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 BIRDSONGS AND TRAFFIC NOISE ON PEDESTRIAN WALKING SPEED DURING DIFFERENT SEASONS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The article uses clear and technically correct text. The introduction was reorganized and it is clearer now. My only suggestion is to join the sections “Auditory and visual interactions: Sounds can change perception of environment”, “Auditory and visual interactions: Environment can change evaluation of sounds” and “Attempts to explain the phenomenon of auditory and visual interactions” in a single section, and include the section “the aims” in the “The current study” section.

The article includes sufficient background to demonstrate how the work fits into the broader field of knowledge, and the results from previous studies were adequately presented.

The structure of the article is conforming to an acceptable format. Figures are relevant to the content of the article, and Figure 1 has now been referenced.

The time of walking for each section and for total route were now presented in the raw data.

The authors claimed that the work is self-contained justifying it in an appropriate manner.
Experimental design: The research question is well defined, relevant and meaningful. The authors intend to fill the knowledge gap regarding the evaluation of direct behavioral responses (walking speed) to noise in specific environments.

The authors had affirmed that there were no known risks for participants in this study. Nevertheless, any potential risks of walking around the route and the discomfort it may entail should be considered for ethical purposes. The authors stated that the place is safe and located around the university campus. They consider that the participants were young healthy adults, who use to walk, and that the short walk on a flat surface did not cause any discomfort.

I still suggest uploading the stimuli to an online repository, to be consistent with the PeerJ’s policies.

The link for the “Forest Birdsong - Relaxing Nature Sounds - Birds Chirping” -https://www.youtube.com/ watch?v=Qm846KdZN_c - is not working because there is a missing letter. Please try this one: https://www.youtube.com/watch?v=Qm846KdZN_c
The authors have presented the effect size value for the analysis. The sample size of each of the three conditions was now specified in the text for the analysis.
Validity of the findings: The authors did not state that the data were made available in an acceptable discipline-specific repository but they uploaded them as Supplemental files.

The data was not fully controlled because the research was conducted in an outdoor environment. Nonetheless, according to the authors they have tried to reduce the effects of the external variables by balancing the experimental conditions during the days on which the experiment was performed.

The results were more robust for the interactions between the effects of the specific environment of the route and walking speed than for the interaction between the presented sounds and walking speed.

In some parts of the manuscript, the authors had attributed that there was a difference between two conditions even if this difference was not statistically significant and had overestimated some results. The authors have now corrected it and considered these statements more carefully. Nevertheless, they still present statistically non-significant results as a tendency to a certain result, not considering that the results are just either statistically significant or not. In other words, either the participants walked faster or not, because there were no statistically difference between groups. Instead, it could be discussed regarding the effect size as they have done for some issues in the discussion section.

The authors have now considered all of the presented hypotheses in their discussion.
Additional comments: Tha manuscript was muc improved. Please see my comments above for a few suggestions.


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 BIRDSONGS AND TRAFFIC NOISE ON PEDESTRIAN WALKING SPEED DURING DIFFERENT SEASONS
Review round: 2
Reviewer: 2

Output:
Basic reporting: Please proofread the document, as there are some typos (since there are no page numbers or line numbers in the tracked changes version, it is hard to give exact locations of these typos).

I still recommend to for tables on ratings on walking experience to add histograms that show statistical differences between ratings.
Experimental design: no comment
Validity of the findings: I am still not convinced about the claim that walking fast has negative health consequences. I suggest to make more explicit the hypothesised implications of walking faster. Is walking faster bad for health? The authors mentioned some old research that walking faster might have health risks, but this is not enough in my opinion to say walking fast = negative health consequences. It would be ideal to give a figure of what is the ideal average walking speed (from previous literature).
Or, do results imply that since people walked faster in traffic environments, they are less likely to go out and walk in nowadays cities? This is a bit of a long shot. Also, moderate-intensity physical activity has positive health impacts. I suggest to add reflections on the meaning of "walking fast" in the limitations section.
Additional comments: I thank the authors for their hard work in editing the 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: BARK TISSUE TRANSCRIPTOME ANALYSES OF INVERTED POPULUS YUNNANENSIS CUTTINGS REVEAL THE CRUCIAL ROLE OF PLANT HORMONES IN RESPONSE TO INVERSION
Review round: 1
Reviewer: 1

Output:
Basic reporting: - English must be improved.
- References provided but some important refrences must be added for the used programs.
- Too much figures
- Research hypothesis and specific objective must clearly detailed
Experimental design: The experimental set up is clear.
Research questio must be specified in details at the end of the introdution
More details must be added to the used methods
Validity of the findings: Data re robust.
Conlusions well stated
Additional comments: The idea and the set up of the manuscript The manuscript ''Bark tissue transcriptome analyses of inverted Populus yunnanensis cuttings reveal the crucial role of plant hormones in responding to inversion''  are very interesting. The different part of the manuscript were welle structured and illustrated. However, the English should be checked in some parts. Also, some important details are lacking and should be clarified for the readers.

 Comments

Introduction
1 -   The research hypothesis and the specific objectives of the work should be clearly detailed in the introduction
2- Lines 44-45. The authors should explain what they mean by 'developmental significance'.
3 - The authors should provide the name of the botanical family of Populus yunnanensis. Some details about the ecological and economical value may be also interesting to justify the choice of the species in this study.
Materials and Methods
4-   Line 87: the word 'cultured' must changed by 'cultivated'
5- Details about the geographical origin of the samples must be provided
6- For RNA-seq, we assume that mRNA was purified from the total extracted RNA and then used for cDNA preparation. Please provide details about the protocol of mRNA purification (I guess with oligodt beads).
7- Details about the steps followed for cDNA preparation must be provided (mRNA fragmentation, reverse transcription, first and second strand preparation, adaptors ligation ...)
8- Details about the method used for quantify the cDNA libraries must provided. This is an important step before proceeding to the sequencing.
9- Please specify that the sequencing performed is a pair - end (2 × 150 bp)
10- The authors should add details about the quality of the generated transcriptome. Several approaches could be used
- CD-HIT program for contigs clustering and elimination of redundant contigs
-  BUSCO program to align the generated transcriptome with provided databases (in your case; Plants or eukaryotes databases)
- maping back each sample to the generated transcriptome and check the % of similarity
- For all databases used for annotations, programs, parameters used and references must provided.
Results
- More details must provided in the part ' Real-time quantitative PCR verification'. The authors should give value to the results of RT PCR analysis and compare them with the results of RNA-seq approach.
Discussion
The discussion part was well prepared.
- Too much figures in the manuscript! Some of them may be combined in one figure to reduce the total number of the figures.


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: BARK TISSUE TRANSCRIPTOME ANALYSES OF INVERTED POPULUS YUNNANENSIS CUTTINGS REVEAL THE CRUCIAL ROLE OF PLANT HORMONES IN RESPONSE TO INVERSION
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript "Bark tissue transcriptome analyses of inverted Populus yunnanensis cuttings reveal the crucial role of plant hormones in responding to inversion" (#36443v1) by Zhou et al. is a very interesting work concerning the transcriptome analysis of the response to inversion in bark tissues of this species.Professional Engilsh is used throughout the manuscript. The work is well planned, and the technical development of the analysis is excellent, making use of many bioinformatics tools to properly examine the results.Figures are adequately designed and very informative. Authors have generated a vast amount of information that is very useful.I find the work suitable for publication in its current state.

I will only make some minor comments and suggestions:

- The discussion, though correct and well referenced, could have been more sound. In the introduction the authors write about plant polarity, but this issue is not further discussed concerning the results. Indeed, the involvement of the ethylene in the lightgreen module (related to BI) provides interesting information about the implication of this hormone in polarity. In a similar trend, the putative relation of plant-pathogen responsive genes with polarity is neither discussed. Nonetheless, I understand that inversion is a neglected issue in plant physiology and there are not many references about it.

- There is no information about the primers of the PEDI gene.

- Line 311: Nicotiana benthamiana. Names of species should always be in italics. Besides, some references are in italics while others not.

- Line 484: Estelle M.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: No additional comments


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

Input:
Title: POPULATION GENETIC STRUCTURE OF TEXAS HORNED LIZARDS: IMPLICATIONS FOR REINTRODUCTION AND CAPTIVE BREEDING
Review round: 1
Reviewer: 1

Output:
Basic reporting: Satisfactory.
Experimental design: Satisfactory.
Validity of the findings: Satisfactory.
Additional comments: This manuscript meets all of the review criteria required for publication at PeerJ. The basic reporting, experimental design, and validity of findings are all satisfactory.

In general, the paper presents novel results for an interesting study system that is of special conservation concern. There are also practical implications from the study in terms of management of natural populations and implementation of captive breeding projects.

The data are sufficient for answer the basic questions of the study. I only have a few concerns for the authors to consider.

