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

Input:
Title: THE IMPORTANCE OF SPAWNING BEHAVIOR IN UNDERSTANDING THE VULNERABILITY OF EXPLOITED MARINE FISHES IN THE U.S. GULF OF MEXICO
Review round: 1
Reviewer: 2

Output:
Basic reporting: 1. Raw data, writing style, references, and structure of this paper conform standards of the PeerJ journal.
2. However, I find that presentation of data requires some work: e.g., L326-341 could be shortened and listing of species names should be avoided. Some results are uninformative: e.g., L357-360.
3. I like the presentation of Figure 3, which demonstrates disassociation between spawning behavior and life-history traits clearly.
4. Discussion is too long—The scope of information on 6 species (L500-588) is beyond that of this paper. These parts should be shortened or removed.

5. Specific comments
a) L 318-321: This paragraph should be moved to Methods.
b) L321-325: Data on no. of references per trait seem unnecessary. Please remove these.
c) L363-364: The data do not match numbers in Table 5. Please double-check.
d) Table 1: Use consistent notation for max age, max weight, and max length: Amax, Wmax, and Lmax, respectively.
e) Table 6: I suggest to move this table to supplemental material as data of this table are presented in Figure 4.
Experimental design: 1. Species selection: I thank authors for providing detailed procedures of species selection in supplemental material S1. They primarily selected species 1) those form spawning aggregations and 2) those are of conservation or fisheries importance. As a result of these selection processes, these data cannot be considered as random samples.

2. L250-252 and Figure 5: The non-random sampling may have impacted the analysis results in Figure 5. For example, the selected overfished stocks may contain primarily those associated with spawning aggregation. As such, the comparison of magnitudes of PC1 or PC2 between overfished and non-overfished stocks do not help to evaluate the additive explanatory power for spawning behavior traits.

To evaluate the additive explanatory power of spawning behavior traits, authors can conduct discriminant analysis or classification & regression tree with explanatory variables of 1) life-history traits vs. 2) life-history and spawning behavior traits.

3. Table 4: I am pleased to see data of lengths of spawning season for all study species. I am wondering if the results of PCA (Figure 4 and Table 6) may change if incorporating the no. of peak spawning months in this analysis.

4. Vulnerability analysis: I appreciate that authors provide detailed protocols of this assessment. I have two technical comments. First, as this assessment assumes equal weights between the two compound factors: extrinsic and intrinsic indicators, authors should provide reasons to justify this assumption. Second, with the a priori assignment of different weights for the individual intrinsic indicators, it disenables evaluating the species vulnerability with respect to these indicators. That is, this vulnerability analysis quantified stock vulnerability, but it could not evaluate vulnerability in association with spawning behavior. Related to the second comment I cannot find the association between vulnerability scores and overfishing statuses (L39-40) based on Figure 6.

Furthermore, the scope of vulnerability analysis seems to be much broader than the research question in this study. To make a coherent paper, I suggest to either to revise (by addressing the comments above) or exclude this vulnerability analysis.
Validity of the findings: The issues of 1) non-random sampling and 2) vulnerability analysis raised above may have impacted the results in Figures 4-6. In particular, the first issue makes it difficult to generalize the result. Thus, validity of findings cannot be justified at the present state.
Additional comments: Authors present a study that evaluates whether incorporating spawning behavior improves assessment of vulnerability of exploited marine fishes. Overall, this study provides abundant background information and high-quality of biological and management-related data for 28 marine fishes. I am especially pleased to see that authors apply published methods for quantifying spawning behavior traits among species.

However, some problems in the experimental design could have compromised the findings of this study. For the most, the procedures of species selection may have impacted the evaluation on discrimination of overfished statuses using the spawning behavior traits. Also, the design of vulnerability analysis disenable understanding the association between vulnerability and spawning behavior traits.

Although I appreciate authors’ work on data compilation, the species selection issues make it difficult to justify validity of findings. Consequently, I do not recommend to accept this paper. Nonetheless, I suggest that authors may revise or expand the vulnerability analysis on its own into a 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: THE IMPORTANCE OF SPAWNING BEHAVIOR IN UNDERSTANDING THE VULNERABILITY OF EXPLOITED MARINE FISHES IN THE U.S. GULF OF MEXICO
Review round: 1
Reviewer: 3

Output:
Basic reporting: The structure of this paper conforms to PeerJ standards and guidelines. It is very clearly written with objectives/hypotheses plainly stated. Authors stick to these objectives throughout the manuscript and present relevant results and conclusions.
The introduction is well written and documented with relevant data and sufficient, valid references.
Tables 1-3, 5 and 6 are very clear and necessary. (However, I do not see a significant positive correlation between Aggregation type and Am or Lm in Table 5 as reported in text). Table 4 is not necessary since spawning months are listed in Table 3 for each species. Similarly, figures are in general clear and well described however Figure 3 may be superfluous as it appears to be a graphical version of Table 5. Although it shows a clear picture of the numerical table, I did not find it necessary. The authors may consider deleting these two. In addition, the caption in Figure 1 lists the weighted influence on vulnerability for intrinsic and extrinsic indicators. This part of the caption is not necessary and does not add to the graphical representation of the figure. These values are listed in Tables 1 and 2 where they make more sense.
Supplemental literature is supplied. Because this paper does not utilize original raw data but rather constructs indices using data analysis published in previous reports, no original raw data is required.
Experimental design: This paper uses a novel statistical treatment of data that has been collected for 28 recreational and commercially valuable fish in the Gulf of Mexico (GOM) by various researchers and fisheries programs. Initially the paper determines the relationship between life history parameters used in traditional stock assessment, and spawning behavior of these fishes. Once the lack of a relationship between the two is established using correlation coefficients, they use a treatment that addresses the vulnerability of the fish during spawning and compare it to the current status of each species.   Although the methods used by the authors are standard statistical treatments, the addition of spawning behavior and extrinsic indicators into vulnerability models makes their approach unique, and arguably much more realistic and valuable as fisheries management tools. The simplicity of the model renders it potentially useful in places other than the GOM.
Validity of the findings: The Discussion and Conclusions are well stated, supporting and reflecting the Results. The examples of vulnerability for specific species used in the Discussion section were particularly helpful, illustrating the validity, practicality, and need of this new model which incorporates spawning behavior. The lack of spawning aggregation data, and in particular the relative abundance of fish at spawning sites, was highlighted as a gap in the vulnerability model. I found this to be overall an excellent and valuable paper.
Additional comments: No additional comments


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

Input:
Title: THE IMPORTANCE OF SPAWNING BEHAVIOR IN UNDERSTANDING THE VULNERABILITY OF EXPLOITED MARINE FISHES IN THE U.S. GULF OF MEXICO
Review round: 2
Reviewer: 1

Output:
Basic reporting: Comments for Biggs et al. revision:

I am pleased to see that authors have improved description on species selection and the weightings of intrinsic and extrinsic factors, which help to increase clarity of the results. Also, coherence between the PCA and vulnerability analysis is now improved. Furthermore, discussion flows well. As a result, I have no further comments, and would be happy to recommend this paper to be accepted.
Experimental design: I have no further comments.
Validity of the findings: I have no further comments.
Additional comments: I have no further comments.


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

Input:
Title: EFFECT OF MODIFIED POMACE ON COPPER MIGRATION VIA RIVERBANK SOIL IN SOUTHWEST CHINA
Review round: 1
Reviewer: 1

Output:
Basic reporting: The paper proposed to evaluate the use of fruit pomace, naturally and modified with EDTA, in the immobilization of Copper in areas close to rivers in different places in China. It is a valid assessment, in the sense of looking for “ecological” alternatives to minimize the impacts caused by human activities, with the use of residues for the treatment of contaminants and meets the premises of a circular and sustainable economy.
Initially I indicate that I identified several flaws in the writing of the article regarding the English language. Several sentences and words throughout the text are improperly written, which means that the article must undergo a complete revision of English (i.e. L47-48, 50, 60, 62-63, 139 ... and so on ).
The purpose of the work is clear, to test the different compounds of the pomace to assess their ability to immobilize or decrease the flow of copper from the soil to the rivers. However, several flaws are identified in the way that the results were presented and analyzed.
Experimental design: The experimental design used a column experiment, which is a methodology already well used to evaluate experiments of this type. However, some procedures and definitions can be improved.

First, why was lemon pomace used? Is it a common waste at the site? Easy to obtain? Or because other studies have already demonstrated the potential of this material? It is important to write down why this material was used specifically.

Between lines 92-94, is it described the characteristics of what reagents was used? I could not understand this paragraph. I believe it is only a description of the reagents used. It is? If so, write that paragraph better.

L.102 “Within 50 m from the riverbank, a typical area was selected”. What would these typical areas be? What are the human activities at these collection points? Was there the same activity at all points? Agriculture? Industry? Residence? I saw that table 2 indicates the cultures? You should mention this at this point.

L115-116. You don't need to repeat all locations again. Suffice to say that it was mixed with all the samples.

