temperatures. Testing how fluctuations in temperature affect fish distributions requires a more elaborate design.

In summary - the results from this study are compelling in that they support the predictions articulated by the authors.  The scope of the study however is limited.   This research should either be condensed into a short note or submitted to a regional journal.


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: USING THE WAX MOTH LARVA GALLERIA MELLONELLA INFECTION MODEL TO DETECT EMERGING BACTERIAL PATHOGENS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Clear, well-referenced and well structured presentation of fundamental science.

Fig S1: Please could the authors add labels for location key at the end of each line on the graphs, I’m finding it impossible to match most the lines to the boxplots on Fig S2 by colour vision alone, a lot of the shades are very similar.

Proteus clone: I thought it was very interesting that this clone turned the Galleria brown instead of black, could this be noted in the main text as well as in the SI? I don't know what it signifies, but it may be something that another reader recognises as diagnostic of a particular virulence mechanism or immune response.
Experimental design: Lines 120-125: I was interested in why the authors decided remove a single drop of haemocoel from the infection site, rather then homogenising the whole larva to sample bacteria from throughout the haemocoel. I guess there are arguments for and against both methods – the method used may fail to sample pathogens which have moved away from the infection site (as systemic infection can proceed rapidly in wax moth larvae), but will reduce sampling of “native” bacteria present in and on the larvae. As homogenisation of whole worms seems to be more typically used in the literature, please could the authors use a couple of sentences to justify their choice of this technique? I guess a lot depends on whether you expect infection to be caused by single pathogen strains present in the environmental samples, or whether high virulence is a function of the sample community. Fig S3 is broadly consistent with the former, which might justify the authors' choice? Also, could the authors specify whether the inclusion criterion for this experiment was melanisation at the infection site, or any melanisation at all?
Validity of the findings: Results are robust. Raw data is provided.
Additional comments: A small number of typos:

Line 73: “in laboratory” should be “in the laboratory”
Line 76: add plural “s” to “supernatant”
Line 82: erroneous . after “in”
Line 130: missing space after “37°C”
Line 188: Citations of Fig S3 and S4 should be Fig S2 and Fig S3, respectively.
Line 246: remove – after “environmental”


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: USING THE WAX MOTH LARVA GALLERIA MELLONELLA INFECTION MODEL TO DETECT EMERGING BACTERIAL PATHOGENS
Review round: 1
Reviewer: 2

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

 Literature references, sufficient field background/context provided.

Fig 1 it is not sufficiently clear: label on the X-axis (presumably hours ?) is missed on all three graphs. Besides, an interruption on the scale on the second third of the X-axis help to make be more readable the left side of the graf.
Experimental design: Hernandez et al. here report an alternative model of potential use for the screening of environmental pathogens.
The idea is interesting and original.
nevekess due the higt varibilty of this model
Validity of the findings: The idea is interesting and original.
l nerveless due to the high variability of this model some concerns must be clarified.

as read out the mortality it is weak and not fully appropriate,
a scoring scale similar to  doi:  [10.3390/jof4030108] it wilòl improve the word and the data
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: USING THE WAX MOTH LARVA GALLERIA MELLONELLA INFECTION MODEL TO DETECT EMERGING BACTERIAL PATHOGENS
Review round: 1
Reviewer: 3

Output:
Basic reporting: The publication of Hernandez et al. describes potential use of Galleria mellonella infectious model to detect pathogens from water. The paper is written in clear unambiguous professional English. The “Introduction” clearly explains research area and provides enough background information which are supported by appropriate references. Another sections are properly structured as well. Figures are relevant to the content and authors also provides 3 figures, row data and 9 tables in supplemental material. I appreciate such amount of data in supplemental material and row data as well, however I would like to suggest authors to include figures from supplemental material to main paper, it would be more clear to reader for better understanding of methodology and results. The authors also should provide better and more detailed description of all supplemental figures.
Experimental design: Methods are well described and provides sufficient details and enough information to replicate. I have only those minor points:
Material and methods, line 77: Please write more information about M9 buffet. Please, specify composition of buffer or company which distribute it.
Material and methods, Lines 79 – 82. Extraction of bacteria from sediment samples: After twice spinning down of sediment samples (first: 3000 rpm, 2 min; second: 500 rpm, 15 min) you collected supernatant with bacteria. Are you sure that you separated all bacteria from sediment? Is it possible that some of bacteria remained in pellet with sediment particles after spinning of tubes?
Validity of the findings: Data is robust, statistically clear and well described. I have only one question: How did you analyze results from G. mellonella killing assay? Did you use any statistical program to compare survival of larvae after inoculation of different water samples or different CFU of bacteria?
Figure 1. Please, add description of X axes in G. mellonella survival assay. What does X axis represent (hours, or days of survival)?  I also suggest to add curve for controls with 100 % of survival as well, it would be better for illustration of the virulence power of each bacteria sample or inoculated CFU.
Additional comments: The main point of this manuscript is to use invertebrate model organisms Galleria mellonella to detect emerging pathogens. Survival of G. mellonella larvae infected with samples from water and sediment was measured by killing assay. Water samples with the highest lethal impact on larvae has been further identified by another methods confirmed presence of emerging bacterial pathogens and identified their pathogenic potential. Results shown that in vivo model organisms - G. mellonella can be used for such a first screening of pathogens from environmental samples. This method represent easy, low cost and replicable novel approach of testing water from environment. I find this idea very interesting and I have doubt only with some minor points listed upper.


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: SECRETED FRIZZLED-RELATED PROTEIN 4 INHIBITS THE REGENERATION OF HAIR FOLLICLES
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: In this manuscript, Guo at al. demonstrate an inhibitory effect of sFRP4 on hair growth. Although this result is interesting, I have issues with insufficient and superficial data analysis and interpretation.
1.	This manuscript could be sufficiently improved if authors demonstrate that the effect of sFRP4 on hair growth is mediated via inhibition of Wnt pathway. Expression of cytoplasmic and nuclear beta-catenin after sFPRP4 injections would provide a good evidence of activation/inhibition of the Wnt signalling.
2.	Authors should describe more clearly the effect of sFRP4 on hair growth. Quantifications of hair cycle stages as per Muller-Rover at al. (2001) would help to clarify the effect of sFRP4 on anagen initiation and progression.
3.	In the results section, the authors state that sFRP4 does not affect the structure of hair follicles (lines 109-113). However, in the discussion section, the author’s claim that “The structures of ORS, IRS and matrix were smaller than the control” (lines 127-128). Furthermore, “sFRP4 may … inhibit the structure formation of hair follicle” (lines 129-130). These parts of the results and discussion sections need improving and clarification.