Population groupings - The choice of the 16 population groups seems arbitrary and subjective. What is the justification for these groupings, since they guide all of the descriptive statistics and population comparisons? The groupings have labels that are completely separate from the main document figures. There is no way to identify where these 16 places are in relation to one another without searching through supplement materials. The 16 groups in the PCA plot (Figure 2) have no geographic context without a map. If 16 arbitrary groups are used, then they should be plotted on the map in the main article. However, perhaps the pop. gen summary statistical comparisons should be conducted for the populations detected from the microsatellite data (indicating K=3), instead of determined subjectively to be K=16.

MtDNA data - The mtDNA data alignments are not sufficiently described. For example, the program MEGA is used to manipulate sequences, but this program does not conduct multiple sequence alignment. It uses other algorithms (programs) to perform alignments. This is an issue because the ND4 protein coding gene has gaps that are not in multiples of 3 (corresponding to amino acid indels), indicating an alignment problem or perhaps an erroneous sequence.
It is not clear if the mtDNA stats considered the outgroup, or if only the ingroup was used.
There is no real reason why the two mtDNA genes shouldn't be linked, since they share the same genealogy. Perhaps this was a logistical choice because of the different levels of sampling?
Why wasn't an outgroup used for the control region sequence? A complete mitochondrial genome for a coast horned lizard is available on GenBank. The phylogeny for control region seems relatively useless without branch lengths or an outgroup. This phylogeny figure should be redone to match the style of the ND4 phylogeny. The ND4 tree provides adequate information. The circle tree for control region does not.
I recommend adding a Bayesian skyline analysis to estimate population trajectories, a more modern approach that is based on a coalescent population model that is relevant for these types of data.

Structure analysis - what is the justification for designating population membership values of q>0.1 to signify admixture?
The colors for the structure plot do not match the colors for the groups on the maps. There needs to be some coordination between the colors across the various figures.


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: POPULATION GENETIC STRUCTURE OF TEXAS HORNED LIZARDS: IMPLICATIONS FOR REINTRODUCTION AND CAPTIVE BREEDING
Review round: 1
Reviewer: 2

Output:
Basic reporting: Overall a well-written and easy to read manuscript. Though generally clear, there were only a couple of points (maybe in Results) where a sentence was confusing. The structure and flow of the article was simple to follow and seemed well set out. However, whilst the appearance of the figures was professional and high-quality, I tended to find a few figures, and associated captions, could be adjusted to improve clarity. Whilst the context and aims of the work are clear, it felt that a little more detail could be included in the Introduction outlining any previous genetic work and understanding of the focal species, so as to further elucidate how this work was to build upon that knowledge and fill a gap in understanding; something that was only touched on very briefly in the Discussion. Furthermore, although the study on the genetic structuring within the focal species seemed to be reasonably sound and thorough enough to address the genetic aims of the work, more could have been done to address the question of any relationship between genetic divisions and ecoregions, which was raised as the secondary aim of the work. Whilst this would not necessarily require additional analyses, further exploration into this element would be desirable.
Experimental design: The authors have conducted a solid study with an impressive number of study animals and comprehensive sampling of the species’ range. Sufficient and clear information was detailed in the methods, and multiple comparable analyses were conducted on different genetic datasets, which is great practice. Despite my initial concerns the genetic sampling used was modest, analyses appear sound and the authors were able to produce relevant results with which they make informed recommendations as to future management strategies, as per the aims of the study. As mentioned in the section above, a small amount of additional background in the Introduction section clearly identifying the knowledge gaps the genetic work in this study would fill compared to prior work would further strengthen this manuscript.
Validity of the findings: The genetic findings of the study are fairly well presented and put in context. Observed patterns are discussed concisely and relevant possible explanations are provided. The conclusions and associated management recommendations made by the authors are clear and reasonable. I would have liked to see the authors further explore the question of potential adaptation of different population units to specific ecoregions than what is currently presented, particularly given it is stated as an aim of the study.
Additional comments: Major missing details:
As raised in the above sections, I had two main areas that stuck out as warranting further detail.
The first, to really ensure the research is placed in the context of the broader research area, a brief mention in the Introduction of the current standing genetic knowledge of the Texas horned lizard would go a long way. There was one sentence (Ln 340-343) in the discussion, which could be raised in the Intro. for further context, e.g. 'there was known to be an east-west divide but sampling was not geographically comprehensive', or 'we only had an idea of mtDNA patterns, nuDNA would be useful to gain better understanding of the genetic diversity of the species'. Even the potential this represents one panmictic population due to the known history of human translocation vs. the general theory that these are low-dispersal critters, could be good to bring up in the Intro., as it then presents two quite contrasting patterns that could be predicted within the study, that can then be compared to the findings.

The second issue was that aim 2 (relation of genetic divisions to ecoregions) was not quite explored to its potential and I was left wanting more. This is a particularly interesting area of investigation and could be done greater justice, even in terms of fleshing out the detail within the text of how these ecoregions differ. There were no methods/analyses laid out to directly test for an association between genetic groups and ecoregions, which are not absolutely necessary, however, testing for correlations of environmental/habitat variables or significant differences in such variables between regions occupied by different genetic clusters is one example of how this could be done. Alternatively, and regardless, I would have liked to see the authors delve into the differences they believe are present between the broad regions occupied by these different clusters. In particular:
-   Results section: - Ln 252-262 – This section seems to be the only part of results where ecoregions are discussed in regards to genetic clusters, but there is still no clear indication there is any preference for particular habitat types from the information provided, simply that different clusters occupy multiple different ecoregions, which could simply be geographic.
-   Discussion section: - Ln 363 – “…which represents a much different habitat than…” – Could this be elaborated as to how the ecoregions are different, to better justify that the differences may indeed be relevant to these lizards?
-   Ln 385-386 – Possibly the clearest point offered that the habitat types may be strongly different.
-   Ln 403-405 – Again, what makes these two broad “regions” so different as habitats, rather than simply divided by a barrier?
-   Ln 453-455 & Ln 464-465 – I agree that regional adaptation could be an important factor, but more justification is required to convince me how these broad regions differ in consistent ways.

Defining/referring to structure:
It can fast become tricky to define what clusters or clades are being discussed at any stage when patterns within genetic datasets are not 1:1 concordant. Suggestion is to be consistent in terminology and as clear as possible whenever units are discussed, e.g. be explicit when discussing mtDNA clades vs. nuDNA clades. For example:
-   Abstract, Ln 32 – perhaps ‘mitochondrial’ genetic groupings, but could see the argument for without.
-   Discussion, Ln 294 – “There was no evidence for separate mitochondrial clades…”
Similarly, consider bringing up the regional divisions (North, South, West) as early as Ln 225-229 within the Results, so the reader can follow that there is a consistent pattern of clustering throughout the analyses. Along similar lines, Ln 246-247 – maybe raise this point initially, so the reader can follow the concordance.

Tables & Figures
-   Tables 1 & 3 – consider indicating (e.g. via colour shading or grouping under sub-headings) which sites correspond to which genetic clusters (West, North, South).
-   Fig. 2 – Is it possible to include % loadings on PCA axes to indicate the variation? Maybe also consider incorporating colours on the points, or ellipses; some way to indicate the clusters coincide with West, North and South nuDNA clades.
-   Fig. 3 – Probably could be in supplementary material.
-   Fig. 4 – Slightly confusing information display, with too much division of unclear units. Without more information regarding what the three clusters or panels correspond to location-wise the division seems either arbitrary or slightly misleading. If possible, perhaps have all panels connected.
-   Fig. 5 – Fairly confusing and possibly not critical for the main text. The associated caption was also challenging; perhaps could be clearer if names in legend indicated the genetic data involved. Would be great if it was evident where these admixed individuals are with respect to the broader clade distributions on the map. Would it be possible to have the different maps – Figs. 1 & 5, even Fig. S1 – all incorporated into a single figure? Either a single map that indicates mtDNA and nuDNA clade limits, and perhaps admixed individuals, and the 16 sites (may be getting too busy…), or alternatively a figure with the different map displays featured as separate panels?
-   Fig. 6 – could do with labeling the mtDNA clades West and East so as not to be misled by the variation in coloured symbols causing it to appear that there is no reason to the structuring. How many samples are represented in either clade (i.e. how many samples comprise the 10 West haplotypes)?