L120. “The column experiment was used to simulate the migration and morphological characteristics of copper”. The authors write "copper morphological characteristics". I think the most appropriate term would be geochemical characteristics.

L 130-131. “Then the exchangeable copper, carbonate, iron_manganese oxide, and organic matter of soil parts were determined.” After all, were four or five fractions analyzed? In addition, the method proposed by Tessier is an already established method of sequential extraction, but there are many other more modern methodologies in the literature that could have been considered.(i.e. (Gimeno-García et al., 1995;
Siregar et al., 2005; Silveira et al., 2006, Perlatti et al., 2014).
Validity of the findings: L. 140-141. The authors use the term "lemon dregs". Standardize the identification in the text. Pomace or dregs?

L.142-144. “The adsorption performance of lemon dregs modified by EDTA was stronger for copper ions, because of the acidic nature of lemon dregs after modification will enhance its adsorption capacity for copper ions Shen et al. (Shen and Lei, 2016; Shen et al., 2017b) ”. This statement is not based on the literature, since it is known that the greater the acidity, the greater the mobility of copper in the soil. Perhaps should be better to emphasize the chelating capacity of EDTA rather to address the greater acidity.

L 147-150. Very speculative stretch. It is very difficult to evaluate this statement without having provided the complete physical characteristics of the soil, such as clay, silt and sand content. These elements have the greatest influence on the dynamics of the soil solution and adsorption processes.

L172-173. “The results showed that the addition of lemon pomace could enhance the ability of soil to accumulate copper”. This seems a little obvious, given the well-known affinity of copper with organic matter. The authors could better explore the correlation between the increase in TOC levels with the addition of lemon pomace to identify if there was an increase in carbon levels, as it is a component with high affinity with Cu.

L 176-178. “EDTA modified fruit residue can form soluble metal complexes with heavy metals, which improve the migration rate of heavy metal ions in soil (Dariush et al., 2017), and ultimately enhances the adsorption capacity of copper”. This statement presents antagonistic data. If EDTA favors metal solubilization, how can it favor adsorption? The right thing would be to increase mobility and consequent leaching. I suggest that the authors explain this phenomenon better.

L187-188. Considering that the pH has a lot of influence on the mobility of copper, the authors should better explore this statement. Perhaps the fact that all soils are slightly alkaline may have aggravated this process.

L 188 - 191. This passage is confusing and difficult to understand. What would be a moderate correlation between Cu and carbon?

In general, I consider the section 2.4 very confusing. I suggest rewriting, pointing out specifically the role of each property (TOC, Surface area, carbonates, oxides) in Cu geochemical behaviour.

L 198-199 - “The absorption of copper by natural soil depends on the physical and chemical properties of soil and clay content.” I agree with that statement. But where are the data on soil clay content? And wouldn't it be adsorption instead of absorption?

L 201-205. This statement is very speculative. It must be associated if the presence of lemon pomace increases the contents of TOC in the soil, which in turn can help in the adsorption and immobilization of the metal.

Conclusions: The conclusion is not supported by the results presented. It is clear that CEC has a great influence on the adsorption of ions in the soil, since it is the general capacity of the soil to retain not only contaminants, but also macro and micro-nutrients. The authors must better correlate the natural components of the soil, the changes provided by the addition of the materials and the specific effects of these on the geochemical processes that influence the mobility of copper.
Additional comments: I believe that the paper has the potential to be published, as it presents interesting data and addresses a relevant topic.
However, the analysis and discussion of the data must be improved, with a more robust scientific basis. There are many studies that demonstrate the effects of pH and organic matter content on the geochemical dynamics of copper, and I believe that the authors should focus more on that.

With a little more depth discussion and better organization of topics, I believe it can be published.


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: EFFECT OF MODIFIED POMACE ON COPPER MIGRATION VIA RIVERBANK SOIL IN SOUTHWEST CHINA
Review round: 1
Reviewer: 2

Output:
Basic reporting: The article should include a sufficient background to demonstrate why author choose modified materials for solidification of copper in a riverbank soil. I suggest that you improve the description at lines 63- 68 to provide research significance for your study (specifically, you should expand upon the practical value or research significance of your work). In addition, the English language expression should be improved to ensure that an international audience can clearly understand your text.
Experimental design: The manuscript "Effect of modified pomace on copper migration via riverbank soil in southwest China" is a study about leaching behaviour and remediation of heavy metal in riverbank soil in southwest China. The authors attempted to suggests a new modified materials for solidification of copper in a riverbank soil. This paper falls within the subject of this journal and in spite of the interest of present study, the manuscript needs to be improved. I doubt if a PVC column with a diameter of 1.6 cm and a length of 20 cm in the experiment can be used to simulate actual soil conditions. Is it a reference to the existing standard soil column simulation method or self-designed, if it is a self-designed experimental method, please explain why you used this method. Is it reasonable？
Validity of the findings: In results and discussion section,  I suspect that pH value is not a key factor affecting migration of copper in soil? At the same time, I am very confused that the adsorption capacity of the original soil to the copper is weak, is it compared with the addition of modified materials? The amended soil has a high adsorption capacity for copper, the authors should give the mechanism explanation what is the form of EDTA modification pomace? I suggest that infrared spectrum analysis should be used to explain the reaction mechanism of functional group participation before and after adsorption, as well as before and after modification of pomace.
Additional comments: The manuscript "Effect of modified pomace on copper migration via riverbank soil in southwest China" is a study about leaching behaviour and remediation of heavy metal in riverbank soil in southwest China. This paper falls within the subject of this journal and in spite of the interest of present study, the manuscript needs to be improved:
1. The authors attempted to suggests a new modified materials for solidification of copper in a riverbank soil. Nevertheless, the authors do not explain in the Introduction Section whether the pomace or modified pomace application in copper remediation has practical value or research significance?
2. Is there heavy metal pollution, especially copper pollution, in the research area? Are raw materials (pomace) fully available?
3. Is it meaningful to use ID1.6cm, and length 20 cm of soil column experiment? The authors should give an sufficient explaination why they used this soil column and experimental method.
4. What is the form of EDTA modification pomace? how EDTA modified or combined to raw materials (pomace)?
5. Inaccurate professional descriptions of discussions (Line 156). the exchangeable state or form?
6. The initial pollution copper concentration is set to 2000 mg/L, at a 20 cm length of soil column, it is difficult to make a sufficient reaction between pollutants and soil particles, thus, the further discussion is meaningless.
7. There are some language expressions that are difficult to understand, please revise them carefully in the entire manuscript.
After these improvements the paper could be reviewed again to assess if it is suitable for publication in Peer J, however, in the present conditions I recommend Major Revisions.


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: EFFECT OF MODIFIED POMACE ON COPPER MIGRATION VIA RIVERBANK SOIL IN SOUTHWEST CHINA
Review round: 3
Reviewer: 1

Output:
Basic reporting: See comments to the editor
Experimental design: See comments to the editor
Validity of the findings: See comments to the editor
Additional comments: See comments to the editor.


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: EFFECT OF MODIFIED POMACE ON COPPER MIGRATION VIA RIVERBANK SOIL IN SOUTHWEST CHINA
Review round: 3
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: After the author's revision, the methods  are described with sufficient information.
Validity of the findings: After the author's revision and explanation, the result of this paper is reliable and the conclusion is appropriately stated.
Additional comments: After the author's revision and and explanation, the manuscript of article meets the Peer J. criteria and can be accepted for publication in this 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: PYDAMAGE: AUTOMATED ANCIENT DAMAGE IDENTIFICATION AND ESTIMATION FOR CONTIGS IN ANCIENT DNA DE NOVO ASSEMBLY
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript by Borry et al. is well presented. It is written in straightforward and clear English. The authors introduce the manuscript with ample background information and context so the non-expert reader can follow what is going on.

The paper is coherent and well-structured in the conventional and unsurprising article format, and figures and tables are beautiful and mostly easy to understand and relevant. The working hypotheses, while implicit, are clear enough. Overall, I applaud the authors for writing such an easily-understandable article on a technical topic.

There is plenty of relevant references with few exceptions
Experimental design: Generally, the central research question is well defined, meaningful and fill a clear and explicitly stated gap in knowledge. Technically, the paper is fairly clear in its intentions and methods, and described in detail enough to replicate. Code and data is publicly and easily available.

Comments:
Line 148: It is not clear why the lambda value approaches a chi-square distribution with two parameters. If this is well-known, a reference would be needed. If this can be deduced mathematically, please include a proof or explanation.

Line 169: In the model selection, the use of Nagelkerke’s R-squared raises some issues. Its concrete value is difficult to interpret, and it’s not clear why it is preferred over simpler measures like receiver operating characteristic (ROC) area under curve (AUC), F1-statistic, or similar. In particular, given that Table 1 shows that the selected model is inferior according to the balanced accuracy – a measure which is much more straight forward and presumably more relevant, the discrepancy between R-squared and accuracy should at least be investigated and discussed.