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: SECRETED FRIZZLED-RELATED PROTEIN 4 INHIBITS THE REGENERATION OF HAIR FOLLICLES
Review round: 1
Reviewer: 2

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: In this study, the authors want to identify the role of Sfrp-4 in hair regeneration cycle. Through observing the expression pattern of Sfrp-4 and functional assay, they found that Sfrp-4 can inhibit but not block the regeneration cycle. Although the topic is important, there are several issues that the authors should take care and address to confirm their hypothesis.

1. Previous study had demonstrated that Sfrp-4 was expressed during the refractory telogen period (Chen et al., 2014). However, in this study, the author only showed the expression pattern during the anagen period. To evaluate the expression pattern of Sfrp-4 during the whole hair regeneration cycle period, including anagen and telogen is recommended.
2. If the Sfrp-4 did inhibit the hair regeneration, why is it expressed strongly in ORS and IRS during anagen period? According to this, the expression of Sfrp-4 should express strongly in telegen period which inhibit the hair stem cell entering anagen period.
3. In figure 3, the author measure the length of regenerated hair follicles to prove that Sfrp-4 did inhibit the regeneration of hair follicle. Can the author explain when they measure the length? Day 4 or the end of the anagen? To measure the full anagen hair follicle length is recommended to determine if Sfrp-4 diminish the period of anangen which let the hair follicle length become shorter.

Chen CC, Murray PJ, Jiang TX, Plikus MV, Chang YT, Lee OK, Widelitz RB, Chuong CM. Regenerative Hair Waves in Aging Mice and Extra-Follicular Modulators Follistatin, Dkk1, and Sfrp4. J Invest Dermatol 2014; 134(8): 2086-96.


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: PRIMULINA ANISOCYMOSA (GESNERIACEAE), A NEW SPECIES WITH A UNIQUE INFLORESCENCE STRUCTURE FROM GUANGDONG, CHINA
Review round: 1
Reviewer: 1

Output:
Basic reporting: No critical comments.
The manuscript is well-written. Language usage is clear, with only a few points requiring clarification which are detailed below.
Literature cited is appropriate to the topic and the introduction provides suitable context.
The article is suitably structured and novel data are made available in appendices.
Results and discussion are suitably correlated.
Experimental design: No critical comments.
The manuscript is well conceived with respect to methodology, analyses and conclusions.
The paper sets a high standard for the recognition of a new species.
Validity of the findings: No critical comments.
The conclusions of the paper are sound and the recognition of a new species is well-supported.
Additional comments: The following minor points are noted for consideration or correction:

Line 27: ‘generic’ is not needed here as it is followed by ‘in the genus’
Line 59 and elsewhere: Check the journal style as to whether initials in citation are appropriate, or if repeated dates with the same surnamed should be lettered to distinguish different authors.
Lines 72–80: This information is generally assumed to be true, so would usually be left out, but it may be in keeping with journal standards to include these statements?
Line 84: Change ‘these’ to ‘this’
Line 103: Change: ‘the newly acquired sequences of’ to: ‘sequences were newly acquired for’
Line 10: Change: ‘reduce’ to ‘reduced’
Line 113: ‘trn’ should be in italics.
Lines 167 and 168: Change ‘triangle’ to ‘triangular’
Lines 172–173: It would be good to clarify the position of the vestigial flowers on the inflorescence further. Lines 228–229 present this more clearly.
Line 175: You vary whether or not a space is used to separate bracketed measurements through the description – please check the recommended journal style.
Line 179: I suggest changing ‘sect’ to ‘partite’ or ‘merous’
Lines 201–202: change ‘caves entrance’ to ‘cave entrances’
Line 202: Add: ‘the’ before ‘two localities’
Line 215: Add: ‘for’ before ‘P. anisocymosa’
Line 217: Delete ‘the’ before ‘inflorescence’
Line 231: Change: ‘that of other’ to all’
Line 334: ‘Petrocodon’ should be in italics.
Line 338: The closing bracket is missing.

Figure 1 caption
Line 1: Change ‘shoeing’ to ‘showing’; delete second ‘i' in ‘populatiions’

Figure 2 caption
Line 1: Change ‘Hand-‘ to ‘Line ‘
Note that the angle of presentation of the inflorescence makes it difficult to determine the extent of the ‘zig-zag’ arrangement that is diagnostic for the species, so this may be best updated.

Figure 3 caption
Line 2: ‘sp. nov.’ is not required here – otherwise also add to Fig. 2 caption.
Line 6: Change ‘Photoed’ to ‘Photos’ or ‘Photographs’

Figure 4 caption
Line 2: Change ‘Habitat in flowering’ to ‘Habit when in flower’. As A2 in photographed in a cultivated environment, it is not suitable to describe this as ‘habitat’.

Table 1
Text flow would be clearer if all aligned to the top of the cells.


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: PRIMULINA ANISOCYMOSA (GESNERIACEAE), A NEW SPECIES WITH A UNIQUE INFLORESCENCE STRUCTURE FROM GUANGDONG, CHINA
Review round: 1
Reviewer: 2

Output:
Basic reporting: This is a well written and very comprehensive piece of research.  It is well referenced and the authors are aware of the relevant literature in the field.

The legends for the figures are not as well written. The first part of the legend for Figure 1, for example, is not necessary as the results are discussed in the main text and more information about what type of tree it is would be useful eg maximum clade credibility tree.
Experimental design: This is a comprehensive study allowing the authors to confidently describe this as a new species and place it in the correct genus.
Validity of the findings: Although placement of this new species in Primulina is well argued, a comment is made in the introductory section about the highly variable nature of the inflorescence, even within an individual and, yet, it is an inflorescence character that is selected to differentiate this species from all others in the genus.  Although I feel confident that the authors are correct, and the character is striking, I think it would be worth adding a statement about the apparent lack of variability in this character in the specimens seen of this new species.
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: IDENTIFYING ACCURATE METAGENOME AND AMPLICON SOFTWARE VIA A META-ANALYSIS OF SEQUENCE TO TAXONOMY BENCHMARKING STUDIES
Review round: 1
Reviewer: 1

Output:
Basic reporting: English used throughout is mostly fine. Would have the following comments:

1.	Often the word “method” is thrown around too loosely to refer to the bioinformatics software being evaluated, where a better word would be “tool”, “software”, or “program”, to differentiate from the methods that these tools might actually implement, and which might be shared by more than one tool. Examples: twice in the abstract, last column header in Table 1, lines 100, 372, 404, and likely many other places.
2.	Line 50, other places: “reads” are mentioned, though contigs and other types of sequences are possible. Consider replacing “reads” with “sequences”.
3.	Line 74: “k-mers frequencies”: should be “k-mer frequencies”. Also check consistency of spelling and capitalization of “k-mer” throughout document, it is currently inconsistent.
4.	Figure 1: “Metagenomic sequencing” is referenced in the second box from the top, though this is not a good descriptor (everything under discussion is metagenomic sequencing). Better would be to use the term used in the figure legend, “Shotgun metagenomics”.
5.	Lines 111-112: sentence is not grammatically correct, please revise.
6.	Lines 201-202: make “Pearson’s correlation coefficients” singular to agree with the other items in the series.
7.	Line 208: not sure what a “taxonomic threshold” is in this context or how it can vary. Perhaps this should be described in more detail.
8.	Line 218: “dependant” should be “dependent”.
9.	Line 232: “GoogleScholar” should be two words.
10.	Line 232: “7th July 2017” should be either “July 7th 2017” or perhaps better, “7 July 2017”. In any case, all dates referenced in the manuscript should be consistently formatted (according to journal standards, if there are any).
11.	Line 249: the word “illustrated” would be better replaced with “shown”.
12.	Line 250: “re-samplings” doesn’t need to be hyphenated.
13.	Line 252: double space where there shouldn’t be. Other instances of this throughout manuscript as well, should check the whole thing.
14.	Line 255: ‘metacont’ is in single quotes. I don’t see a need to sometimes use double quotes and sometimes use single quotes throughout the manuscript. I would pick one or the other and use it consistently. If you want to make function calls stand out as different from regular text, perhaps a different font can be used (e.g., Courier).
15.	Lines 267-268: “we found that 45% … of all published tools have been evaluated”. Though the authors go on to say it, “evaluated” in this context really means evaluated to their standards. There are other instances in the manuscript where “evaluated” is used in this strict sense. I would make this very clear the first time “evaluated” is used this way, and include the additional explanation given in lines 269-277 as well. Then subsequently the reader will know what “evaluated” means without further explanation necessary.
16.	Line 301: “the Sczyrba study” vs e.g., “Sczyrba et al.” elsewhere – studies should be referenced in a consistent manner throughout the manuscript. Also, format of “et al.” should be checked throughout the manuscript as sometimes it is italicized and sometimes it isn’t (whatever the journal standard is should be followed, I think not italicized is better English).
17.	Line 302: extra space between the words “a  much”
18.	Line 338: “, stemming from a diverse intersecting sets of evidence”. This sentence needs to be revised to make sense.
19.	Line 342: remove comma after “performance”
20.	Line 343: unnecessary space in “random- effects”
21.	Line 382: “sequence“K-mers””: needs a space
22.	Line 413: comma needed after “i.e.”
23.	Lines 413-415: sentence phrased as a question should end in a question mark.
24.	Line 420: ampersand looks odd but maybe this is the journal standard.
25.	Line 421: extra space after comma
26.	Line 428: “higher taxonomic levels”: higher than what? Could be phrased more specifically.
27.	Lines 433-435: “Spike-ins … could be very useful for this.” This is not a complete sentence, please revise.
28.	I noticed Fig./Figure are sometimes used inconsistently.

Literature references are mostly adequate. However, I would recommend the following changes:

1.	The metagenomic assembly references on lines 57-58 are not great. The 2008 review is before any sort of true metagenomic assemblers were available. The Nagaranjan and Pop reference is mostly about generic genome assembly, not necessarily metagenome assembly. And the Magoc et al. study is not really about metagenomics either. Better references would include the following, for example (and there may be others): (1) Breitwieser, Lu, and Salzberg (2017): A review of methods and databases for metagenomic classification and assembly. (2) Olson et al. (2017): Metagenomic assembly through the lens of validation: recent advances in assessing and improving the quality of genomes assembled from metagenomes.
2.	Please add the MEGAN assembler (Huson et al. 2017) to the list of targeted gene assembly references, which currently includes SAT-assembler and Xander. MEGAN assembler was shown to have some performance advantages over Xander.
3.	Very few specific references to metagenomic assembly tools are given. Currently, only reference-guided “gene-centric” or “targeted gene assembly” tools and references are given. No specific references to whole-genome metagenomic assemblers are given. Two of the best current de novo whole-genome metagenomic assemblers are MEGAHIT and metaSPAdes; these should be mentioned. There are also reference-guided whole-genome metagenomic assembly tools, such as MetaCompass, that could be mentioned. This current manuscript is not targeted at metagenomic assembly and currently does a poor job of characterizing this body of work. While I understand the authors are trying to describe the whole metagenomic workflow, either it needs to be done more thoroughly, or it should be streamlined so that steps are described more succinctly and generically.
4.	I don’t think all of the tools listed in Figure 1 are cited in the manuscript; they should be (somehow/somewhere). For boxes that are given multiple times (e.g., taxonomic assignment and functional assignment), are the tools listed always specific to that “path” through the workflow, or are sometimes different tools listed arbitrarily? (I suspect the latter, which in my mind is somewhat misleading.)
5.	Line 268: “Metagenomics – Tools, Methods and Madness”: I don’t really know what this refers to and does not look like a proper citation. Perhaps more info should be included in the text (author, URL, etc.).
6.	Lines 348-349: citations for these software tools are missing here.

Field background/context provided:
In general, I think a decent effort is made to give background. However, the meta-evaluation in the paper concerns only a very specific part of a metagenomic workflow (taxonomic assignment), so much of the background (sketched out in Fig. 1) is not really necessary and better left to a review paper. Partly because of this, sections such as metagenomic assembly are not described very accurately or thoroughly. Because the core problem this paper is concerned with, taxonomic assignment, is in some ways independent of the workflow it’s embedded in (from a collection of sequences, either return a sequence-by-sequence taxonomic assignment or some percentage-based profile), a comprehensive description of all possible metagenomic workflows in which taxonomic assignment is performed is not really necessary. On the plus side, background concerning taxonomic assignment specifically is good: Table 1 is mostly very good, the section on positive and negative control dataset selection is good, and issues regarding metrics used for benchmarking are treated well. Here are some specific recommendations:
1.	I think the current description of the global metagenomic workflow (i.e., Figure 1 and associated text) should be streamlined and shortened, but alternatively it could be fleshed out (I gave ideas regarding how the metagenomic assembly section could be improved, for example).
2.	Table 1: I think the “Positive Control” column descriptions could be described more consistently. A bolded number draws your attention (e.g., 742, 417, 846, 11, 689, 125) but these refer variously to taxa, genera, species, and other things. Perhaps more effort could be made to describe the data analyzed in these studies in such a way that would facilitate easy comparison. Also, capitalization on Table 1 column headers should be made consistent.
3.	Figure 2: I only just noticed the subtle difference between the “Simulated evolution” diagram and the “New genome sequences” diagram (aside from the color of the dotted lines), namely that “Simulated evolution” could extend from a tip on phylogeny whereas a new genome sequence wouldn’t. I think this should be made more obvious.
4.	Lines 182-197: I would lead with the two major analysis paradigms (distributional recapitulation and read-by-read), and then talk about implications for definitions of TP/FP/TN/FN in each of these contexts.
5.	Line 190: “This approach”: starting a new paragraph this way, I’m not sure which approach is being referred to.