Minor comments:
•	Add a comma: Ln 37 – after ‘urbanization’, Ln 40 – after ‘fragmented’, Ln 220 – “similar across sites, with”
•	Spell out numbers: Ln 62 – ‘nine of ten’, Ln 215 – ‘six’, Ln 299 – ‘seven’
•	Ln 70 – Add full-stop after ‘spp’
•	Ln 91 – could samples be elaborated here to indicate what type of sample, e.g. tissue samples? It is clear a few sentences later that some are tissues, but for readers who are unsure what type of sample is being collected by the cloacal swab method, this would be useful.
•	May not always be critical, but consider spelling out acronyms at first mention, e.g. Ln 94 – IACUC and TCU, Ln 96 – EPA
•	Ln 117 – “and electrophoresed on an ABI”
•	Ln 132 – “manufacturer’s protocols”
•	Ln 136 – Add reference/citation for Sequencher
•	Ln 185 – “MrBayes”
•	Ln 224 – This seems like a very low Fst value regardless of p-value, if an Fst of zero implies complete panmixis.
•	Ln 241 – Fig. S3 appears to be missing? Elephant Mountain WMA is mentioned in the text but not featured in any tables or figures.
•	Add semicolon to maintain consistent style: Ln 242 – after ‘83%’, Ln 243 – after ‘95%’.
•	Ln 259 – move ‘(q > 0.9)’ after ‘ancestry’ to minimize interruption to sentence flow. Could also remove ‘(q > 0.90)’ from Ln 261 as already defined at Ln 259. Furthermore, is ‘high ancestry’ the standard wording used with these types of measures; would it be clearer to say, ‘high ancestry assignment’?
•	Could remove ‘horned lizards’ from Ln 263 and 264 if desired as not strictly necessary.
•	Ln 275 – consider alternative word to ‘diverse’ such as ‘high’. Sounds odd as a describer of diversity.
•	Ln 293 – maybe be cautious in referring to areas as hybrid zones or discussing hybridization, if within a species (at least as currently recognized) it is probably considered a region of admixture between populations.
•	Ln 297-298 – Does Fig. 6 support this sentence? The wording is a little confusing.
•	Ln 313-318 – from “There was a well-supported clade…” to “…also found in some of the same areas.” – These lines may not be necessary. The level of divergence between these groups of haplotypes is minimal and discussion of them potentially clouds the stronger findings that you want readers to take away.
•	Ln 328 – some readers would expect a P-value to be provided with a statement indicating any “significant” statistics, e.g. “(P < 0.05)” or similar.
•	Ln 337 – keeping consistent style, suggest move ‘(eastern and western)’ to end of sentence after ‘mtDNA loci’.
•	Ln 343 – a little pedantic, but maybe rephrase this sentence – the presence of a lake poses a potential biogeographic barrier, but the formation of the lake would be the possible vicariant event.
•	Ln 361 – suggest “that is also broadly concordant” or something similar. Consider the presence of northern microsat cluster individuals with eastern and western mtDNA haplotypes?
•	Ln 364 – clarify what type of genetic distance, e.g. mtDNA? And what is this level high compared to; what are the divergence levels like between closely related species?
•	Ln 410 – consistent unit notation, ‘~800 m’
•	Ln 417 – higher or deeper?
•	Ln 442 – “expected to come into”
•	Ln 464 – Is “strong genetic breaks” reasonable wording, given the level of admixture occurrence? Seems like broadly concordant genetic disjunction, but strong breaks almost implies absolute deep reciprocal monophyly.
•	Ln 481 – This sentence is great and an intriguing area to be considering further. If the authors are predicting that different habitats do indeed play a role in shaping divergence and structuring diversity (hinted at in the mention of xeric vs. mesic regions), it could be expected that differences in morphology (e.g. overall body size, scale size) and physiology (e.g. thermal tolerance, water balance) might also arise as mechanisms for dealing with pressures of water loss or extreme temperature variations.


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: POPULATION GENETIC STRUCTURE OF TEXAS HORNED LIZARDS: IMPLICATIONS FOR REINTRODUCTION AND CAPTIVE BREEDING
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have done a great job incorporating further information into the Introduction to better set the scene as to the prior genetic knowledge available on the focal species, which emphasises the need and importance of their study. Likewise, the manuscript is much enhanced by the addition of detailed information on the environmental differences of the broad ecoregions inhabited by the different genetic divisions within the Texas horned lizard. Other minor changes throughout have also further improved the clarity and ease of following which genetic divisions are being discussed. The simple addition of genetic cluster identity within Tables 1 & 3 aid a lot in understanding and quick comparison of statistics across clusters. Adjustments to figures, particularly Figs. 1,3, & 4, have very much improved clarity; though there are still a couple of quite minor details that could be altered.
Experimental design: As mentioned above, additional background in the Introduction section now clearly identifies the knowledge gaps this study fills, adding to the importance of this work. Further minor adjustments and additions to the Methods section have also further improved clarity of exactly what was done during data collection and analysis.
Validity of the findings: The additional information and clarification added to the Discussion regarding differences in ecoregions, and possibilities for adaptations &/or fitness costs associated with different genetic groups and their crossings, adds to the conclusions and associated management recommendations made by the authors. It is clear from the added information that this work sets the stage for future avenues of study to investigate these issues.
Additional comments: Minor comments:

• Add a comma: Ln 33 – after ‘clusters’, Ln 125 – after ‘populations’, Ln 220 – after ‘stable’, Ln 283 – after ‘PCA’, Ln 294 – after ‘Plains’, and Ln 328 – after ‘haplotypes’.
• Change comma to semicolon: Ln 129 & 147 – after ’15 min’, Ln 130 & 148 – after ‘72°C’, and Ln 431 – after ‘Haldane’s rule’.
• Spell out numbers: Ln 190 – ‘ten independent’, Ln 193 – ‘ten runs’, Ln 321 & 344 – ‘ten unique’, and Table 1 & 2 captions – ‘ten microsatellite’.
• For consistency, either remove ‘v’ for version at Ln 135 (Genemapper), Ln 154 (Sequencher), Ln 215 (MrBayes), Ln 222 (DnaSP), or add to all other cases where the version number is provided for a program.
• For clarity, Ln 168 – add ‘(Ho and He)’ after ‘heterozygosity’, Ln 169 – add ‘(AR)’ after ‘richness’, and Ln 205 – add ‘(HR)’ after ‘richness’.
• Could remove ‘6.5’ after GenAlEx at Ln 177, 180, 203, & 207, as already listed at Ln 167; and ‘10’ after MEGA at Ln 210 & 211, as listed at Ln 154.
• Ln 29 & 529 – “…especially between the western and eastern clades…”
• Ln 143 – add a space between the hyphen and primer sequence for Leu.
• Ln 152 – Could remove “(ThermoFisher Scientific, Waltham, Massachusetts, USA)” as already listed at Ln 134.
• Ln 160, & Fig. 2 & 3 captions – for consistency in formatting, add a space between ≥ and 10.
• Ln 181 – “… STRUCTURE results (see below) which utilized all samples”.
• Ln 185 – for consistency, change ‘100,000’ to ‘104’, or change ‘106’ to ‘1,000,000’; also check Ln 217 depending on format decision.
• Ln 187 – ‘(K; Puechmaille, 2016; …’
• Ln 189 – Change ‘ALPHA’ to ‘α’ or ‘α value’, since α was previously used in this sentence.
• Ln 208 – as was explained to reviewers, perhaps add something to the end of this sentence as justification for not concatenating mtDNA loci, e.g. “… and the control region due to the difference in sampling for each marker”.
• Ln 212 – ‘branch swap filter’.
• Ln 222 & 224 – ‘DnaSP’.
• Ln 223, 311, 336 (x3) – italicize the h for haplotype diversity ‘(h)’.
• Ln 230 – Could just have either ‘tau’ or ‘τ’ given it is listed in Ln 228.
• Ln 287 – Could remove ‘ecoregion’ after ‘Southern Texas Plains’?
• Ln 311-314 – It took a few reads and looking back at Tables 1 & 3 to understand what these sentences were getting at. I would suggest at least referring the reader to both tables (Table 1, 3) either at the end of the sentence at Ln 312 or Ln 314 so it is clear that values from both should be compared to get this point.
• Ln 322-324, 326, 345-348, & Table 1 NA column – consistency of number of decimal places quoted. If standard error is to two decimal places, mean should also be to two decimal places, similarly with ranges.
• Around Ln 324 – Could you maybe include the divergence between P. cornutum and P. blainvillii, like is provided in the ND4 section for P. asio?
• Ln 343, & Fig. 5 & 6 captions – remove hyphen in ‘maximum likelihood’.
• Ln 346 – Remove ‘=’ and the space between ‘1.7’ and ‘%’.
• Ln 359 – would again provide a P-value with the significantly negative statement, simply (P < 0.05), and reference (Table 4) at the end of this sentence regarding Tajima’s D results.
• Ln 368 – Minor but consider reordering the listing about mtDNA groupings (western and eastern) for consistency, this reinforces in the reader’s mind that west matches west and east matches north + south. Often throughout the text west is discussed before east because it is the more straightforward division.
• Ln 371 paragraph – This section is excellent. You could even remind readers as it goes which genetic cluster is associated with each region being discussed, for example, Ln 377: “The Chihuahua desert ecoregion (west clade)”, Ln 380: “The South-Central Semi-Arid Prairies ecoregion (northern cluster)”, etc. But that is not at all critical and it would be perfectly fine without also.
• Ln 454 – (MUs; Moritz, 1994)
• Ln 463-4 – If citing chronologically, swap Endriss, 2006 and Stark et al. 2005.
• Ln 476 – ‘effective population size’?
• Table 2 caption – remove comma after (q).
• Fig. 1 & 2 – These figures are much improved and easy to understand. It would be nice if it were possible to have them combined in a single page figure somehow as two panels, Fig. 2 doesn’t even necessarily have to be so large. But it would be understandable if this forced a reduction in size of either map that would compromise the clarity of the information.
• Fig. 3 caption – state somewhere in this caption that these values are from the nuclear microsats to ensure it’s very clear to readers what data the values are from. It also says, “dark grey and red on #1 indicates…”, should there be grey on the symbol for site 1 which is not currently there?
• Fig. 4 caption – again, state something like “… clustering of nuclear multilocus genotypes…”
• Fig. 5 caption – ‘well-supported’. Are the nodes equal to 80 or equal to/greater than? Only one clade seems to be indicated as being supported with 80 bootstraps. It is tricky to see because of the circular format of this tree where exactly the eastern clade is delineated. It would likely be much easier to distinguish the clades and branchlengths if the tree were presented like that in Fig. 6. A bootstrap value of 80 however could be argued to not be a particular well-supported node, and if there is not a clearly supported node at the base of the other clade then the results section for this particular locus should probably be updated to be clear that there appear to be two clades, east and west, but that neither is strongly supported by this particular analysis.
• Fig. 5 & 6 – whilst it now feels clearer that the symbols indicate the different nuclear clusters both figures would be further enhanced by clearly labelling the two mtDNA clades somehow. For example, in Fig. 6, the branches leading to either clade could be labelled as ‘West’ and ‘East’, or alternatively could have labelled vertical bars to the right of each clade.