Furthermore, there is no discussion about recall/precision tradeoff. Any choice here would be arbitrary, and many choices could be defended, but it is still relevant. For example, suppose different models could tradeoff recall/precision at different rates between recall and precision. Then different tradeoffs would favor different models to appear superior. The authors could address this by either deciding balanced accuracy was the relevant criterium and fit the models to optimize this instead of sum of squared error, or by showing AOC curves or similar for each model to explicitly show the tradeoff.
Validity of the findings: Underlying data has been provided, and reproducibility is greatly improved by a code repository containing all relevant code.
Overall, the conclusions are well supported, with some caveats.

Comments:
Lines: 209-221: While the contigs assigned as ancient were analyzed, there is no mention of the remaining contigs. According to the implicit hypothesis, these should presumably be from clades associated with soil metagenomes, and not gut metagenomes, but this is not investigated. A simple comparison between the ancient versus modern contigs in order to show that the populations are clearly different would greatly strengthen the author’s argument.

Lines 254-256: It is not clear from the article that PyDamage is “highly reliable”, in the sense of having a near-100% accuracy. The only place where I can find direct measures of accuracy is Figure 4, from which it is very difficult to tell e.g. 95% accuracy from 99% accuracy. If this statement should be included in the conclusion, there should be at least one place in the article where the authors directly measure accuracy and show it in a clear figure or in text.

Lines 273-275: This is not clear from the figure. If the figure is cited for this claim, I would re-make figures 3 and 4 in a way where it is possible to read this more precisely.

Figures 3-4: Besides the general comments above, it is not clear from either the figure nor the legend that the numbers above each of the 10 boxes represent damage frequency. Please add this to the legend or the figure itself.
Additional comments: Lines 48-50: Is this supported by a reference? If so, please add it. If not, please explicitly state it is a hypothesis.

Lies: 94-96: For clarity, and if word count allows it, consider mentioning explicitly why GC content could be an interesting variable for PyDamage. Considering that the conclusion explicitly mentions that it turns out to not be important, it would be nice it you explicitly stated why you investigated it. Perhaps something like “because PyDamage predicts based on C->T transition frequency, we expect GC-content to impact the available number of possible C->T transitions, and hence could influence the predictions of PyDamage”.

Line: 198: Specify that “filter for” means retain, not remove. This is ambiguous in English. In this particular case, it could mean either retain long contigs (because PyDamage works better on long contigs), or retain small contigs (because you expect damaged ancient DNA to result in short contigs).

Lines 199-200: It is not clear what you mean by successful estimation. Do you mean that the program worked? Or that it was correct? And how do you know it was correct?


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: PYDAMAGE: AUTOMATED ANCIENT DAMAGE IDENTIFICATION AND ESTIMATION FOR CONTIGS IN ANCIENT DNA DE NOVO ASSEMBLY
Review round: 1
Reviewer: 2

Output:
Basic reporting: Basic reporting adheres to journal requirements.
Experimental design: No comment
Validity of the findings: No comment
Additional comments: This paper by Borry et al. tackles a really key issue in ancient metagenomics, and as such is clearly of very high interest to the community at large. As the authors state, there are a number of existing software that attempt to tackle this type of issue, but none that do this terribly well, particularly on meta-genome de novo assemblies. I was really impressed with the software, and the paper is written well. I don't have any major critiques, but have listed a few minor questions/comments below:

Line 129: 'non-default k-mer lengths...' How were these chosen?
Lines 172-173: This is a bit of an awkward sentence
Table 1: I can't seem to find where you give a justification of retained model choice.
Figure 3: The values in grey boxes need to be mentioned in the legend.
Lines 262-263: I'd be interested in more delimitation of the parameters affecting prediction accurracy. Some of this can of course be pulled from the figures, but it would be nice if these were explicitly stated.

Lines 76-77: You mention in the introduction some of the existing software, and some of the issues associated with them. Particularly HOPS and mapdamage2. I'd be interested to see some comparison of the sensitivity/scaleability issues you mention.


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: DISTRIBUTION, SOURCE, WATER QUALITY AND HEALTH RISK ASSESSMENT OF DISSOLVED HEAVY METALS IN MAJOR RIVERS IN WUHAN, CHINA
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: This manuscript deals with seasonal variation of heavy metals using various assessment developed by other researchers as indicators to evaluate pollution status of river pollution, China. By analyzing and interpreting the metal concentrations in surface water samples, the manuscript presents metal distributions and conclusive pollution indices to show the current status of this region. Major revision of this manuscript is needed and some information needs to be added.
Abstract: looks like reporting the results based on various analysis and it doesn't give any significant findings through this research. It is even better to show some analytical values (ranges), which give some highlight to this study.
Introduction part doesn’t sufficient, concentrates on the methods used to identify this research and at the present state reads as a methodology. This also does not seem to be a thorough literature review and the provided methods that they have adopted in the study. If the authors could attempt to improve the introduction by an in-depth review of these methods, this study will serve as a valuable reference to the scientific community. Any type of metals contamination studies didn’t done this study area? Then how this study will make a difference from the previous works. Add some research cap to justify this current research.
The precision of the analytical process is not shown. Also, is this dataset adequate to describe the temporal changes of the water quality? In terms of sampling strategy, I would like to understand the reasoning behind stations location. Are these stations related to point and non-point pollution sources? A table with the location of each sampling station and possible impact on the water quality contamination will be helpful for the readers not familiar with the study area. Also locate of point sources in Fig.1 with different symbol.
Seasonal variation and statistical analysis of metals important so, author need to discuss in detail about this process and bring more evidences. Is there any lithogenic impact to the metals concentration? Is the rainfall on that particular season will contribute to the dissolution of minerals during weathering? How about the summer season? Another aspect is the lack of correlation between metals with the geology as well as contamination sources. Many of the sentences relating these metals need to justification or scientific discussion. PCA – explain accumulation of metals (3 groups) with different sampling stations.
Conclusion need to rewrite by showing salient observation through this research. Author need to add some statement on the quality management strategy plans based on this assessment.
I recommend from the authors to read and cite the following articles that will improve the manuscript drastically

Figures 7 station id not clear.


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: DISTRIBUTION, SOURCE, WATER QUALITY AND HEALTH RISK ASSESSMENT OF DISSOLVED HEAVY METALS IN MAJOR RIVERS IN WUHAN, CHINA
Review round: 1
Reviewer: 2

Output:
Basic reporting: This manuscript presents interesting data on the heavy metals at 30 sampling points in major rivers in Wuhan. The authors interpret their heavy metals results by applying a couple of statistical techniques to better understand their data along with distribution, sources, water quality, and health risk assessment. Although the statistical analysis method is nothing new, the study presents the health risk directly and vividly via GIS technology. For the sensitive water quality protection, this study states important data not only at a local scale but all over the world and the amount of data about the new river system is worth publishing. However, there are some issues needed to be considered prior to publication.
Experimental design: In statistics analysis section, Spearman’s correlation analysis was performed. However, the correlation coefficients mentioned in Line 243 are different from it in the figure 4. Please do check and classify.
Validity of the findings: 1 The discussion section in the present form is relatively weak and should be strengthened with more details and justifications.
2 When using acronym for the first time, complete spelling should be written first. It is mentioned in line137 that "… inserted into the surface rivers of Wuhan by IDW method to show the spatial distribution. …." IDW should be used complete spelling first.
3 Inverse distance weighted method is not detailed enough, should be appropriate reference to the relevant research literature.
4 The language and writing should be further improved across the whole manuscript.
Additional comments: 1 The abstract should present the data summary of the study results, such as the content, HPI and HI values of heavy metals over the surface river.
2 In the introduction section, heavy metals need more illustration, which is in direct connection with the aim of the study. Moreover, the description of Wuhan should be merged with the study area section.
3 The paper deals with the heavy metals with little description of the pH, DO, EC, TN, and TP, ignoring the importance of these parameters for the distribution of heavy metals in the rivers. I recommend a more appropriate description of the physicochemical characteristics and its influence on heavy metals.


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: GENOME-WIDE ASSOCIATION STUDY OF YIELD COMPONENTS IN SPRING WHEAT COLLECTION HARVESTED UNDER TWO WATER REGIMES IN NORTHERN KAZAKHSTAN
Review round: 1
Reviewer: 1

Output:
Basic reporting: Wheat is one of the most important crops in the world. water stress has a large effect on total grain yield and yield components. It has been widely studied in Wheat. In this work, Amalova et al. performed the genome-wide association study on the basis of 179 wheat genotypes which were genotyped based on a 20K SNP chip. They identified several QTL responsible for eight yield components and further revealed the homology of highly stable candidates. However, I do not think the current manuscript is suitable for publication. The identification of marker traits association is interesting but the writing and description quality need to improve.
Experimental design: Experimental design and analysis are well designed and described. Hence I would like to suggest adding the candidate gene annotation analysis if possible.
Validity of the findings: The results are good and innovative
Additional comments: The yield and yield components are the well-studied topics in wheat, while the review literature did not summarize the already known genes and QTLs for those yield components. hence it is sugested to add a table for known genes/QTLs for already known genetic information of yield components and cite in the introduction section.