The article structure, figures, and tables are mostly fine. I would perhaps put less effort into trying to describe metagenomic workflows in all their possible permutations. Furthermore, there is some information in the Conclusions that is not really germane to the study at hand or supported by the results, namely the final paragraph on spike-ins. I did not see it mentioned anywhere in the results that comparative evaluation studies that used negative controls were more fair, produced better results, etc. Perhaps this paragraph should be incorporated into the introduction/background material instead.
Experimental design: This is original primary research within the scope and aims of the journal.

The research question is well defined and fills an obvious knowledge gap (conflicting reports between different comparative evaluation studies).

I believe the meta-comparison was done rigorously and ethically, though I have a few concerns:
1.	The rankings in Figures 4 and 5 are currently by median F-measure. However, the confidence interval is not currently taken into account in the ranking. For example, in Fig. 4, TACOA has a huge confidence interval whereas the three tools below it (DiScRIBinATE, commonkmers, and CARMA3) have almost the same median F-measure but much tighter confidence intervals reflecting the fact that they perform more consistently and/or were evaluated more thoroughly. Based on this data I would be inclined to choose any of tools ranked #2, #3, or #4 over the tool ranked #1. Might it be possible to come up with some ranking scheme that is a composite of median F-measure and size of confidence interval? This is important for Figure 4, anyway; the confidence intervals in Fig. 5 are all pretty darn big and all about the same size, making me question how much real information is contained in that analysis.
2.	A very big issue is how the five “consistently highly ranked” performers (CARMA3, DiScRIBinATE, MetaPhlan2.0, OneCodex, and TACOA) were chosen. These represent five of the top seven tools in Fig. 4, and five of the top nine tools in Fig 5. However, by this criterion (ranking within the top nine in both analyses, say), commonkmers, DUDes, and MetaBin should also be included. This is important because people are going to remember the tools that were stated by name to be top performers, and it would seem that these five were arbitrarily selected. Not only is the cutoff (top nine) arbitrarily selected, but three tools that make the cut in both analyses were omitted. I think a more principled way of selecting top performers is needed. For example, look for a break in each distribution or state explicitly where the cutoff is (top five, top 10, etc.), and then name all the tools that make the cut in both analyses, not just some of them. Moreover, see my previous point about how the rankings might change if confidence interval were somehow factored in. This has lots of implications because much of the ensuing discussion treats the five selected tools specifically.
3.	There is obvious concordance between the lists resulting from the two ranking approaches (Figures 4 and 5) as they’re currently ordered; however, it might be nice to quantify this somehow (e.g., calculate “mean rank discrepancy”, i.e., what is the average difference in rank for a given tool between the two approaches?)

Methods are described in sufficient detail to replicate, especially since the scripts used to scrape data from the existing studies are provided via GitHub.
Validity of the findings: I find the methods applied to be appropriate and statistically sound. However, I take issue with some of the conclusions (specifically, the five tools selected as top performers), as noted in my previous comments.
Additional comments: Overall, I like this study --- it’s a good concept, the stats developed and methods of analysis used seem appropriate, there are some very nicely drawn figures, etc. I feel like the manuscript could be improved, however, by focusing more specifically on taxonomic assignment (much of the background or supporting information may be interesting, but isn’t necessarily relevant to the results or conclusions of the present study – e.g., information about metagenomic workflows generally). Also, the discussion of negative controls, types of metrics, potential usefulness of spike-ins, etc. is interesting and useful information, but not really necessary in order to understand the results of the study. The manuscript is a little weird in that respect --- it feels like a hybrid review paper/new results paper. Also, and most importantly, I think the rankings and subsequent selection of “top performers” needs to be reevaluated, as this takeaway is what most people will be interested in.
Here are some additional specific comments:
1.	Abstract, line 21, and main text, line 98: should also talk about robust Z-scores in addition to network meta-analysis. I actually think the robust Z-scores approach contains more data and is likely to be more informative than the network meta-analysis, and is weighted just as heavily in the conclusions drawn.
2.	Line 231: should there be a link to the project referenced?
3.	Lines 310-311: “The different software versions do not explain this discrepancy.” How do you know this? What sorts of analyses were performed to determine this?
4.	Figure 4A: in the legend, circle size is defined as “proportional to median F-measure value”; at the top of the figure, circle size is defined as “Number of F-measure estimates”. These sound like different things to me, I think this definition needs to be made clearer.
5.	Lines 350-352: “small numbers of direct comparisons between these methods” --- does this mean direct comparisons between these methods and something else? If so, what is that “something else”? Or do you mean small numbers of direct comparisons among methods (either these five or all methods more generally)?
6.	Supplementary figures 1 and 3 are never referenced in the main text.

In summary, I enjoyed reading this manuscript and I think the data analysis could eventually be useful to the community. I encourage the authors to spend time revising the manuscript, think about how the findings can be best presented in context (and providing only the most relevant context), and be very careful about how the findings are interpreted. In particular, I worry a little bit about the amount of information contained in the network analysis given the consistently large size of the confidence intervals in Fig. 5.


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: IDENTIFYING ACCURATE METAGENOME AND AMPLICON SOFTWARE VIA A META-ANALYSIS OF SEQUENCE TO TAXONOMY BENCHMARKING STUDIES
Review round: 1
Reviewer: 2

Output:
Basic reporting: The paper is generally clear. There are some problems with the structure as I describe below.
Experimental design: The experimental design is generally sound. There are some inconsistencies as i describe in my comments below
Validity of the findings: The paper fails to achieve a real conclusion. The conclusion can probably be either the recommendation of a tool (or set of tools) to profile microbial communities, or the striking inconsistency of available evaluations. The main conclusion can only be one of the two, but at the moment it is unclear what is the conclusions of the paper.
Additional comments: - Given the title of the paper and several of its paragraphs (incl "Our aim here is to make non-arbitrary software recommendations"), the reader will expect to find in the paper a recommendation based on the evaluation meta-analysis about which computational tools for his/her research. However, the work does not provide any real advice for this but it rather push the message that the evaluations are inconsistent and that no "winner" can be established. I think the paper should be either restructured to provide clear recommendations on the methods to use, or it should focus (starting from the title) on the message that the evaluations are inconsistent.