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: VALENCE MAKES A STRONGER CONTRIBUTION THAN AROUSAL TO AFFECTIVE PRIMING
Review round: 1
Reviewer: 1

Output:
Basic reporting: This work is nicely grounded in the antecedent literature and was thoughtfully controlled for many possible confounds as indicated by previous work. I was able to easily recapitulate all three analyses in JASP, and I appreciated the clear and concise descriptions of the relatively complex designs.
Experimental design: The present manuscript describes three affective priming experiments independently manipulating aspects of valence and arousal of the primes and targets in a lexical decision task, with a blocked design (where every block began with the same class of prime word, e.g. positive high-arousal). Pseudowords comprised half of the experimental materials and only ever occurred in the target position. The first experiment held the arousal level of the non-pseudoword prime-target pairs constant (at high or low values) and manipulated the positive/negative valence of the prime and target in a fully crossed design, nominally finding a facilitative effect of valence congruity given positive primes and an inhibitory effect of valence congruity given negative primes (though see #1 in the "Validity of Findings" section). The second experiment held the valence of the non-pseudoword prime-target pairs constant (at positive or negative valence) and manipulated the high/low arousal of the prime and target in a fully crossed design, again showing slower responses to negative trials but additionally showing an arousal congruity effect for positive pairs only (with incongruous trials slower than congruous trials). The third experiment mirrored the valence-priming design of experiment 1, but replaced all valence-incongruous targets with neutral words, again finding that negative targets elicited slow responses and additionally that the same neutral targets were responded to more slowly when preceded by a negative prime than a positive prime. The authors conclude that valence is a stronger contributor to the automatic processes elicited during reading, as both valence effects and valence-congruity effects were robustly observed at every opportunity to find one in the present experiments.
The number of repetitions was kept relatively low (the paper says 5, though see below), and word properties were matched across visual (e.g. # of strokes in the characters), lexical (frequency) and conceptual (concreteness, very important to control in this type of research) features.Though there are a few minor aspects of the analysis that could be improved (see below) and some instances of unclear text, possible typos, or minor omissions that could be cleaned up, the experiments are thoughtfully designed and competently executed.
1. I recommend using omega-squared as the effect size estimate instead of partial eta squared for samples of this size in order to correct for the optimistic bias almost certainly present here (see, for example, Okada 2013 in Behaviormetrika).
2. It’s not clear what the two values are on line 188, particularly since they extent beyond the lowest possible rating. These relatedness value could be more clearly summarized – perhaps a range of items-level proportions of “1” ratings would make sense? Or a cutoff threshold for the proportion of non-1 ratings?
3. Results section starting on line 214 should state that a repeated-measures ANOVA was used. If possible, however, a mixed-effects regression approach is likely better suited than an ANOVA, given the bounded set of possible prime-target pairs presented repeatedly to each participant. The present analysis can be thought of as assuming a representative sample of participants from which population inferences can be drawn. Also of interest to readers is the assumption that the particular items used are also representative, from which inferences about language processing more generally may also be drawn. The random effects of subjects is presently reflected in the analysis, but the random effects of items is not. See Barr et al 2014 in JML (the supplemental materials contains an extensive discussion related these regression models to ANOVAs).
4. Line 183 states the emotional words were repeated 5 times (3 in the prime position and 2 in the target position) but I’m counting 6? For example, the 30 PL words in experiment 1 appear twice  in PL-PL trials (once in the prime and once in the target position), once in PL-NL trials, once in NL-PL trials, and twice in PL-pseudo trials. Could these numbers be double-checked and/or the relevant text about the trial composition clarified?
5. In the description of study materials, please state the cutoffs used for the different valence and arousal categories (including the “neutral” category).
6. Please provide part of speech numbers in the table of descriptive statistics for the study materials.
7. Please provide 95% confidence intervals in the figures. I also don’t recommend bar plots in cases where the low range of responses are implausible and/or artifactual (e.g. response times less than 100ms). A swarm plot or boxplot may be more appropriate for these data.
Validity of the findings: I find the interpretation somewhat un-parsimonious. Blocking by prime type, and centering the factorial analysis and follow-up simple effects on the prime valences (rather than target valences, as is common in the other behavioral research and ubiquitous in the relevant ERP research) somewhat obscures the fact that the primary valence and valence congruity results in the present experiments could largely be accounted for by a slowing of lexical decisions to negative targets. Addressing this alternative explanation is necessary for the present manuscript, as this alternative explanation does not actually require priming at all (though the arousal congruity effects are not subject to this possible alternative and are fine as is).
1.	The block-based design implemented here very nicely reflects some of the observations made in the Herring et al metanalysis on experimental factors moderating affective priming effects, setting up a nice opportunity to make some meaningful contributions. However, the focus on the prime position here can make it difficult to parse some of the resulting effects given the literature on unprimed responses of those word classes. For example, the present paper reports a classic affective priming facilitation given a positive prime in all 3 experiments and a reverse valence priming effect (where congruous trials were slower) given a negative prime in two experiments. But this set of simple effects is also equivalent to simply concluding that “negative targets elicit slower lexical decision responses than positive targets”, a framing that parsimoniously accounts for the present data and also has some support in the literature (see for example Estes and Adelman 2008 in Emotion, and also Larsen et al 2008 in Emotion). This possibility is especially important in the present case because A) the primes are not task relevant (strategic participants would ignore them, and while the evidence here for prime-driven effects is sound, this design aspect may increase the importance of target-driven effects) and B) because of the blocking, the emotion aspects of the primes is perfectly predictable given a few trials exposure to the block. I’m actually preferentially sympathetic to the affective priming interpretation, but this view of the results (and design of the blocking) does carry this important consideration, and the issue of effects of the words that participants are making responses to needs to be addressed. I highly recommend a supplemental experiment showing lexical decision reaction times to the same positive and negative targets used in these experiments. If these targets show no difference in lexical decision times when presented in isolation, but do show differences in the priming experiment, it would be important evidence that the direction of the valence congruity effect may depend on numerous contextual factors (such as valence, arousal, blocking, and task) when the present results are interpreted in light of the broader literature. Without such an experiment, it’s unclear why the less parsimonious interpretation would be preferred.
2. The arousal difference between nominally high and low arousal words was significantly smaller than the valence difference between positive and negative words. While articulating the conclusions about the relative “strength” of valence and arousal priming effects, I would recommend addressing the possibility that these may have been influenced by the strength of the manipulations.
3. I think the reaction times to the pseudowords given different primes could be interesting. I recommend reporting these (in supplementary materials would be fine if the manuscript is desired to remain concise). There’s a possible concern here that deriving the pseudowords from the experimental materials could have left some residual possibility of activating at least the orthographic recognition of the emotional words, but confounds in the lexical decisions could be ruled out by assessing the marginal effects of the word type (e.g. PL, PH etc) each pseudoword was derived from.
Additional comments: Thank you for the thoughtful and concise manuscript. It was easy to read, addressed intuitive issues, and didn't get lost in the weeds. I rarely recommend collecting additional data, but in this case a very small additional experiment (of lexical decisions to these emotional words in isolation) bears significant implications to how the present results should best be interpreted, and I think would considerably strengthen the arguments in the paper. All my other points were minor. I look forward to seeing this manuscript published after 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: VALENCE MAKES A STRONGER CONTRIBUTION THAN AROUSAL TO AFFECTIVE PRIMING
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: I’ve reviewed this paper already and thought it should have been published the first time.  I’ve looked back at my old review and have found that the author’s addressed many of my original concerns.  Below are some areas the authors could improve upon.  Like I said to the authors over a year ago, the authors are commended for their systematic investigation of valence and arousal contributions on evaluative priming.  Unkelbach has been working to update priming accounts that more clearly involve the hedonic valence and this study is noteworthy to this end.