To improve the readability of the manuscript, it needs extensive editing for linguistic corrections for spelling mistakes, ambiguous sentences, and grammatical mistakes. For example
L48 “umber”=number, L158-161 and so on.
L54 “QTLs were identified for…..” need to make clear as “identified as stable QTLs” otherwise need to write criteria that how were they identified.
L134 what A.I. stands for? It is suggested to write in full
L141: “shown in Raw data”, Please make a table number and indicate that table number here as “supplementary Table 1” etc.
The units should be in international standard format for example L181 “P<1E-03”=P < 1x10-3


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: GENOME-WIDE ASSOCIATION STUDY OF YIELD COMPONENTS IN SPRING WHEAT COLLECTION HARVESTED UNDER TWO WATER REGIMES IN NORTHERN KAZAKHSTAN
Review round: 1
Reviewer: 2

Output:
Basic reporting: The article is poorly written, and ambiguous words have been used throughout the manuscript.
Experimental design: The Authors used a Randomized design instead of a Randomized Black design which is recommended for the field experiments. I do not think their design is appropriate for this type of field study.
Validity of the findings: The findings are valid but the writing style does not meet the minimum requirements of scientific publications.
Additional comments: A major modification is needed to accept it for publication.


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

Input:
Title: GENOME-WIDE ASSOCIATION STUDY OF YIELD COMPONENTS IN SPRING WHEAT COLLECTION HARVESTED UNDER TWO WATER REGIMES IN NORTHERN KAZAKHSTAN
Review round: 1
Reviewer: 3

Output:
Basic reporting: The present study aimed to explore the QTLs associated with yield traits in Spring wheat collection. Authors conducted experimentations in under irrigated and rainfed conditions in Northern Kazakhstan. Such types of studies are present-day requirements. I found this study informative and should be considered for publication by addressing my few minor changes.

1. Manuscript needs moderate language control.
2. In title authors are saying they performed GWAS, while in the abstract and whole manuscript, They are stating the identified QTLs. I will suggest the authors to use the word "genetic basis" in place of QTLs.
3. In the abstract Authors stated that "The study suggested
4. Please add this statement at the start of line 109: Characterization of germplasm is a prerequisite for the breeding activities as it provides novel variations that can be used for the marker-assisted breeding of crops (10.3390/genes11010036).
9 QTLs for HD and 11 QTLs for SMD were presumably novel genetic factors" I will suggest the to replace QTL with SNPs
4. I will suggest the authors construct Manhattan plots for HD, SMD, PH, PL, SL, NPS, NKS, and TKW.
5. Conclusion section should be reconsidered
Experimental design: Experimental design is fine.
Validity of the findings: Satisfactory
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: GENOME-WIDE ASSOCIATION STUDY OF YIELD COMPONENTS IN SPRING WHEAT COLLECTION HARVESTED UNDER TWO WATER REGIMES IN NORTHERN KAZAKHSTAN
Review round: 1
Reviewer: 4

Output:
Basic reporting: The presented idea is not very clear and written in a dispersed way. The manuscript needs extensive English editing. I must suggest reviewing the manuscript from English native speaker.
The manuscript provided sufficient literature that helps the reader to know the background of the conducted study.
The manuscript structure is poor and need extensive changes. The portions (Materials and methods, Results and Discussion) of the manuscript must be presented in the order like; description of the field data, genomic DNA isolation, diversity parameters, structure analysis and finally association and MTAs analysis.
The manuscript presented the relevant results to the hypothesis.
Experimental design: The conducted study falls into the aims and scope of the journal “Peer J”.
The presented research question is well defined, relevant and meaningful. The conducted study identified genomic regions that will be beneficial to the wheat breeders working in the dry arid zones in Kazakhstan.
The proposed investigations are conducted rigorously as they repeated the field experiments for three years. Other ethical standards are not applicable to the conducted study.
The manuscript does not describe the methods with sufficient detail and are also presented randomly. Materials and method section of the manuscript need to be improved. It is suggested to perform normality test regarding the field data. It is not described that how differences among the tested accessions estimated and which software was used.
Validity of the findings: The current study reflected the identification of candidate parents for wheat breeding activities in the dry arid zones of Kazakhstan. It also identified the genomic regions related to important agronomic traits. The findings of the conducted study will pace wheat breeding activities and will be helpful in the food security.
The manuscript presented and provided all the underlying data related to support the hypothesis. The data is presented randomly in an inaccurate way. It is always suggested to present the data in a systematic way. We must present the field data first and then molecular data. In molecular data, first describe diversity parameters, STRUCTUE, UPGMA, PCoA analysis etc, and then perform QTLs/MTAs analysis. It is suggested to revise the materials and method, results and discussion section of the manuscript in the proposed pattern.
The conclusion portion of the manuscript can be improved.
The authors proposed the identified QTLs as novel factors but they are not validated yet. First they should recommend it for validation and after that they can be used in molecular wheat breeding programs.
Additional comments: Your manuscript entitled "Genome-wide association study for yield components in spring wheat collection harvested under two water regimes in Northern Kazakhstan" has now been reviewed. Overall, the manuscript does not look sound and suitable for publication in the present form and need major revision but I suggest some major changes that must be properly addressed before acceptance/publication. The manuscript provided sufficient data but presented inaccurately. The manuscript is poorly written and extensive English editing is suggested. The data in the manuscript is presented randomly in an inaccurate way. The discussion portion needs to be revised. The conducted study observed no QTL for PH although PH and 1000 seed weight are the traits that most of the times showed QTLS in most of the studies. This is very interesting to me. It is suggested to read 2 or 3 good quality papers and observe how the data is presented. Some of the minor changes are also highlighted in the manuscript that needs to be addressed properly.


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: PROTEOMIC ANALYSIS REVEALS SOME COMMON PROTEINS IN THE KIDNEY STONE MATRIX
Review round: 1
Reviewer: 1

Output:
Basic reporting: The study by Yang et al., conducted a meta-analysis of previously published proteomic studies of urinary stones and crystals to identify the most common stone matrix proteins. They classified these common proteins according to their predicted functions and pathways/biological processes, and, finally, examined the over-expression of three of these proteins in the renal tissues of kidney stone patients using immunohistochemistry as a complementary technique to proteomics.

Generally, the manuscript is well-written, and the authors used clear, intelligible, and professional English for the majority of the manuscript. There have been, however, a few places where the authors need to express themselves more clearly. I have listed these cases for the authors under the “Comments for the author” section.

The presented study is short in its format, which is acceptable given the goals presented by the authors in their abstract and introduction of summarizing the literature and presenting common biomolecules to enable future nephrolithiasis research. The authors provided a sufficient background for their work and described how their work fit with the previous knowledge in the field. Overall, the study represents a cohesive body of work and is self-contained, with the presented results directly linked to the study goals described by the authors in their introduction.

The manuscript also follows the “Standard Sections” structure of the journal to a large extent except for the “Conclusions” section which was forced into the main text with a single sentence (please refer to my comments under the “Validity of the Findings” section for more details). The figures are of sufficient resolution, labelling, and description (except for the cases that I have listed for the authors under the “Comments for the author” section).

I commend that the authors shared the raw data in the main text tables and in the supplementary tables; the data were clearly described in the manuscript.

The authors have provided a “Statement of Ethics” in their manuscript and placed this statement under the “Materials and Methods” section as well per the journal’s instructions. The experiments in this study seemed necessary (to provide complementary data for the proteomic inferences) and ethical (the tissues were only obtained from patients undergoing nephrectomy) and the authors removed patient-identifying information from the text and supplementary/raw data. However, the “Informed consent form” and the “The approval document” are provided in Chinese. Please provide translated versions of these documents in English.
Experimental design: The present study represents an original primary research and fit under the “Biological Sciences” and “Medical Sciences” scopes of the journal. The authors explicitly stated their research questions, how their study contributes to filling the gap in previous knowledge of the field, and hence how their results are meaningful and relevant to the field.

The authors sought to follow high technical standards with regard to their immunohistochemistry experiment, controlling for baseline expression of their target proteins by including renal paracancer tissues in their protocol. The authors however didn’t justify the exclusion of uromodulin and albumin from the same experiment. Even if the patterns for these proteins were lower expression relative to the paracancer tissues or no expression at all, these results are useful for the comprehensiveness of the study (and at least serve as additional controls for the experiment). If conducting these additional experiments is not possible, the authors should mention this as a caveat of the study.

The study is generally meeting the ethical standards and the screening of >3,000 publications for the authors’ meta-analysis represents a rigorous investigation.