- The statistics in Figure 3A and Figure 3B are interesting and provide a nice summary of the available methods. It would be even more interesting if the statistics (specifically citations) of Figure 3B are correlated with the actual performances in the evaluations.

- The paragraph between lines 370 and 390 is weird to me. Here you describe how some methods work, which is disconnected with the rest of the results. I think this part should be removed and replaced with a better attempt at interpreting the results of the meta-analysis.

- I think that also the intro is a bit weird because it reports a mini-review of what amplicon sequencing and metagenomics are, whereas the focus of the paper is on a meta-analysis of microbiome profiling tools. I think the authors should focus the intro on the need to comparatively evaluate computational profiling tools and what are the challenges in doing it. There are many reviews of the microbiome sequencing field you can just cite for the interested reader.

- I very much agree with the three listed in the section "Overview of eDNA analysis evaluations". However, principle number 2 is not satisfied by most of the papers used in the meta-analysis. I think this is a very crucial point. Actually, it would be very interesting to highlight the performance of a tool in the evaluation in which the developers of the tool are in the author list with respect to the evaluations in which the developers of the tool are not in the author list. In any case, the authors need to be more consistent: if they truly believe that principle number 2 is right (and I fully agree with that), then they have to explain why they still use evaluations breaking this principle.

- I could be wrong, but the evaluation by McIntyre et al (for which a journal publication is available and should replace the bioRxiv citation) includes datasets used by other studies, and maybe even other considered evaluations. This would not be ideal and should be discussed. Again, I could be wrong here, but please double check.

- The use of "eDNA" is not standard and I think should be avoided. If you want a term covering both shotgun sequencing and amplicon sequencing you can just use terms like "microbiome sequencing"

Figure 1. There are several inconsistencies in this figure:
-  The "metagenomic reads" to "binning" link misses the metagenomic assembly part which is instead mentioned downstream of the binning. This is incorrect and misleading. CONCOCT and MetaBat requires contigs (and reads) as input, not binned reads only.
- not sure what "amplicon contigs" mean? If you refer to merging the paired end reads for the amplicon, it is not very correct to call it "assembly"
- closed-reference OTU assignment does not need OTU clustering as starting point
- I would not call PICRUST a functional assignment tool. Maybe "functional potential prediction tool"?

Introduction:
- when introducing metagenomic assembly, the authors do not cite the state-of-the-art tools (as reported by some of the benchmark papers they consider later on in the paper)


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

Input:
Title: IDENTIFYING ACCURATE METAGENOME AND AMPLICON SOFTWARE VIA A META-ANALYSIS OF SEQUENCE TO TAXONOMY BENCHMARKING STUDIES
Review round: 2
Reviewer: 1

Output:
Basic reporting: The English used throughout the manuscript is fine, although there were many more grammatical mistakes, formatting issues, etc. than I would have liked to have dealt with as a reviewer.
Experimental design: No comment
Validity of the findings: No comment
Additional comments: All-in-all, the manuscript is much improved — in particular by removing much of the attempt at a comprehensive review of metagenomics workflows, and by improving the methods used to select well-performing tools (and subsequent discussion of those tools).

I’m attaching a combined annotated PDF of the main manuscript and the supplementary material that the authors should review carefully. I’ll reiterate a couple of the more important points here, though.

1) The F1 measure fundamentally weights sensitivity and PPV equally. Thus, tools that have been developed with high PPV in mind (not necessarily sensitivity), such as marker gene-based approaches, suffer using the current ranking methodology. Indeed, it’s no surprise that the three top-ranked tools by PPV in Fig S5 are all marker gene-based (GOTTCHA, MetaPhlAn, and mOTU). I think the authors need to make it more clear that tools that are not "balanced" with regard to the sensitivity/PPV tradeoff are not going to do well in their ranking, and they may wish to call out top-performing tools (either by sensitivity or PPV) in their conclusion. They touch on this to some extent in the final paragraph of the conclusion, but I don’t think it’s explicit enough with regard to the points that need making here.

2) The colors used to represent the various benchmark studies in Fig 4 are different from those in other figures — confusingly so. This should be corrected for consistency.


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: LONG-TERM EFFECTS OF STRAW AND STRAW-DERIVED BIOCHAR ON SOIL AGGREGATION AND FUNGAL COMMUNITY IN A RICE–WHEAT ROTATION SYSTEM
Review round: 1
Reviewer: 1

Output:
Basic reporting: This manuscript is generally well-written using clear and professional English. The manuscript can be further improved regarding the following aspects: 1) define abbreviations when the term is firstly used, such as C (line 67) and TN (line 138). These terms might be commonly used in this area but it would be better to get consistent as the authors did a good job defining other abbreviations throughout the manuscript; 2) be explicit when describing methods or results. For example, in the abstract, the author said "soil samples of different aggregate fractions...". It should be explained what are different fractions. Is it based on sizes? Another example is "...CO2-emission-related fungi in specific aggregates (p<0.05)." What are the specific aggregates. 3) a few confusing statements containing misconceptions. In Line 64-66, it reads like bacteria are a branch of fungi, which is not the case.
Experimental design: The reviewer has a major concern about the experimental design because: 1) the author did not clearly describe how many types of biochars were applied in this study and how frequently applied. The reviewer guesses there should be two biochars according to the supplemental table, and the biochars were yearly buried instead of burying them only during the first or first two years as the crop biomass wastes were yearly generated so they will be yearly sent back to land as amendments. If so, the study would be interesting but becoming complicated as there are two types of biochars making influences at the same time without knowing which one had more impacts than the other. Also, as time goes on, the biochar/straw application rate, on an area basis, would increase since the carbon stayed on the land and accumulated year after year. These factors should be considered and discussed in this manuscript.