Primary concerns

-I think the authors are sill diving into the affective priming paradigm too quickly.  Why not add a little pizzazz to the very beginning by talking about why anyone would be interested in indirect measures in the first place?

-Here were my comments from a year ago that the author’s did well in addressing:

“…On the same page it seems the rationale that is provided is that LDT allows for elucidating purely valence or arousal dimensions given it doesn’t provide a particular mindset of either valence or arousal.  But, given an attentional sensitization account (see Spruyt’s work), it does not appear that a strong prediction of affective priming can be made using an LDT provide the participant is in more of a semantic mindset.  I encourage the author’s to spell out the rationale more clearly for using the LDT and pit it against Spruyt and Kiefer’s work.  Similarly, an addition to the discussion (p. 19, para 1) is in order to compare the current results to a recent meta-analysis showing null effects of the LDT.”

Is that accurate the LDT creates a relative neutral and not a semantic mindset? At any rate, I still wrestle a bit with the fact that one wouldn’t then anticipate any behavioral effect given the Herring meta-analysis.  This might be a nice spot to mention where using ERPs in this area is particularly well suited.  That is, we know the evidence for evaluative priming is weak for the LDT behaviorally, but ERPs offer an opportunity to investigate whether arousal or valence effects are stronger.

-Table 1: please provide superscripts to where the four words types (i.e., PH, NH, PL, NL) differ in valence and arousal.

Minor concerns

-effect size estimates are missing in Experiment 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: VALENCE MAKES A STRONGER CONTRIBUTION THAN AROUSAL TO AFFECTIVE PRIMING
Review round: 2
Reviewer: 1

Output:
Basic reporting: - New figures look great.
- Was able to reproduce all analyses with the shared data of all 3 experiments during my previous review.
- Recommend running the manuscript by a copy editor to better align the professional English with the style of the other publications in this journal.
Experimental design: - All typos and omissions noted in the previous review have been addressed.
Validity of the findings: - Authors graciously included additional data and analyses of interest in the supplemental materials after the last round of reviews.
- Experiment is well controlled and precisely described.
- The discussion of the possibility that the arousal manipulation was not as "strong" as the valence manipulation could be made much simpler. The U-shaped relationship between explicit valence and arousal ratings seems a bit tangential to the primary concern on this point, as does the temporal resolution of ERPs. It seems fine to simply present the limitation, perhaps noting that future attempts could be made to achieve valence and arousal manipulations of similar magnitudes.
Additional comments: - I appreciate the authors' considered edits and gracious response to reviewer comments.
- Some of the intro and discussion, particularly the newer sections that likely haven't had the benefit of quite so many editing passes, occasionally structured information in ways that were hard to follow. Specifically, it was not always clear what argument was being served by a reference to concepts or findings from the broader literature. I recommend one more pass to check that each reference isn't just topical, but connects to a clearly articulated argument. And additionally, that those arguments have a logic that is transparent and well-structured.


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: VALENCE MAKES A STRONGER CONTRIBUTION THAN AROUSAL TO AFFECTIVE PRIMING
Review round: 2
Reviewer: 2

Output:
Basic reporting: meets standards
Experimental design: meets standards
Validity of the findings: meets standards
Additional comments: The authors did an excellent job addressing my concerns.  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: THE ASSOCIATION OF DEPRESSION WITH LOWER URINARY TRACT SYMPTOMS: DATA FROM THE NATIONAL HEALTH AND NUTRITION EXAMINATION SURVEY, 2005–2008
Review round: 1
Reviewer: 1

Output:
Basic reporting: The writing is generally fine, but there are some instances where the writing is a little awkward or slightly unclear (e.g. Lines 129–130, 132). Careful proofreading should find these.

The authors need to be careful to avoid making causal statements from observational cross-sectional surveys (including “impact” in the title and on Line 239, and “cause” on Lines 18 and 64).

Lines 16–18: Because the authors use logistic regression for their adjusted analyses, the question being investigated here is whether these variables are associated with the risk of LUTS (despite the data being cross-sectional, there is still a direction in the statistical models which generally becomes the implied direction for causality in adjusted models—while causality cannot be confidently determined from such data, we can, and do, test the consistency of the observed data with an assumed, global null, causal model). This is not meaningfully different to the question as stated here for unadjusted analyses (comparing means and comparing odds would lead to similarly worded questions with basically the same intent), but in the adjusted analyses, the effects are conditional on other variables’ effects on the dependent variable and so the two approaches (comparing means and comparing odds, each time adjusting a different dependent variable for the other variables) diverge in terms of their research questions. See also Lines 25, 27–28 (but note that Lines 28–29 is correct as are Lines 29–32), and elsewhere for this. If all results are presented in the same way (with LUTS as the dependent variable), I think the reader will find the results easier to follow.

I wouldn’t describe depression and sleep disorders as demographics (e.g. Lines 16–17) but rather as measures of health or as morbidities.

Line 20: 2005 not 2006.

Line 37: “discussed” or “investigated”? This doesn’t seem to me to hold though as you are modelling unemployment as a statistical predictor of LUTS, rather than LUTS as statistically predicting unemployment. I think you could note that the association between unemployment and LUTS and that this could motivate investigation into the economic burden of LUTS. (See comments about the underlying causal model below, particularly the possibility that unemployment is a collider in the fully adjusted models here.)

Readers might be interested in the number of men excluded due to each criterion listed on Lines 79–80.

Lines 129, 130, 131, 132, etc.: “multivariable” is for multiple independent variables; “multivariate” is for multiple dependent variables. Similar for univariate and univariable. Please check your usage of these terms throughout the manuscript but note that your usage on Lines 24 and 29 was correct.

Line 127: Are all continuous variables approximately normally distributed so as to justify using means and SDs (rather than medians and IQRs or geometric means and geometric SDs)? Weight and BMI tend to be positively skewed and CRP (Line 211) is always highly positively skewed, as is the case here.

Lines 132–135: This text read to me initially as if you would model the symptoms as statistical predictors of LUTS. This does not match the models in Table 4 and so the text may need to be reworded to make the intent clearer.

Normally, where a mean is presented, a standard deviation will also be included, e.g. Line 146.

Lines 146 and 147: The decimal places for this are inconsistent with other percentages. There may be other instances of this and the manuscript should be checked carefully.

Lines 157–158: The decimal places for ORs and their associated 95% CIs are inconsistent. I would suggest that two decimal places would be sufficient in all cases here.

Line 157: If this is per year of age, it must be made clear to the reader (also Line 165). The level of interest (the non-reference level), e.g. for employment status, must also be clear to the reader.

For Tables 2 and 3, ORs, 95% CIs, and p-values should be reported for both unadjusted and adjusted analyses. As discussed below, the selection of variables for the adjusted model cannot be entirely empirically-driven due to the need to still have a sensible causal model underlying these cross-sectional analyses.

For Table 4, if effect modification by depression status is of interest, p-values for the appropriate interactions should be added after the first three results columns.

I’d like to see the authors’ thoughts on the next steps needed for research in this area added to the discussion or conclusions.

While the data is not provided directly, it can be obtained easily enough through the NHANES website.
Experimental design: The single most important part of a study such as this is identifying the necessary set of variables (confounders) to adjust for in order to estimate the association(s) of interest, without including variables that should not be included (colliders and variables on the causal pathway should the total effect and not the direct effect be of interest). This part of the study needs to be entirely clear to the reader in both the abstract methods and in the body of the manuscript. It is good that these variables are clearly listed in the body of the manuscript, although they are not clear in the abstract’s methods, but they still need to be put into context with an underlying causal model. Identifying the necessary variables cannot be done by looking at unadjusted models (as in Tables 2 and 3), for example, due to the potential for colliders and variables on the causal pathway being included, and the possibility that other important confounders were not examined or not available. Preparing a directed acyclic graph (DAG) for this purpose is often useful to the reader (perhaps included as a supplementary file) in understanding the modelling, as well as to the authors in planning their statistical analyses and in interpreting their results. For example, depression may lead to sleep disorder, which in turn leads to LUTS, i.e., sleep disorder could in theory be on the causal pathway between depression and LUTS, making its inclusion an overadjustment for the purposes of estimating the total effect of depression. Alternatively, you might have a different DAG in mind. Similarly, unemployment might increase the risk of depression, in turn increasing the risk of LUTS. Alternatively, unemployment might be a collider if the DAG was drawn differently.