The authors need to describe their methods to sufficient detail and provide relevant information for other investigators to reproduce their results. For example, the authors need to provide more details on how they did the enrichment analyses and how they calculated the false discovery rate, indicating the specific software functions used for the different steps. They also need to clarify the criteria of exclusion for the 3,115 records displayed in Figure 1. Finally, the authors need to provide references for all the entries in Table 2 (as an additional column in the table).
Validity of the findings: As I have discussed under the “Basic reporting” section, the conclusions of this study were partially discussed by the authors at the end of the “Discussion” section (Lines 280-285), while the “Conclusions” section itself was forced into the main text with a single sentence. While the authors explicitly state their conclusions and link them to their original research questions, one of these conclusions is not really addressed by this study (beyond referencing literature). To elaborate, the authors didn’t do any experiments that address that there is a “common process” (Line 280) during stone formation. If the authors were referring to “inflammation and immune response” (Line 288) as the “common process”, then how would they fit the role of albumin (as a nucleator of calcium oxalate dihydrate) in this “common process”? If the authors chose to provide this “common process” as a speculation in the conclusion section, they should explicitly state so.

The authors provided all the underlying data, their experiment was controlled (although other controls could have been provided as discussed under the “Experimental design” section), and their bioinformatic analyses seemed thorough. However, in the absence of more details on the methodology behind the enrichment analyses (as discussed under the “Experimental design” section), it’s hard to assess the soundness of the statistics applied.
Additional comments: Line 136: What is the meaning of “protein files”?
Line 185: The citation linked to “Thurgood et al.” is missing from the text. This happened in several places throughout the manuscript. Please make sure to provide the citation numbers in the text.
Line 208: What is meant by “calcium iron”?
Line 235: “It was surprising that only four of the 20 proteins showed medium to high expression in kidney”. Which table/figure is referred to here?
Line 258: The sentence should start with “In virto”
Lines 258-266: Please use the past tense when reporting the results from previous studies (in these lines as well as other places throughout the manuscript). Also, please revise the text for the cohesiveness between the sentences.
Lines 280-282: The expressions in these lines are very vague; please rewrite more clearly
Figure 2: Panel A should read “Detection frequency”
Figure 3: Please spell out “BP” and “MF”. Also, please describe in the legend the “Rich Factor” and the “FDR”, indicating the FDR cut-off used on the figure.
Figure 4: the x-axis should read “-log(Pvalue)”. Why the FDR was not used here? What is the cut-off?
Figure 5: Please spell out “Ctrl”


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: PROTEOMIC ANALYSIS REVEALS SOME COMMON PROTEINS IN THE KIDNEY STONE MATRIX
Review round: 1
Reviewer: 2

Output:
Basic reporting: No commenrts
Experimental design: No comments
Validity of the findings: No comments
Additional comments: The manuscript entitled "Proteomic analysis of stone matrix: A window to the pathogenesis of urolithiasis' is very well defined, written, and presented.  The authors used bioinformatic tools and identify 20 top proteins to explore their biological function and performed Immunohistochemistry of the three main matrix proteins.

The results are very nicely presented, and I don't have much to add however, the author may like to reduce the introduction section of the abstract and may increase the results section.

 Thank you


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: PROTEOMIC ANALYSIS REVEALS SOME COMMON PROTEINS IN THE KIDNEY STONE MATRIX
Review round: 1
Reviewer: 3

Output:
Basic reporting: Introduction:  The introduction suffers from some grammatical errors.  It should read “Kidney stone disease is a common…” and “10 to 12% of people”.  I will not comment further because this text will likely change with the restructuring of this paper.
Title:  The title of the paper appears to be quite similar to reference 26.  The title does not describe the work done by the authors, but work performed in other labs.  A better title may be “Calgranulin and osteopontin in calcium oxalate urolithiasis: Roles for inflammation and immune response.”
Overall review: This paper begins and reads as a review article with the inclusion of primary histologic data on osteopontin and S100 proteins in kidney biopsies of only calcium oxalate stone patients.  The paper would be better restructured to start with the author’s primary immunohistochemistry findings in the renal biopsies from calcium oxalate patients.
The authors probed for calgranulins and osteopontin, likely due to prior interest in these critical proteins in their research lab (References 28, 36).  These proteins have been studied for quite some time for their role in kidney stone disease and inflammatory diseases throughout the body (calcific-arthritic conditions, cardiac calcifications).  The primary findings of increased expression of these proteins in the kidney tissue of calcium oxalate stone patients have been largely overshadowed by the extensive literary review of kidney stone matrix proteins.
The articles reviewed should only include those that look at the stone matrix from calcium oxalate kidney stones because this is consistent with the immunohistochemistry study population.  Thus, there would be 12 studies of interest (References 8, 9, 10, 15, 16, 19, 20, 21, 22, 24, 25, and 26), choosing to study only the calcium oxalate data only when a paper reports more than one stone type. Comparing all types of stone matrix into one summation obscures the differences that are present in varying stone disease states. It actually impedes urolithiasis research and the goal of understanding disease pathology by assuming that all stones are formed due to the same type of inflammation and immune responses.  The authors might also consider the removal of the protein-crystal binding studies (Ref 13, 14) from the comparison since these are derived from non-stone forming participant urine samples.
Again, the authors hide their primary findings behind the review of proteomic papers.  This is doing a disservice to the actual findings in the paper. Little data has been generated on kidney stone patient biopsy samples due to the limited availability of such samples.  Is there any other quantitative data that may be relevant to the patients studied in this paper (Demographics, comorbid health histories such as hypertension, diabetes).  Additionally, is there any way that the immunohistochemistry work could be quantified.  Regions of interest (ROI) quantification is commonly available in software packages.  This would strengthen the visual observation of increased expression of osteopontin and S100 proteins.
Line 204:  Biologic GO and KEGG annotation is a good set of analysis.  It should be repeated with the proteins derived from studies relevant to your calcium oxalate stone forming study population (Likely the 12 studies highlighted above).  This new comparison may or may not change the proteins listed in Table 2.
	Line 274:  Osteopontin’s role in stone disease is confounded by the level of phosphorylation of the protein which may be decreased in kidney stone formers.  Tamm Horsfall protein similarly will have a different behavior depending on the level of glycosylation (sialic acid) on the protein (Viswanathan, et.al.).
	Lines 276-277:  The discussion of albumin as a monomer or a polymer is a confusing way to describe a moderate sized protein.
Experimental design: Seems OK.
Validity of the findings: Suggestions have been made above.
Additional comments: This manuscript needs a major rewrite and restructuring before it should be published.  Suggestions have been made above.  Grammar and incorrect or missing words need to be cleaned up.


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: PROTEOMIC ANALYSIS REVEALS SOME COMMON PROTEINS IN THE KIDNEY STONE MATRIX
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have adequately addressed my concerns, provided additional experimental results and supplementary data, and made appropriate and thorough revisions in the resubmitted manuscript.
Experimental design: No further comments.
Validity of the findings: No further comments.
Additional comments: Thank you for providing this resource and for thoroughly addressing my comments.


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

Input:
Title: PROTEOMIC ANALYSIS REVEALS SOME COMMON PROTEINS IN THE KIDNEY STONE MATRIX
Review round: 2
Reviewer: 2

Output:
Basic reporting: No further comment
Experimental design: No further comment
Validity of the findings: No further comment
Additional comments: The manuscript is quite improved now and I don't have any other 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: BETWEEN DARKNESS AND LIGHT: SPRING HABITATS PROVIDE NEW PERSPECTIVES FOR MODERN RESEARCHERS ON GROUNDWATER BIOLOGY
Review round: 1
Reviewer: 1

Output:
Basic reporting: An interesting systematic review of the literature about obligate fauna in springs. The goal of the authors is to discuss the potential role of springs as an integrant part of the subterranean realm.
Experimental design: In general, I think that the systematic review part is methodologically sound. I have just a few suggestions/requests for clarification:

1) I don’t think it is appropriate to call this study a meta-analysis (e.g., in the abstract “In this systematic evidence review and meta-analysis ...”). My point is that the authors didn’t combine the results of multiple scientific studies, e.g. providing a weighted average from the results of the individual studies in the form of a forest plot or similar.
Also, the aim of the GLM analysis is to assess the relationships between features of the selected documents and the occurrence of stygofauna. This is, to my view, an analysis that falls within the domain of scientometrics/bibliometrics.
Long story short, to avoid misleading a reader, I would refer to the study as a ‘systematic literature review’ throughout the text and in the abstract.

2) Since both authors performed the “screening” of literature (sensu PRISMA, see Line 143), it would be important to report a measure of inter-operator agreement (e.g. Cohen’s Kappa)-

3) In the PRISMA diagram, I would separate “Screening” and “Eligibility”.

4) The different GLM model structures are not very clear (to me) from reading the text. Could the authors please report them also as formulas to help the reader visualize the models? See, e.g., Point 5 in Zuur & Ieno 2016

Zuur, A. F., & Ieno, E. N. (2016). A protocol for conducting and presenting results of regression‐type analyses. Methods in Ecology and Evolution, 7(6), 636-645.
Validity of the findings: (-L190): Please briefly report on model validation (see point 7 in Zuur & Ieno, 2016 mentioned in the previous comment).

-(L224-254): The entire first section of the discussion is a repetition of the result. Please omit/integrate it in the result section.

-(L263-266): Can the authors support this statement with data? How many studies have been missed by excluding these keywords?

-L230: “Our results revealed that typical stygofauna have been reported in springs more frequently in recent years.”