2) The manuscript lacks some important soil property descriptions as baseline or background data. For example, the soil texture plays an important role in soil aggregation. If the soil is clayey, many studies showed that soil amendment could not significantly increase aggregation. Is the soil used in this study sandy or silty? It would be interesting to know. Also, it would be better to have some data about the soil microbial data in year one or before the crop wastes were applied. Basically, the authors were comparing CS and CB with CF and CK during the sixth year and concluding what kind of impacts biochar or straw had on the microbial parameters. Without knowing the initial baseline data, the conclusions can be misleading.
Validity of the findings: The results and discussion are generally well-written and closely correlated but in some statements, it would be better to point out which figure or table supports such sentences. For example, line 266-268, "C:N ratio was among the strongest correlates of fungal community..." Which figure could prove this?
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: LONG-TERM EFFECTS OF STRAW AND STRAW-DERIVED BIOCHAR ON SOIL AGGREGATION AND FUNGAL COMMUNITY IN A RICE–WHEAT ROTATION SYSTEM
Review round: 1
Reviewer: 2

Output:
Basic reporting: The research topic is very important. Understanding the long-term impacts of biochar at field scale is critical for determining whether biochar can be used as an soil management tool in the future. At the same time, there is very few research looked into the impact of management practice on microbial community in soil water-stable aggregate fractions.
The manuscript is written in fine English. The related and latest references were sufficiently and properly cited in this manuscript.
The results of this study is presented clearly and well discussed.
Experimental design: The research question is clear and well defined. It's helpful to fill the current knowledge gap in biochar field application.
Methods are well described and sufficient details of methods were provided.
Validity of the findings: The experiment was properly conducted including control and replicates. Data provided is robust and can support discussion and conclusion in this manuscript.
Additional comments: Here a couple of specific comments:
Specific comments:
Line 64-66. Seems like some part of this sentence is missing or half modified. Please check.
Line 66. Provide nutrients for?
Line 87. The impact of biochar on soil properties and microbial activities were not always positive. Please modify this paragraph and include some literature with negative impacts, too.
Line 103. Please also provide some hypotheses here.
Line 116. Please state what pure nitrogen meant here. Was it total N or plant available N?
Line 138. Was soil inorganic C tested here?
Figure 1. This figure looks redundancy to me since the mean weight diameter of soil aggregates were reported already.


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: FUNCTIONAL POLYMORPHISMS OF THE APOA1/C3/A4/A5-ZPR1-BUD13 GENE CLUSTER ARE ASSOCIATED WITH DYSLIPIDEMIA IN A SEX-SPECIFIC PATTERN
Review round: 1
Reviewer: 1

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


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

Input:
Title: FUNCTIONAL POLYMORPHISMS OF THE APOA1/C3/A4/A5-ZPR1-BUD13 GENE CLUSTER ARE ASSOCIATED WITH DYSLIPIDEMIA IN A SEX-SPECIFIC PATTERN
Review round: 1
Reviewer: 2

Output:
Basic reporting: The present article aimed to investigate the association between six polymorphisms located in APOA1/C3/A4/A5-ZPR1/BUD13 gene cluster and dyslipidemia in a sex-specific pattern. I will list some important topics that should be taken into consideration by the authors and be better addressed before publication.
Introduction/Background Section has recent references from literature contextualizing the importance for the studied theme. However, the authors failed in explaining why they have chosen these specific genes and/or polymorphisms for further investigation. Lines 216-227 and 247-266 (Discussion Section) should be better replaced within the Introduction Section because these paragraphs could better explain the function of the selected genes and how they influence lipid levels contributing to the risk of dyslipidemia.
In line 112 (SNP Selection Section), the authors mentioned that these SNPs were selected according to minor allele frequency and based on previous studies; however, they did not mention the allele frequencies observed for these polymorphisms in the literature and did not provided references for this sentence.
The English language is adequate and clear in most of the text, but the authors should be more objective and concise mainly in the Results Section. Sometimes, it is difficult to follow the description of the results because of the amount of detailed information that was presented. The authors should try to reduce both the text size and the number of Tables by focusing in the presentation of the most relevant results. It would be easier to understand if the authors joined both the results of Chi-Square tests (Supplementary Tables 1 and 2) and logistic regression models with adjusted OR values (Table 2) in one single Table.
Table 1 should present the names of the genes together with the polymorphisms. It would be also interesting to have a complete description of other diseases and/or serum levels collected from the same individuals.
Experimental design: The authors focused in showing the results separately by gender, but it should be interesting to present the results in the total sample before stratifying it in males and females groups. They could be missing important information that is not gender-related.
The sample size is big (N=3850) and individuals were randomly selected from another study containing 21435 individuals. The authors did not explain if the sample size was previously calculated considering the frequencies of the studied SNPs.
The studied population is part of a project that aimed to evaluate the prevalence and risk factors associated with chronic diseases (as mentioned in line 93 – Materials and Methods Section). Therefore, the authors probably have access to more detailed information regarding the patients included in this study (e.g. other co-morbidities or serum levels). They should also provide the genetic association results for these other studied variables (mainly those related to dyslipidemia, such as cardiovascular disease, obesity, stroke or other metabolic-related diseases).
I would suggest for the authors to analyze the Hardy-Weinberg Equilibrium (HWE) both in the entire sample and separately by gender. Also, the results of HWE should be presented as Supplementary Table.
Validity of the findings: Regarding the Statistical Analysis Section, lines 127 to 129 described that the authors chose statistical tests considering distributions that followed or not the normality of the continuous variables. However, once they have previously transformed the numeric variables to logarithmic equivalents, they could use only parametric tests. Furthermore, lines 132-133 and lines 136-138 described the same analyses (Chi Square test + Logistic Regression Models with OR and 95% CI adjusted for covariates) and both sentences could be joined in one single sentence. Moreover, once there are different polymorphisms under investigation as well as several independent tests being performed for each SNP, the authors should have performed a correction for multiple testing (e.g. Bonferroni Correction).
The authors showed the association of the polymorphisms with Dyslipidemia (as categorical variables), but also the association results with lipid levels as numeric variables. However, Dyslipidemia was characterized considering the altered lipids levels and, in my opinion, it is redundant to present both results. Therefore, it would be better if the authors could focus their analyses by choosing only one type of variable (categorical or numeric). For example, it was confusing when comparing the results presented in Tables 2 and 3, not only because of the lack of concordance between the presented results, but also because they were not performed using the same genetic models.
The authors should better explain the genetic models that are under investigation for the association analysis. It was not clear why they are presenting different genetic models for different analyses. They should choose the best genetic models and use the same ones for all the analyses. For example, Supplementary Tables 1 and 2 showed the results of Chi-Square test associating polymorphisms and dyslipidemia in males and females, respectively, using Genotype and Allelic Models. However, Table 2 showed the adjusted OR values (and 95% CI) for dyslipidemia considering Dominant and Allelic Model. Also, while Table 2 presented the results using the Dominant and Allelic Models, Table 3 used only the Genotype Model.
The authors only performed association analysis considering the individual polymorphisms. Once they are located within the same gene cluster, they should provide the distance among them and also perform linkage disequilibrium and haplotype association analyses.
Additional comments: There are several points that the authors should address to make the manuscript acceptable for publication. I pointed some concerns and limitations and gave some suggestions for the improvement of the presented article.