Some of the models in Table 4 should be identical to models in Tables 2 or 3 once the appropriate underlying causal model is identified, unless there is a very good reason for these to differ. Can the authors comment on this point?

You look at a large number of independent variables. Did you consider interactions between any of these? Model specification tests might be useful in identifying any omitted interactions, but better would be to address this based on the plausibility of the effect modification suggested by the interactions.

The level of detail in the statistical methods should be enough for a (bio)statistician to be able to replicate your results with the data sets downloaded from NHANES without needing to resort to trial and error. This includes all model diagnostics and decisions made on the basis of these.

Lines 71–72 could be misread as saying that you obtained this consent for this particular project.

Lines 128–131: While unadjusted logistic regression and Chi-squared tests will give comparable p-values for binary dependent variables, as noted above, it doesn’t make sense to use the latter for the unadjusted analyses (with no measure of effect or 95% CI provided) and then the former for the adjusted analyses. A simpler approach is to use logistic regression for all models with a binary dependent variable, making a smoother transition from unadjusted to adjusted models and providing effects and CIs for all of these results.

Given the number of cases, and note that the effective number of cases will be reduced by design effects, the authors need to outline how they have controlled for excessive model complexity (see, for example, Peduzzi, et al., 1996's guidelines about sample sizes and logistic regression; if alternative approaches are used, they should be justified).

Note that given that this is not a case-control study, the authors are not required to use logistic regression, and so can use modified Poisson regression to produce relative risks, or could instead justify using odds ratios to estimate these.

The reader might wonder why you used NHANES 2005/6 and 2007/8 and not a more recent survey/surveys.

In Tables 2 and 3, it is not clear why you have not used logistic regression for all independent variables (providing ORs, 95% CIs, and p-values in each case irrespective of statistical significance, as you do in Table 4 for the ORs and 95% CIs, with p-values still needing to be added there). Note that it does not make sense to both compare mean age and use age as a predictor of the odds of LUTS, for example. The linearity (on the log-odds scale) of associations will need to be checked, with this process explained in the methods, for all continuous variables. It is useful to show both unadjusted and adjusted results (and in some cases, partially adjusted results are also useful where there is some uncertainty about the underlying causal model that is informing the analyses, e.g. whether something is a confounder or potentially on the causal pathway).

While I appreciate that the number of cases and non-cases is out of the authors’ control, I would still hope that they performed some sample size adequacy checks prior to analyses and, if so, these should be presented here. If this was not done, this fact should still be noted.
Validity of the findings: All statistical models make mathematical assumptions. How were the assumptions for the t-tests, Chi-squared tests, and logistic regression models checked? These diagnostics need to be clearly explained in the statistical methods in order for the reader to have confidence in the analyses. Note that I am also suggesting that you use logistic regression (or perhaps better, modified Poisson regression) for all analyses involving LUTS, which would simplify the set of assumptions that need to be discussed here.

It is good that the authors have described the NHANES (Lines 68–74) but please note that NHANES is a complex survey and as its analytic guidelines state: “Survey sample weights should be used, and the complex survey design must be accounted for in the estimation of variance.” Such methods are not described as part of the statistical methods here. As you are looking at a subset of the data, please also note the instruction: “An analyst may have a certain demographic subgroup of interest, such as a particular age range or gender, or a subsample of participants who received a particular laboratory test. For proper variance estimation, the entire set of data containing the appropriate weights must be used. The estimation procedure must then indicate which records are in the subgroup of interest.” (Both quotes are from the National Health and Nutrition Examination Survey: Analytic Guidelines, 1999-2010.) This has important implications for statistical analyses, especially for prevalences and other descriptive measures. While the statistical methods are silent on these, the legend to Tables 1, 2, and 3 says that percentages (but does not mention means) are weighted and the legend to Table 4 mentions sampling weights (but not clustering, etc.). Could the authors please explain, and if necessary, justify, what was done in this regard?

How has multiplicity been addressed?

Note that ORs and 95% CIs are required irrespective of statistical significance (Tables 2 and 3).

The ORs and 95% CIs should be used to interpret clinical/practical significance as well as whether further research is needed for certain questions.
Additional comments: While there is certainly value in the research question posed here, there are some significant issues that I feel need to be very carefully addressed. Especially important is establishing an underlying causal model so that the appropriate variables are included in the appropriate statistical models (which have been checked for their mathematical assumptions), leading to full and complete reporting and interpretation of the results.


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 ASSOCIATION OF DEPRESSION WITH LOWER URINARY TRACT SYMPTOMS: DATA FROM THE NATIONAL HEALTH AND NUTRITION EXAMINATION SURVEY, 2005–2008
Review round: 2
Reviewer: 1

Output:
Basic reporting: While the authors say they have removed causal language, “impact” remains in the title (this was noted previously) and on Line 242. See also “increased” on Lines 178–179. It would be worth checking the entire manuscript for any other language that alludes to causality rather than association. The limitations of cross-sectional data are appropriately noted on Lines 244–245.

Line 95: Repeated “as a as a”.

The details on Lines 119–120 (about measuring blood pressure) might fit better on Line 113.

Line 126: “…t-testS WERE used…” or “…THE t-test was…”

Line 134: Missing space in “andLUTS”.

Line 150: It would be helpful to specify “mean ± SD” on Line 150. (I appreciate you mention this in the methods on Line 125, but some readers will skim/skip that section.)

Line 182: “more significantly associated” isn’t a meaningful description. You could note that the odds were higher in one group. This numerical comparison would be strengthened by statistically comparing these two groups and noting the statistical significance in the text around here. Also Line 221.

Line 189: I’m not sure what you mean by “depression as the outcome of interest” here. Do you mean study outcome rather than outcome variable in the statistical models? If so, that wasn’t clear to me.

Lines 210–211: I think there is some unintended repetition in “in the pathogenesis of major depression in the pathogenesis of depression and LUTS”.

Table 1 talks about “weighted percentages” but you do not otherwise talk about the complex sampling in NHANES. Is this spurious? The percentages I checked did not appear to be weighted (or if they were, the effects of weighting were negligible). Line 252 suggests that weights were not used at all.

Table 1: Note that there is clearly some skew in age (a 95% reference range would be 33.8 to 80.0 which doesn’t make sense given the minimum age of 40 here). I would also expect some skew in weight and BMI. This might make arithmetic means and SDs inappropriate descriptive statistics for such variables and it would be worth checking that medians for these are not meaningfully different to arithmetic means, with medians and IQRs or geometric means and SDs used as appropriate if this is the case. The approach used to determine appropriate descriptive statistics should be mentioned in the statistical methods.

Tables 1 and 2: You mention “weighted percentage” for both of these, but, as mentioned above, I’d understood that the sampling design was not incorporated into the analyses. If they were, it’s unclear why means were not similarly weighted. See Line 252 again.

Table 2: Given the skew in age, t-tests might be questionable here. Similarly for any other variables with substantial skew.

Table 3: You report unadjusted p-values, but not Wald test p-values for the adjusted models. Is there a reason for this? Personally, I’d recommend using a univariable Wald test p-value rather than the Chi-squared test p-value here just for consistency (so using a logistic/modified Poisson regression model with depression category only, then this with age, and finally adding the other variables).

Table 3: Refers twice to using “sampling weights”. These were not mentioned in the statistical methods and based on Line 252 and your rebuttal response, I had understood that these were not used here. The Chi-squared tests, however, do not mention weights (note b). Could you please clarify?

My comments about multivariable and multivariate might not have been clear. A common convention is that multivariable is the correct term here as it is multiple independent variables being modelled. See Lines 128 (twice), 130, 133, 176, and Table 3. Similarly, “univariable” is correct when one predictor (including groups) is being looked at, e.g. Lines 126, 129, and 168. See https://dx.doi.org/10.2105%2FAJPH.2012.300897 for arguments in favour of this nomenclature.

Following on from my comments about Tables 1 and 2 above, I had previously asked about whether some variables were too skewed to warrant using means and SDs as descriptive statistics (suggesting geometric means and SDs or medians and IQRs) and I could not follow your response: “Thank you for your comments. The size of the study population was large enough to represent the population and in the clinical perspective. According to recently reported demographics of the United States population, the value of continuous variables were not skewed, and we think that using means and SDs is justifiable.” While a larger sample provides some protection with the sampling statistic being more likely to be approximately normally distributed, and so the p-values being appropriate despite the underlying distribution, this does not address the issue around descriptive statistics. The demographics of the US population are also not relevant to your sample here, unless these were for men aged 40+.
Experimental design: I still find the causal model here to be, in part at least, unexpected, but not unreasonable. By looking at LUTS as the outcome in logistic regression models, it is implied that you’re interested in whether depression leads to LUTS, or at least whether depression can be used in screening for LUTS, and you are adjusting LUTS for the covariates rather than adjusting depression for these variables. From a strictly association perspective, which is the dependent and which the independent variable is not too important, but the variable that is being adjusted for is important. It seems to me that depression is potentially an outcome of LUTS, although I’m also open to arguments that depression could be causally linked to LUTS beyond just nocturia, and I appreciate that investigating patients with depression for LUTS is also an option. The studies cited here (3, 10–13; as referenced on Line 55) appear to be a mixture in terms of the direction, and my comments on this point can be seen as inquisitive rather than directive. One option when there is genuine uncertainty about the causal direction is to look at models in both directions and it is not uncommon, in my experience, for one direction to be statistically significant even after adjusting for potential confounders and the other direction to not be so, providing some guidance in thinking about the causal direction of the association. This comment is just following up on my previous comments on this point and I am not requesting further changes here.