Maybe I’m missing something—see my previous doubt on the model structure—but I don’t think the authors have tested this. To test this, one should summarize the proportion between the paper reporting stygobionts in spring and the total number of papers for each year, and model this proportion over time (proportion ~ year).
Additional comments: I found the text generally well-written, except for the discussion that has somewhat less structure (... also note that the entire result section is repeated once in the discussion). I would suggest rethinking it by discussing each of the hypotheses tested in the same order as these are reported in the results. Also, I provided below additional minor suggestions.

I hope this revision will be useful.

Yours sincerely,
Stefano Mammola

-L77: About ecotones underground, I suggest also cite the classic:

Prous, X., Ferreira, R. L., & Martins, R. P. (2004). Ecotone delimitation: Epigean–hypogean transition in cave ecosystems. Austral Ecology, 29(4), 374-382.

-L83-84 the sentence is not fully clear, I suggest rephrasing to “the etymology of the word ...”

-L88-L91: I don’t fully understand the link between the first and the second sentence (i.e. permeability of the ecotone at night; presence of stygobionts) unless the studies mentioned (Bressi et al. 1999; Fišer 2019; Manenti & Barzaghi 2020) refer to nocturnal samplings. For example, in terrestrial subterranean systems, we observed a similar pattern, but only after confronting diurnal and nocturnal occurrence patterns (https://subtbiol.pensoft.net/article/28909/).

-L94: Can the ‘use of habitat’ be considered as a ‘trait’?

-L105: Within the frame of ‘caves as laboratory’ topic, references cited are very old. This subject has been covered extensively in these works:

Mammola, S. (2019). Finding answers in the dark: caves as models in ecology fifty years after Poulson and White. Ecography, 42(7), 1331-1351.

Mammola, S., Amorim, I. R., Bichuette, M. E., Borges, P. A., Cheeptham, N., Cooper, S. J., ... & Cardoso, P. (2020). Fundamental research questions in subterranean biology. Biological Reviews, 95(6), 1855-1872.

( I know, it’s shameless self-promotion... but see an interesting read:
https://scientistseessquirrel.wordpress.com/2020/02/04/what-should-you-do-when-reviewer-2-says-cite-my-papers/  )

-L128-133: I would suggest omitting this introduction to systematic literature in the methods. Doesn’t add much to the story.

-L140-142: Could worth mentioning also the computer/and internet browser, to ensure theoretical reproducibility of the search. See:

Pozsgai, G. et al. 2020. A comparative analysis reveals irreproducibility in searches of scientific literature. BioRxiv.

-L204: is 45,375 species correct? Does it refer to unique species or 45,375 mentions of species (mostly repeated)? Please clarify

-L208: faunal assessments → faunistic?

-L209: multiple fields of study → do you mean the “Multidisciplinary” category in WoS? Or an ad hoc definition for this paper when a paper deals with, say, ecology and evolution?

-L307: there is also the classic case of proteus washed out from caves in Carniola, that could worth mentioning. See:

Aljančič G. (2019). History of research on Proteus anguinus Laurenti 1768 in Slovenia. Folia Biologica et Geologica 60, 39-69.

-L317-318: Developments in this area have been very fast, and I think could worth mentioning more recent literature, e.g.:

West, K. M., Richards, Z. T., Harvey, E. S., Susac, R., Grealy, A., & Bunce, M. (2020). Under the karst: detecting hidden subterranean assemblages using eDNA metabarcoding in the caves of Christmas Island, Australia. Scientific reports, 10(1), 1-15.

White, N. E., Guzik, M. T., Austin, A. D., Moore, G. I., Humphreys, W. F., Alexander, J., & Bunce, M. (2020). Detection of the rare Australian endemic blind cave eel (Ophisternon candidum) with environmental DNA: implications for threatened species management in subterranean environments. Hydrobiologia, 847, 3201-3211.

Yonezawa, S., Nakano, T., Nakahama, N., Tomikawa, K., & Isagi, Y. (2020). Environmental DNA reveals cryptic diversity within the subterranean amphipod genus Pseudocrangonyx Akatsuka & Komai, 1922 (Amphipoda: Crangonyctoidea: Pseudocrangonyctidae) from central Japan. The Journal of Crustacean Biology, 40(4), 479-483.

-L365-366: This happens also with terrestrial organisms (e.g. Dolichopoda). See the previous point on comparing day/night

-L392 romero → Romero


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: BETWEEN DARKNESS AND LIGHT: SPRING HABITATS PROVIDE NEW PERSPECTIVES FOR MODERN RESEARCHERS ON GROUNDWATER BIOLOGY
Review round: 1
Reviewer: 2

Output:
Basic reporting: This is a clear review of the literature on subterranean organisms in springs.  I do think that the review would be improved by at least a basic review of springs, especially the types of springs, e.g. artesian, gravity, etc.  This is not to broaden the scope but because I think spring type has a major impact on what orgnaisms one can find in the spring, and the reader should at least be made aware that not all springs are alike.  In my detailed comments on the manuscript, I suggest some major references.
Experimental design: The study design is fine but I wonder if it would be possible to add country or continent or latitude to the information.  Just as surface and subsurface biodiversity varies geographically, so too should spring biodiversity and may be a major factor.
Validity of the findings: See above
Additional comments: None of these comments should be taken to mean the study is not worthwhile as it stands, and they are offered as possible improvements


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: BETWEEN DARKNESS AND LIGHT: SPRING HABITATS PROVIDE NEW PERSPECTIVES FOR MODERN RESEARCHERS ON GROUNDWATER BIOLOGY
Review round: 1
Reviewer: 3

Output:
Basic reporting: I deeply regret to say that the manuscript is not correctly written. It should require a profound revision of the whole text. There are several grammatical errors throughout the text, and several sentences are unclear, and inserted in parts of the manuscript where the final conceptual frame is going definitively lost. Literature references are not sufficiently provided, and some seminal works on groundwater-fed springs not mentioned at all. The title is catchy, but the Introduction section is not clearly arranged. The whole Results section is doubled in the Discussion, generating confusion to the reader. Several statements are not adequately supported in the Discussion and Conclusion sections. The hydrogeologic dynamics governing the groundwater flow discharge at the spring outlet is neglected, as well as its strong effects on the presence of stygobites at the spring "surface". Under my opinion, and based on my knowledge of stygobites, some concepts are wrong, or incorrectly given to a reader, and body of evidence of such statements not given.
Experimental design: No comment
Validity of the findings: I regret again to say that I was unable to find any positive impact or novelty into this manuscript. The results remain to me inconclusive, and predominantly based on speculation.
Additional comments: Dear Authors,
I think that much more effort is needed for a good Opinion Paper, together with a profound knowledge of the groundwater biology and ecology. Unfortunately I was unable to read a well told story.
I would suggest the AA. to better address their discussion, by providing at first robust bodies of evidence when basic concepts related to the Groundwater Ecology are razed to the ground, which are not only coming from past research, but also from very recent scientific works.
I have provided a .pdf file by given some suggestions and improvements.
Best regards


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: BETWEEN DARKNESS AND LIGHT: SPRING HABITATS PROVIDE NEW PERSPECTIVES FOR MODERN RESEARCHERS ON GROUNDWATER BIOLOGY
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Thanks for this revision. My main concerns on the reporting of methods have been addressed. A few additional suggestions:

L88: the the

L91: during night → at night

L96 please remove spp.

L144: change “high-impact” to “peer-reviewed” (WOS also contain papers published in rather low impact journals)

L162-167 Please consider breaking down this very long sentence

L172 please remove also.

L243: conservation concerns → conservation issues?

L296 what is a “major subterranean biologist”?

L297: please consider spelling out the name of the lab

L368-369: To me, the example of marine caves comes a bit out of the blue. Please consider rephrasing (e.g. “A parallel can be made between springs and...”)

L391: GDE is mentioned only once. No need to define the acronym

- Figure 1: Besides the change in light conditions, don’t you expect differences in predation risk between day and night? At night there can be a largely new set of nocturnal predators.