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: FUNCTIONAL POLYMORPHISMS OF THE APOA1/C3/A4/A5-ZPR1-BUD13 GENE CLUSTER ARE ASSOCIATED WITH DYSLIPIDEMIA IN A SEX-SPECIFIC PATTERN
Review round: 1
Reviewer: 3

Output:
Basic reporting: Some sections in your manuscript need more detail and my suggestions can be found below.
- In line 66 it is stated that “genetic factors could also partially explain the development of dyslipidemia”. This has been already proven, probably not in Chinese population, but the genetic control of lipid parameters is well-documented (for example by Weiss et al (Nat Genet. 2006;38:218) or Dron et al (Curr Genet Med Rep. 2016;4:130 and Curr Opin Lipidol. 2018;29:133). Therefore, the statement should include this information.
- In the Background section we can find the description of the gene cluster APOA1/C3/A4/A5-ZPR1-BUD13 (from line 68 to 86) particularly involved in lipid metabolism. In the paragraph the cluster is written in different ways (in upper- and lower-case letters, with more or less components) and it should be uniformed. Upper-case letters are commonly used when referring to a gene and lower-case are used when referring to proteins.
- The first time a contraction is used, it should be described. Despite that, the description of a gene name may also help understanding its role, location, etc. For example, ZPR1 or BUD13 are used in the Background section and should be described. The APO genes cluster is very well-known but the other two genes are less recognized.
- The Figures should include the sample size per group in order to improve its self-explanation.
Experimental design: In the Methods Section there is a description of the study population, however some useful information regarding the participants is missing.
- when describing the study population it is not clearly stated whether the participants are being treated with any drugs that may affect lipid parameters;
- it would be very informative if the sample size for each group of dyslipidemia was showed there;
- it would be very interesting knowing whether there are any participants in more than one dyslipidemia group (for example presenting with increased TG and lowered HDL, or with high TG and high LDL). How many participants do not have any altered lipidic parameter?
Validity of the findings: Since the genetic control of lipid parameters is very well-known for being complex, and since the authors stated in the Background section that different polymorphisms in the cluster have been already described for being associated with dyslipidemia, is there any participant carrying more than one of the studied genetic variants? Is there an allele-dose effect meaning that those participants with more than one rare allele present with a worse lipid profile than those with 1 or none?
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: FUNCTIONAL POLYMORPHISMS OF THE APOA1/C3/A4/A5-ZPR1-BUD13 GENE CLUSTER ARE ASSOCIATED WITH DYSLIPIDEMIA IN A SEX-SPECIFIC PATTERN
Review round: 2
Reviewer: 1

Output:
Basic reporting: In the second paragraph of the Background Section, the authors cannot say that the polymorphisms are influencing the transcription factors and/or miRNA binding to their corresponding sequences unless they provide an in silico prediction for this inference. The same occurs when they say that the studied polymorphisms located in coding regions could lead directly to missense mutations and influence gene expression. There are available softwares (e.g. SIFT and Polyphen) that allow predicting if a missense alteration is pathogenic or tolerant for the protein function.
The authors should be careful with the gene names, because they must be written always in capital letter and in italic (please, correct both text and tables). Also, in the third paragraph of the Background Section, authors should use capital letters to describe the human protein names.
There are missing references for the last paragraph of the Background Section.
Experimental design: Table S1 describes the genotype and allele distributions of the six SNPs in the overall sample and separated by sex. The authors did not specify if the p-value is for the comparison of the distribution between males and females. Furthermore, there is no statistical description for this analysis. Also, the authors did not mention anything regarding the significant p-value (p=0.032) found for the rs651821 polymorphism. Is it really significant? The distribution of the frequencies between genders did not seem to be different.
Hardy Weinberg Equilibrium (HWE) should not be calculated only considering the individuals without dyslipidemia. The authors should consider the total sample (independently of the dyslipidemia) for calculating the HWE. Also, the authors must correct for “Hardy Weinberg Equilibrium” instead of “Hardy-Weinberg disequilibrium”. HWE could be placed in the same table as the genotype and allele distributions (Table S1), in an additional column.
Regarding haplotype analysis, Table 3 showed the results of the associations between dyslipidemia and haplotype blocks including all the studied polymorphisms, but the authors did not analyze if all of them were in linkage disequilibrium. In Table S4, the authors provided only the linkage disequilibrium results (D’ values) for pairs of polymorphisms, separately.
The authors only showed the association regarding the haplotype blocks and dyslipidemia. Why did they not provide association between haplotype blocks and Hypertriglyceridemia, Hypercholesterolemia, high LDL and reduced HDL, separately?
I strongly recommend the use of Haploview software for linkage disequilibrium and haplotype association analyses. The authors should mention the value of D’ chosen as cut-off for linkage disequilibrium analysis and also the distance among the polymorphisms in the haplotype blocks.
Validity of the findings: In the previous version, I have recommended for the authors to join Table S1, Table S2 and Table 2, but the authors decided to delete the Chi-Square results from the revised manuscript. It would be interesting to keep the results of the Chi-square test showing the associations between dyslipidemia and the polymorphisms (in the Genetic Model chosen for this analysis) and, in the same Table, add the adjusted OR values which were calculated by the binary logistic regression.
In Table 2, the authors could present only the adjusted Odds Ratio values. The footnotes should describe the covariates which were chosen for each analysis. They did not discuss the reasons or hypotheses why the associations of the SNPs with dyslipidemia are different when considering the total sample or males and females, separately.
In my opinion, the authors should investigate if the dyslipidemia-related diseases needed to be included as covariates for adjusting the OR values in the logistic model (even if this association have been previously described in a previous article). In the same way, do the authors have access to information regarding the use of medications? All the mentioned factors can clearly modify association between polymorphisms and metabolic variables and dyslipidemia.
Haplotype analysis was poorly described and discussed.
Additional comments: Once the authors performed other analyses as recommended by Reviewers, other concerns and limitations appeared. There are several genetic conceptual mistakes that should be taken into consideration and improved by the author. E.g. correct the gene names, Hardy Weinberg Equilibrium (instead disequilibrium), correct the calculation of the HWE, analyze other important covariates (e.g. medications and dyslipidemia-related diseases) that should be included for adjustment in the binary logistic model, improvement of the linkage disequilibrium and haplotype analyses, 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: FUNCTIONAL POLYMORPHISMS OF THE APOA1/C3/A4/A5-ZPR1-BUD13 GENE CLUSTER ARE ASSOCIATED WITH DYSLIPIDEMIA IN A SEX-SPECIFIC PATTERN
Review round: 2
Reviewer: 2