Related to this though, the statement on Lines 241–243 is much too strong for a cross-sectional association. Words such as “impact” require justification beyond that possible here.

In response to my comment: “Note that given that this is not a case-control study, the authors are not required to use logistic regression, and so can use modified Poisson regression to produce relative risks, or could instead justify using odds ratios to estimate these.”, the authors responded “Since this was a case-control study, we could not compute the probability of the disease in each exposure group, and RR could not be calculated.” However, this is definitely not a case-controls study, despite Lines 140–142. In a case-control study, participants are selected based on their case or control status and this means that no estimate of prevalence is available (the ratios of cases to controls is fixed by the design of the study rather than able to be determined from the data); here, all men aged 40+ who provided the necessary data were included and their case or non-case status was determined by the data and not the study design. The choice to present relative risks from modified Poisson regression or odds ratios from logistic regression is up to the authors, but it is important that they appreciate that this does not appear to be a case-control study and so RRs are not precluded. For completeness, the statement on Lines 142–143: “As LUTS and depression are not common disease entitie, we used ORs to estimate relative risks.” (note “entitie”) would be true but only requires LUTS to not be common, the prevalence of depression is not relevant here.
Validity of the findings: Thank you for adding details looking at collinearity (Lines 129–132) and for your response about some of the diagnostics you used. I suggest adding a reference justifying your cut-off of 2 for VIFs around Line 131. Diagnostics for the logistic regression extend beyond collinearity, and tests of goodness of fit are common here, e.g. Hosmer-Lemeshow’s test. Some readers will also want to see mention of model assumptions and diagnostics for the t-tests included in the manuscript (Line 126). Note that it is not the normality of the dependent variables that matters, but the residuals of the model, which for a t-test is equivalent to normality within each group, and I think it’s worth being careful when explaining this point in the text.

Because this is observational data, I think more caution is needed in interpreting the results, particularly in the absence of a thoroughly justified causal model. For example, perhaps Line 191 should read something like “…our study suggests that multiple factors MIGHT be involved…” Similarly, the “should” on Line 192 is very definite, more than I feel is justified here. There is no evidence here, after all, that LUTS would not predict depression after adjusting for plausible available confounders (and indeed, you discuss this causal direction for the association on Lines 201–203), and both could be being caused by a third variable (as you discuss on Lines 205–206). I think the results here justify looking at interventions, but caution is definitely needed without those RCTs.

Lines 251–252: While this is true in terms of designing the study, the actual power of the study is reflected in the widths of the 95% CIs and so the actual adequacy of the sample size can be evaluated here based on these widths and their relationship to clinically important effects.

The point on Lines 254–255 does not make sense to me. If there are interactions that exist in the data generating process and these are unmodelled, this makes the estimates suspect (the model is mis-specified).
Additional comments: The manuscript has been appreciably improved and reads much more clearly now.


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: INVESTIGATING THE RELATIONSHIPS AMONG LUNG FUNCTION VARIABLES IN CHRONIC OBSTRUCTIVE PULMONARY DISEASE IN MEN
Review round: 1
Reviewer: 1

Output:
Basic reporting: There is only one figure in the manuscript.
The figure is of medium quality and contains unnecessary fragments - e.g. “give reasons”, when analyzed group is zero.
It raises my reservations. Figure 1 is described as a flow diagram of 107 people with COPD, while the graphics and text show that the number of registered people was 105. How many people actually were?
It should be more precise and clear.
Experimental design: The inclusion and exclusion criteria you refer to at work should be clearly and precisely described. Now I cannot see exclusion criteria at all.

In line 111 you write about FEV1/FVC ratio, but I cannot be sure if it is pre or postbronchodiator measurement.
In line 111 you write you have patients enrolled with FEV1/FVC ratio between 0.7-0.75.
We should know how many. Can you check if these patients have FEV1/FVC ratio below lower limit of normal (LLN)? How you comment it if you are asked for GOLD document point of view?

Aaron et al (Am J Respir Crit Care Med. 2017 Aug 1;196(3):306-314. doi: 10.1164/rccm.201612-2531OC.) described the problem of diagnostic instability and reversals of Chronic Obstructive Pulmonary Disease diagnosis in individuals with mild to moderate airflow obstruction. Why haven’t you taken it into consideration?

In line 111 you write about definite obstructive pattern in spirometry. You should define it adding correct reference.
Importantly, I would suggest that the information in Table 1 should contain more detailed information characterizing the group of patients. I advise that the table show what were the number of groups for each interval of degrees of obstruction that you use.

Mannino et Biust (Mannino DM, Buist AS. Global burden of COPD: risk factors, prevalence, and future trends. Lancet 2007;370:765–773) have shown that prevalence of COPD among men and women e.g. in China. Your enrolled group described in line 113 is deeply not consistent with above. Could you explain it, please?
If you decide analyze men COPD population only, you should think about changing the title by adding "in men".

In line 119 you write long-acting beta agonists were stopped 12 h before measurement. Haven’t your patients used ultra-LABA e.g. indacaterol?
In line 133 word “to” is used twice.
In line 144 you decide to exclude the patients because of co-existing asthma. It is deeply not precise. What kind of criteria have you used to do it. It should be described.
In line 145 and 146 you write about moderate airflow obstruction in most patients. You should precisely inform about numbers and you should define which division of obstruction you use giving adequate reference.

There is a lack of information if DLCO measurement was correlated with hemoglobin level.
Validity of the findings: no comment
Additional comments: Dear Author,

It was nice to get the opportunity to review the manuscript
"Investigating the relationships among lung function variables in chronic obstructive pulmonary disease".
I was reading it with interest however I need to share my honest opinion.

Undoubtedly, the weakness of this work is the size of the group. However, I am still open to analyze such small population. But in such case all aspects of the methodology of work must be perfect. This is not here in my opinion, what I have included in detailed comments.

For example, I have serious doubts that all patients actually had COPD. I do not know how asthma was diagnosed.
I see a lot of inaccuracies that require unquestionably improvement.

In my opinion, the manuscript has potential and should be published after the major amendments.

Sincerely,

Reviewer


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

Input:
Title: INVESTIGATING THE RELATIONSHIPS AMONG LUNG FUNCTION VARIABLES IN CHRONIC OBSTRUCTIVE PULMONARY DISEASE IN MEN
Review round: 1
Reviewer: 2

Output:
Basic reporting: - English language (expression & grammar):
The following lines need revision: 41-42, 88-89, 210-211, 252 & 265-266.
- Literature references:
All citations within the text should be revised. Please note that when using “et al.”, only the name of the first author should be mentioned.
- Professional article structure:
Acceptable.
Experimental design: - The research question:
Acceptable.
- Investigation & ethical standard:
Acceptable.
- Methods:
Well described.
Validity of the findings: - The findings are original & valid. The conclusion is well stated.
Additional comments: - The third objective (lines 43-44) is not clear.
-  The statements mentioned at lines 212-215: (… in relation to exercise capacity as RV/TLC, RV% and FEV1% belong to different components, denoting that air trapping played a more important role in exercise capacity than hyperinflation and airflow obstruction is not supported by convincing findings). Even the reference cited (Zhang Y et al., 2013) reports a different indicator (IC/TLC rather than RV/TLC).


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: INVESTIGATING THE RELATIONSHIPS AMONG LUNG FUNCTION VARIABLES IN CHRONIC OBSTRUCTIVE PULMONARY DISEASE IN MEN
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: You have significantly improved the quality of work. The message is currently more readable and comprehensible.


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: PLANT COMMUNITY RECOVERY FROM INTENSE DEER GRAZING DEPENDS ON REDUCTION OF GRAMINOIDS AND THE TIME AFTER EXCLOSURE INSTALLATION IN A SEMI-NATURAL GRASSLAND
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: General comments:

Below you find the review of the paper. However, during literature search I found the following publication: (Nagaike, Ohkubo & Hirose, 2014). This publication uses a survey from 2008 until 2012 and the 1981 plots as reference. This study has one author in common and was not even mentioned in the current submission. Why was this paper and its findings not mentioned given that it is dealing with the same question? I don’t think it is publishing the same data here, however I find this very suspicious.