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: BETWEEN DARKNESS AND LIGHT: SPRING HABITATS PROVIDE NEW PERSPECTIVES FOR MODERN RESEARCHERS ON GROUNDWATER BIOLOGY
Review round: 2
Reviewer: 2

Output:
Basic reporting: Pass
Experimental design: Pass
Validity of the findings: Pass
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: CLINICAL AND PERINATAL OUTCOMES OF FRESH SINGLE-BLASTOCYST-TRANSFER CYCLES UNDER AN EARLY FOLLICULAR PHASE PROLONGED PROTOCOL ACCORDING TO DAY OF TRIGGER ESTRADIOL LEVELS
Review round: 1
Reviewer: 1

Output:
Basic reporting: 1.	Table1 –Table5, the footnotes need to be revised. It was unclear what tests were performed to get these p-values and why some of the groups has more than one p-value. These information should be added to the footnote and the main text. In table3, the numbers for ‘Rate of blastocyst formation’ do not seem right. For continuous variables, it would be better to add the statistics listed in the table, eg.maternal age, mean ± SD.
2.	Figure3 does not have titles or labels.
Experimental design: 1.	In the statistics section, the authors listed all the tests; however, it was not clear enough when the tests were performed. To perform a one-way ANOVA test, there is no need to specify the reference group.
2.	In line 194-195, when stating multiple comparisons corrections, it is not appropriate to say it is the ‘comparison between continuous and dichotomous variables’, instead, using something similar like ‘Tukey’s multiple comparison correction was used for pairwise comparison among groups’. In addition, fisher’s exact test or Chi-sq test is not the appropriate test for multiple comparisons for categorical variables; the authors should consider using other corrections like Holm correction. Moreover, can the authors explain the reason why the criteria of statistical significance was set to p<0.008?
3.	In line 197, the authors stated that ‘multiple regression analysis was used to test the correlation of related variables’, however, it was unclear what type of regression was performed here, and it is confusing about the purpose of performing the regression. If it is to test for the correlation between variables, Spearman’s correlation or Pearson’s correlation coefficient should do the work. If it is to determine the relationship between live birth and its predictors, univariate logistic regression is suitable, but the wording should be revised.
4.	The authors utilized ROC curve to see if E2 levels could be used to predict outcomes, however, this is not appropriate since a Receiver operating characteristics only tells you how the sensitivity and specificity will trade off, and should be thought of as supplemental to the main analysis, rather than the primary analysis itself. The authors might want to fit logistic regression models for these outcomes and calculate the cutoffs based on the predicted probability. The corresponding results should also be revised based on the new results.
5.	It would be interesting if the authors can list the sensitivity and specificity achieved on that optimal cut-off point.
6.	In line 198, it seems like the authors performed logistic regressions to calculate the ORs and their 95% CIs, however, it would be better if the authors not use the ‘rate’ or ’percentage’ word as the outcome, otherwise it will be confusing if this is a continuous rate outcome or binary outcome. This language should be revised throughout the paper.
Validity of the findings: The results after revising the statistical method should be updated accordingly.
Additional comments: This is an engaging article with robust methodology that purposefully questions our knowledge of
the subject, however, further revision is required for this manuscript.


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

Input:
Title: CLINICAL AND PERINATAL OUTCOMES OF FRESH SINGLE-BLASTOCYST-TRANSFER CYCLES UNDER AN EARLY FOLLICULAR PHASE PROLONGED PROTOCOL ACCORDING TO DAY OF TRIGGER ESTRADIOL LEVELS
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: In a multivariate system, often one variable may not be able to predict the system accurately. In the future studies, it would be beneficial to build a predictive model using:

1. more sophisticated modeling approaches capable of capturing non-linear relationships
2. multiple variables
Validity of the findings: no comment
Additional comments: This is a very well written manuscript describing the prediction accuracy of E2 level on various pregnancy characteristics of patients in single-blastocyst-transfer cycles. The results are presented clearly and the scientific English language has added to the strength of the manuscript. This study provides invaluable experimental data for researchers to build more sophisticated models such as neural networks in order to predict different pregnancy factors. For this reason, I highly recommend that the experimental data to be publicly available if possible.


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: CLINICAL AND PERINATAL OUTCOMES OF FRESH SINGLE-BLASTOCYST-TRANSFER CYCLES UNDER AN EARLY FOLLICULAR PHASE PROLONGED PROTOCOL ACCORDING TO DAY OF TRIGGER ESTRADIOL LEVELS
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Thanks the authors for addressing my questions. The current version looks great to me.


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

Input:
Title: A BRIGHT IDEA—METABARCODING ARTHROPODS FROM LIGHT FIXTURES
Review round: 1
Reviewer: 1

Output:
Basic reporting: The article 'A bright idea? Metabarcoding arthropods from light fixtures' is an interesting study that proposes a new tool for identifying dead insects collected from light fixtures in urban environments. The text is well written with a correct English easy to understand. The authors have well-posed the problem, which to my knowledge and in its context is very relevant. The conclusions correspond well to the results obtained. Overall, the figures are well presented, except for figure 4 which should be redone.
Experimental design: The authors have shown clarity in their approach with a level of detail that facilitates its reproducibility. However, the approach has some limitations in capturing the full diversity of species, favoring more flying arthropods and smaller specimens. However, the authors have well discussed its limitations in the discussion. All additional data were accessible and well-informed.
Validity of the findings: The results from the study are robust enough to validate the feasibility of metabarcoding to identify dead species collected in light fixtures.
Additional comments: No general comments for the author as I find the article in its current state to be publishable.

Some minor comments:

Line 52: Remove the comma.
Fig. S5: Give the meaning of the gray color bars


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

Input:
Title: A BRIGHT IDEA—METABARCODING ARTHROPODS FROM LIGHT FIXTURES
Review round: 1
Reviewer: 2

Output:
Basic reporting: I consider the manuscript to be very high quality, well written, and with an appropriate coverage of the literature. The article is well structured with good quality figures and tables.
The raw data is already in suitable repositories, and all the necessary information and metadata, that would allow external validation, is available.
Experimental design: The manuscript is comprised of original primary research that falls within the scope of the journal. The study aims to explore a novel source of biodiversity data and the research question is clear and well defined.

The methods are robust, current, and described with sufficient detail to replicate. The inclusion of the scripts for data analysis further enhances replicability & reproducibility. However, there are a few instances where details are missing that would otherwise allow the reader to better understand the rationale behind certain processes.

1. Why have the author's increased the sequencing length of Read 1 (line 142)? Whilst it's clear that these cycles are available to be used, as no external indexes are involved, the benefit of doing so for a 421bp amplicon is not. Does this lead to an increased number of reads being successfully merged?

2. What is the rationale for deducting 2x the numbers of reads found within the negative controls from each of the other samples? I understand the need to account for background contamination (e.g. errors caused by index hopping) but would be concerned about removing rare taxa. Especially as you highlighted that the methods employed are biased towards the recovery of larger and more abundant taxa.
Validity of the findings: Between the figures for the manuscript and the ample supplementary files, the authors have provided all underlying data from the study. However, as an aside, it would be nice if some of the taxonomic information had made it in to the main body of the manuscript. While the aim of they study is to assess a novel sampling strategy, the underlying purpose is to monitor biodiversity and this seems to have been lost in relegating that information to then end of a table in the supplementary materials.

The conclusions are well stated and supported by the results. The authors haven been open about the limitations of the study and the strength of conclusions that can subsequently be drawn.
Additional comments: This is a nice, well-thought-out study exploiting an overlooked source of urban invertebrate samples that could easily become a large-scale citizen science project.


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: ENSEMBLETAX: AN R PACKAGE FOR DETERMINATIONS OF ENSEMBLE TAXONOMIC ASSIGNMENTS OF PHYLOGENETICALLY-INFORMATIVE MARKER GENE SEQUENCES
Review round: 1
Reviewer: 1