Output:
Basic reporting: The Background section has been modified following reviewer’s suggestions. Information regarding the genes in the apo cluster, which was previously detailed in the Discussion, has been moved to the Background section. However, now there is a clear lack of information describing the other two genes (ZPR1 and BUD13).
Experimental design: Some aspects have been corrected in the Methods section following reviewer’s suggestions. However, the paragraph describing the logistic regression analysis to detect associations with dyslipidemia, now is not clear enough.
In the Results section, the authors added information describing the amount of participants presenting with one or more dylipidemic components, following my suggestion. However the data is not clear enough presented. The authors stated “participants with one to four type of dyslipidemia were 763, 623, 184 and 16, respectively” but it would be very interesting knowing which types of dyslipidemia are shared when presenting either two, three or four.
Validity of the findings: The authors have added some data regarding a dose-allele effect, and apparently presenting with more than one genetic variant is associated with increased risk of dyslipidemia. In fact, in their cohort, a high percentage of participants (84.2%) present with more than 1 studied genetic variants. It would be very informative if the authors could add a table describing the different combinations of genetic variants found in their cohort, and the number of participants sharing each combination.
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: FUNCTIONAL POLYMORPHISMS OF THE APOA1/C3/A4/A5-ZPR1-BUD13 GENE CLUSTER ARE ASSOCIATED WITH DYSLIPIDEMIA IN A SEX-SPECIFIC PATTERN
Review round: 3
Reviewer: 1

Output:
Basic reporting: The authors presented the association results in total sample and also stratified by genders, as suggested before. Therefore, they should include this aim in the Abstract and also in the last paragraph of the Introduction Section (lines 106 -111: “This study assessed the association of tag and well-studied SNPs … with dyslipidemia in overall sample and stratified by gender in a large sample from a Han Chinese population”).
The authors performed haplotype association analysis, but there is no information regarding this topic (both description and results) in the Abstract Section.
The authors forgot to correct the gene cluster name in line 127 of the revised manuscript (Materials and Methods Section). The gene names should be written in italic. Also, in the third paragraph of the Background Section, authors should write the entire name of the human protein names in capital letters, and not only the first letter.
Text and English language should be carefully reviewed because there are several mistakes throughout the text (e.g. words written together; the use of singular instead of plural; sentences with lack of verb; please, correct the word PolyPhen (line 253 of the revised manuscript), etc).
I will point some other revisions to be correct in tables and figures:
- In the footnote of Table 1, please correct “Means (standard deviation)” instead of “Means ± standard deviation”. Please, include the abbreviation meaning of BMI in the footnote.
- In the title of Table 2, please correct “Association between genotypeS of the 6 SNPs and dyslipidemia in overall sample and stratified by genderS”. Furthermore, the word “LDL” is missing in the fourth column (“HIGH LDL”). Please, include the abbreviations meaning (SNP, BMI, OR and CI) in the footnote.
In the title of Table 3, correct “risk FOR dySlipidemia”. In the footnote, please explain the reason for p<0.008 (for example: “p<0.008 were considered as significant after Bonferroni Correction and were represented in bold”). Please, include the abbreviations meaning (SNP, BMI, OR and CI) in the footnote.
In Figure 1, the authors should include the order of the genes analyzed in the haplotype blocks to make it easier for the reader.
In Table S1, significant results (p<0.05) should be highlighted in bold and also be mentioned in the footnote.
In Table S2, please correct the word NORMAL (instead of nomal), in the third column. In Table S3, please include the abbreviations meaning (BMI, OR, CI) in the footnote and also highlight the significant results. Please, correct “number of risk alleleS”, using plural instead of singular in both title and table.
Experimental design: Regarding haplotype analysis, why did the authors not perform haplotype association analysis stratified by genders? In Figure 1, the authors could be less strict when choosing the cut-off value for D’. They chose D’>0.90 as cut off for considering the polymorphisms in linkage disequilibrium and, therefore, they could be losing important associations. If they choose D’> 0.75, rs5104 could be included in the haplotype block 2.
In Table S2, why did the authors present only the association results stratified for genders? They should also present the results found in the overall sample (as performed in Table 2).
Validity of the findings: The authors should compare the frequencies they found with other populations in the literature (for example with the frequencies described in 1000 Genome Project).
Additional comments: The authors have revised the manuscript and answered the suggestions that the reviewers have made. However, I will suggest other revisions to make it suitable for publication in Peer J.
The main concern for me at this point is the correction of the language and the presence of several errors found throughout the text. I will point some of them, but the authors should carefully review the manuscript before publication.


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

Input:
Title: FUNCTIONAL POLYMORPHISMS OF THE APOA1/C3/A4/A5-ZPR1-BUD13 GENE CLUSTER ARE ASSOCIATED WITH DYSLIPIDEMIA IN A SEX-SPECIFIC PATTERN
Review round: 3
Reviewer: 2

Output:
Basic reporting: Due to the changes in the Results section, the English should be revised throughout. For example, the verb tenses used when referring to Tables/Figures should be consistent; and some sentences are not clear or easy-to-understand.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: No additional comments


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

Input:
Title: FUNCTIONAL POLYMORPHISMS OF THE APOA1/C3/A4/A5-ZPR1-BUD13 GENE CLUSTER ARE ASSOCIATED WITH DYSLIPIDEMIA IN A SEX-SPECIFIC PATTERN
Review round: 4
Reviewer: 1

Output:
Basic reporting: The authors should correct the footnotes of both Table S1 and S2 “p values less than 0.05 were presented in bold” in place of “no more than 0.05”. Also, the authors should include a legend for the Figure 1, indicating the meaning of the color and the numbers inside the squares of the figure.
Experimental design: No comments
Validity of the findings: No comments
Additional comments: The authors clearly answered all the pointed questions, corrected the mistakes and improved the language. In my opinion, it is now suitable for publication in Peer J.


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

Input:
Title: FUNCTIONAL POLYMORPHISMS OF THE APOA1/C3/A4/A5-ZPR1-BUD13 GENE CLUSTER ARE ASSOCIATED WITH DYSLIPIDEMIA IN A SEX-SPECIFIC PATTERN
Review round: 4
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The authors have revised the manuscript answering the comments and suggestions made by the reviewers. In my opinion the manuscript has improved and is now ready for publication, therefore I have no additional comments.