Output:
Basic reporting: Writing is hard to follow. Some essential background is missing.
Experimental design: I was not able to understand the goals of the algorithms or the details of how the algorithms work.
Validity of the findings: Some implicit claims are not adequately supported by results.
Additional comments: Abstract
The term "resolution" should not be used here. It implies improved distinction between categories, overlooking the fact that most microbes have not been named by taxonomists, so the default assumption should be that more names would mostly be false positives. Thus, tuning to predict more names is quite likely a bad idea, while it naively sounds good. "Number of predicted clade names" is more understandable, but may still be misleading for readers who are unaware that a missing name is often the correct prediction.
"Fidelity" is an infelicitous term in this context. It is not helpful to define accuracy=fidelity without further explanation.
The claim that users can prioritize "accuracy", however defined, is not supported by the results in the paper. Defining and measuring accuracy of taxonomy prediction from sequence is ferociously difficult and controversial, as shown by several cited papers including a couple of mine.
A more supportable statement would be something like "Thresholds can be set by the user to choose between obtaining more predicted clade names at lower ranks, some of which may be false positives, versus fewer predictions where missing predictions may be false negatives."
However, this trade-off can already be made by using one good algorithm, e.g. setting the bootstrap cutoff with the RDP Classifier, which begs the question: what then is the motivation or benefit of using the ensemble classifier described by the paper? There are many examples in the machine learning literature where using meta-classifiers or ensemble classifiers achieve higher accuracy, but simply implementing an ensemble classifier does not guarantee that better results will be achieved.
The design and reporting of the validation on protist data is problematic. The description reads as a series of software commands without adequate motivation and interpretation. Metrics are not clearly defined, e.g. the crucial concept of a "misclassification" is defined only in a figure caption. It is misleading to define a misclassification as a disagreement with the consensus, because the prediction could be correct and the consensus is wrong, or both the prediction and the consensus could be wrong.
Better would be to start with a discussion of the design of the validation, which metrics are used and why, and a discussion of how the results should be interpreted. However, this probably cannot be done in a very informative way because the correct taxonomy is not known, leaving us with the ferociously difficult and controversial problem of measuring taxonomy prediction accuracy from sequence. Presented as an illustration of how the method can be used in practice, noting conflicts between different classifiers as a plausibility argument for taking a consensus would be fine, but the text should be written carefully to avoid over-stating claims about advantages of this approach which are not supported by the results.
Given the opportunity for misunderstanding, the Abstract and main text should make clear that there is no evidence that the described ensemble classifier is more accurate by any metric compared to using one algorithm such as the RDP Classifier.
I had some difficulties following what exactly the package does and why it does it, so I may have missed some salient points. With that said, it seems to me that the ensemble classifier described in the paper adds a layer of complication for no benefit to the user. If so, perhaps the paper could be improved by focusing on nomenclature reconciliation.
There are many possible approaches to combining databases with different sequences, different nomenclatures and different annotation file formats. One is to convert annotations into the format required by particular software packages (say, the RDP Classifier) then run a package on the same set of query sequences with all databases, and as a final step attempt to reconcile the predictions. Another is to reconcile the databases into one set of sequences with a harmonized set of annotations. Then a package such as the RDP Classifier can be run directly on the combined database. Some consideration of these alternatives and motivations for the authors' choices would be useful.
As best I understand it, the authors take the first approach, with the additional feature that multiple classification algorithms can be combined (I suspect this is overcomplicating the solution). I suggest that the second approach would be a more valuable contribution, because it would be plug-and-play in a wide variety of pipelines. I'm a good example -- I'm a command-line / C++ hacker and occasional Python coder with a strong aversion to R because I find it deeply obscure, so I would need a very strong reason to consider using an R package. By contrast, combined and reconciled databases for popular markers such as 16S, 18S and ITS with a choice of annotations in widely-used formats (SILVA, usearch, RdRP Classifier...) would be plug-and-play replacements that could be instantly adopted by users of many different software environments. I would happily recommend such databases to usearch users.
[82] internal transcribed spacer gene (ITS)
ITS is not a gene, it is a transcribed but non-coding region between genes, similar to an intron.
[85] inferred operational taxonomic units (OTUs) or amplicon sequence variants (ASVs)
An OTU as such cannot be "inferred" as it is an artificial construct. An ASV is a special case of an OTU construction where the sequences are clustered at 100% identity, not a separate category.
[86] as representatives of individual microbial species within a community
An OTU or ASV may represent multiple species having the same sequence, this is common with shorter ASVs such as 16S V4.
[88] Because an organism’s taxonomy is often linked to its ecological niche within an ecosystem, assigning taxonomic identities to inferred OTU/ASV sequences imparts ecological significance to genetic data. Taxonomic assignment thus represents a critical component of all marker gene sequencing studies.
This is doubtful in the case of most prokaryotes and fungi. Consider for example E. coli -- two strains of the same species may share only 60% of their genes with radical consequences for phenotype, e.g. pathogen vs. benign or symbiote. This distinction cannot be made on the basis of a typical 16S amplicon, or even with the full-length 16S gene in many cases. Since taxonomy can often be predicted only to around genus or family rank, I doubt that much can be inferred about ecological role from marker gene taxonomy prediction. This should be re-written, or citations provided to support the claim.
Also, most microbes in databases such as SILVA and UNITE are known only from marker gene sequencing, for the large majority there are no direct observations of phenotype other than a sequence. This issue should be noted, and also that it exacerbates the limitations of inferring anything reliable about phenotype or ecological role by comparing amplicons with sequences in a reference database. Further, most microbial species (genera...) have not been named, which limits the ability of taxonomy prediction to illuminate characteristics of microbes in the sample. This should also be mentioned.
[97] However, different representatives of a single marker gene are often found in different reference databases
This sentence confused me for a while. I think it is trying to say that if two databases for a given gene are compared, they will have some sequences in common and others that are found only in one database. Re-word for clarity.
[102] Meanwhile, a large collection of 18S rDNA reference sequences, some of which are not included in pr2, is also included alongside 16S rDNA reference sequences
This seems irrelevant. The fact that SILVA has both 16S and 18S does not create practical problems for the user as far as I can see. If you have 18S data, use the SILVA 18S reference. Or, use both -- maybe your primers targeting 18S weakly amplified some 16S as well.
[104] Unfortunately, the disparate naming and ranking conventions employed by silva and pr2 force analysts of 18S rDNA data sets to use either pr2 or silva exclusively for their taxonomic assignments.
Not if the user has some coding skills. The goal is for the authors' package to make it easier for the user to combine multiple databases.
[132] ...flexibly compute ensemble taxonomic assignments
While the term "assignment" is commonly used for computational taxonomy predictions from sequence alone, I suggest that "prediction" is better to distinguish a tentative prediction by an error-prone algorithm from expert examination of phenotype observed in vivo or in vitro.
[186] RDP 16 train set
Should be "16S rRNA training set", though "training set" is their jargon and simply "reference" might be better.
[187] Additional assignment algorithms and/or reference databases can be incorporated so long as they are formatted according to the documentation provided with the ensembleTax R package.
Should say more about what "formatted according to the documentation" entails here. Isn't incompatible formatting exactly the problem the ensembleTax package is intended to solve? If the user has the skills for this, why would they use ensembleTax -- they could e.g. run the RDP Classifier in the data2 package.
[135] databases'
Misplaced apostrophe, should be database's.
[Table 1] Taxonomy. An organisms taxonomic affiliation
Missing apostrophe, should be organism's. This definition introduces a new undefined term (how is affiliation different from assignment?) and is regrettably circular.
[203-218]
I was completely baffled by the description of the taxmapper algorithm. I was not able to deduce what problem(s) it is designed to solve -- does it try to reconcile (a) nomenclature only without looking at the sequences, or (b) nomenclature+sequence?
This section should be re-written to give more background. Describe the problems to be solved in reconciling taxonomic nomenclatures. Given illustrative examples, and explain how your approach solves each case. If the method does not consider sequence, then this should be mentioned because sequences introduce new types of conflict between databases which are not apparent from nomenclature alone.
The term "ASV" should not be applied to reference databases, because they were not necessarily obtained by amplicon sequencing (e.g., could be obtained using an HMM to find the gene in a full-length genome assembly).
Rank names are capitalized (Genus, Species...); should be lower case.
If the goal is to reconcile nomenclature+sequence annotations, then presumably, the first step is to identify reference sequences in common between two databases. How is this done, exactly -- presumably by alignment, but what is the method? What happens if one sequence is a substring of the other (i.e., the match has 100% identity but is not full length)? What happens if the best match is close but not exact, say 99%, or the best match is much lower, say 80% id?
[206] What is "lowest possible rank"? There is the lowest rank generally in a given database (e.g., species for SILVA or genus for the RDP training set), but this is not necessarily the same as the lowest rank annotated for a given reference sequence, which might be say family because genus and species are not named.
Note that the interpretation of a blank name is usually not clear -- is this is a positive prediction by the database that the sequence belongs to a novel clade, or was it left blank because there were two alternative named clades and the confidence was too low to distinguish? Databases such as SILVA and Greengenes do not tell us one way or the other.
What about placeholders such as "sp." for an unnamed species, e.g. if two sequences are annotated as "Chroococcidiopsis sp." they could be different species in the same genus. What about "incertae sedis", square brackets to indicate unofficial names, and so on.
Does the method identify conflicting annotations of the same sequence? Unambiguous conflicts do occur in practice (see "Conflicting annotations of the same sequence" in Edgar2018b).
Does the method identify hierarchy conflicts where a clade at a lower rank appears under different parent ranks (e.g., a given genus appears in different families)? If so, how are they resolved? These also happen in practice (see "Nomenclature hierarchy conflicts" in Edgar2018b).
I was equally baffled by the description of the ensembleTax algorithm. In line [248], what is a "taxonomy table"? I suspect this is a table of predicted frequencies for named clades in one sample, but this is not clear from the text. Frequencies cannot be predicted from amplicon sequences because of strong amplification biases such as multiple operons, primer mismatches and so on; see e.g. my paper https://doi.org/10.1101/124149. Would "consensus" classifier be better than "ensemble"? As with other sections in the paper, this reads as a stream of disconnected facts which assumes the reader is already familiar with the problems to be solved and the authors' jargon. It needs better structure and more careful definition of terms and concepts. Lines [280-288] are a good example of a paragraph that is hard to follow with much distracting detail and too little background.
[262]
The terms "under-classification" and "over-classification" should be explained.
[171]
"Data pre-processing" should be explained in more detail.
[183]
abbreviation "nr" should be explained.
[183]
Why were these reference databases chosen? For 16S, using SILVA and Greengenes seems unfortunate because they are mostly based on predicted rather than authoritative taxonomy, and as a consequence have very high error rates as I showed in Edgar2018b. Using the 97% clustered version of Greengenes makes the problem worse because clustering discards many named isolate strains, which are the only sequences with authoritative classifications. Predicting taxonomy from a reference set annotated by unreliable methods and clustered at 97% is asking for trouble in my opinion. Edgar2018b recommends using named isolate strains only as a reference. The authors may disagree, but at a minimum these issues should be raised for the benefit of readers who may not be familiar with these complications.