The study has a sound data set and ask what factor might hinder a plant community to recover within a deer exclosure. Very nice is the fact that data from 1981 of the plant community is available and it is assumed these plots have not experienced very high deer grazing pressure. Besides many specific comments I have some major recommendations for the ms. First, I would recommend to include the fact that there was a full exclosure while 1981 was not a full exclosure in the discussion on why there were differences between 1981 and the recent survey. 1981 was not a exclosure meaning there was still deer herbivory, right? Second, and that’s my major criticism, the study should not aim at determining the factor that may have hindered the community to “reach” the 1981 status. It’s only the observational data of the above ground plant community available, and in order to determine what drives the system data on at least abiotic (eg.:nutrients) and/or maybe biotic (eg.:root competition, seed dispersal) and/or their interaction (eg.:plant-soil feedback) is needed. The importance of investigating such aspects has been shown for grasslands (Haynes et al., 2014) as well as forest understory (Stephan et al., 2017). The study is citing examples from grasslands and forest understory quite generally but I think it could be an advantage to better highlight in which system the dominance of a species was observed (eg.Nuttle 2014 is cited as example of a dominant unpalatable plant).
Although stated that the ms had a language check several paragraphs need a language check.

Specific comments:

1: so which factor determine the recovery? It’s better to state the main message of your study

17: is the separation in Background, Methods conform with the journal format?

18: Language: “exclosures that exclude”

20: Language: “However, previous studies shown that the installation of exclosure does not promise the plant community recovery“; from here after may only shortly indicate language issues with an “L”; I strongly recommend a professional language check

30: L increase of year

34: L in outside

50/64: yes, the effect of deer exclosure may not be the same in every case, please elaborate on this point

77: “which has increased since the 1980s” in Japan
79: “most of the semi-natural grasslands in Japan decreased drastically”, what decreased? Their abundance/area/quality…

86: from:” the factors for the plant community recovery” to: “the factors that drive the plant community recovery”
89: from:” coverage of plant” to: “coverage of plants”
91: I would use “recommendations”, rather than “guide”; also: I have not find a guide in the discussion, in fact I have not found recommendations (or maybe they have to more clearly identified. Like: “in cases… we recommend.. and it should be considered that…”)
102: you mean Table S1; “kaempferiand”
132: what does the “[10 outside]” mean? Please rephrase how many outside each; I still don’t know if it is 10 or 22 inside a fence; do you mean 2010: only inside and 2011 inside and outside?? This is very unclear

144: tolerant to…
182: “total number of species in past (i.e., 1981)” defining “past” and use it as name is not very elegant; why not just use the years as names (or “1981 plots”; “2010 plots”,…)
188: figure 1 label: change “species” to “life form”
188: please provide more details in the legend: is only the mean of the original values from all plots shown? (I guess after removal of occurrences below 1??)
191-192 and 192-194: L: please rewrite with professional language help
Table 1 and 2: I think it is not enough to show only the estimates of the minimal adequate model after backwards model selection; I prefer to see what was included from the beginning meaning either you show the starting model in the appendix or show the dropped parameters somehow; you should definitely report p-values (and the AIC itself if starting/intermediate models are shown); “Estimated coeﬃcients of GLMM about dissimilarity index with the lowest AIC” is very uninformative; at least “Mean and standard error of estimated coeﬃcients of a GLMM on the dissimilarity index. Only final model after model selection based on AIC is shown”
I am also confused with “when the coeﬃcients of graminoid was set as 0”. You are playing around with the reference level of the model to get the estimates of the remaining levels? Please explain how and why this is done (I don’t think things were set as 0, but that you use Graminoids as reverence level). Alternatively, I recommend to use the Anova function from the car package in order to get the summary of your model and the emmeans and mulcomp package (glht) in order to compare estimates (for the clmm apparently car::Anova is working, see e.g.: http://rcompanion.org/handbook/G_12.html; for emmeans/ multcomp::glht you may have to check).
194: avoid using species if you have analyzed life forms
201: it is unclear what the “+” means in the figure
208: L: “and that decreased after 2014”; it is unclear if there is a decrease as there are not statistical pairwise comparisons among the estimates (please compute them using emmeans, glht and you can get the letter display using multcomp::cld); given the large sd I doubt at the moment that fence 2010 is decreasing
209: you mean table 2? And fig. 3????

227: are you sure it is resistance to grazing that makes graminoids more successful? I think it is tolerance

233: L
238: now tolerance is cited; please be careful in the hole manuscript regarding which of these fundamental different defense strategies you are talking about

238: L: tolerant to/against grazing

241: please explain; why should colonization be facilitated; I think it more likely they persisted or grown form seedbanks; one thing that I could imagine is that graminoid roots force herbal plants to grow deeper meaning they are less prone to droughts; but again, I don’t see the need to explain why there are some herbal plants left outside

247: I would not say escape grazing; there will still be some; I would rather say may experience less grazing

250: L: “These species contribute the presence”… frequently there is a “to” missing
251: are you trying to say these forbs did not majorly contributed to the dissimilarity?? Please explain
253: after having read the paper I doubt it is possible here to determine the factor explaining the recovery process; if belowground patterns, plant-soil feedbacks, nutrient cycling would have been monitored there would have been a chance to say something about what is more important to determine the observed communities

258: I am not convinced that things would counterbalance; I think it depends on many factors

261: soil compaction in dear presence is assumed to be higher as well as nutrient intake and different life forms respond differently to this; I think concluding that competitive exclusion is hindering recovery is too speculative

271: L

279: I think that hints on a major problem of your study: 1981 plots experience some disturbance (grazing, fire?) but your plots did not had any disturbance at all; please discuss this

References

Haynes AG, Schütz M, Buchmann N, Page-Dumroese DS, Busse MD, Risch AC. 2014. Linkages between grazing history and herbivore exclusion on decomposition rates in mineral soils of subalpine grasslands. Plant and Soil 374:579–591. DOI: 10.1007/s11104-013-1905-8.
Nagaike T, Ohkubo E, Hirose K. 2014. Vegetation Recovery in Response to the Exclusion of Grazing by Sika Deer ( Cervus nippon ) in Seminatural Grassland on Mt. Kushigata, Japan. ISRN Biodiversity 2014:1–6. DOI: 10.1155/2014/493495.
Stephan JG, Pourazari F, Tattersdill K, Kobayashi T, Nishizawa K, De Long JR. 2017. Long-term deer exclosure alters soil properties, plant traits, understory plant community and insect herbivory, but not the functional relationships among them. Oecologia 184:685–699. DOI: 10.1007/s00442-017-3895-3.


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: PLANT COMMUNITY RECOVERY FROM INTENSE DEER GRAZING DEPENDS ON REDUCTION OF GRAMINOIDS AND THE TIME AFTER EXCLOSURE INSTALLATION IN A SEMI-NATURAL GRASSLAND
Review round: 1
Reviewer: 2

Output:
Basic reporting: In this manuscript, the precious data gained by the high school science club in 1981 was utilized effectively, which enables to describe the effect of recent over-grazing by deer on the species compositions straightforwardly, and to indicate the degree of plant community recovery after deer exclusion fence installation by the direct comparison between past and recent data. It is worth publishing after some corrections listed below.
Experimental design: See "General comments" below.
Validity of the findings: See "General comments" below.
Additional comments: 1. I have a concern about the way of use the word "original" because I don't think the situation in 1981 was "original" in this region. The authors should make it clear what is original and add some references, or change it to another word like "past" instead throughout the manuscript.

2. It is interesting that a delay in exclosure installation of only one year could result in a delay in plant community recovery of more than one year. Even though the fact may supply an important suggestion for acceleration of exclosure installation at the heavily grazed region, its reason is not discussed well. The authors partly explained the reason in L272-273 by unpublished data, but should show the data explicitly to discuss how the heavy grazing affect the plant community outside the fence 2010 and why the recovery after the exclosure installation has delayed within the fence 2011, from a plant ecological aspect. This part is especially important in this manuscript. If the authors fail to discuss in this revision, the manuscript must be rejected in the PeerJ.

3. "Conclusions" part is not informative. Add some brief explanations how these factors (such as, the dominance of graminoids and the time after the fence installation, L283) affected in the studied grassland, and make to correspond to the title of this manuscript.

Specific comments
throughout Introduction; Heavy use of "however" in the main text seems to make the logical structure poor. Reconsider the way of using conjunctions and polish up the sentences.
L107; omit either of "in this area".
L108, 110; omit "calculated by".
L120; I think Takatsuki (2009) showed the overall trend of Japanese deer population in Japan. Does this reference include the authors' studied area?
L124-127; "Although ~ construction." Unnecessary information. Omit it.
L131-132; Change to "We deployed 22 1 × 1 m quadrats inside each of the exclosure areas (fence 2010, 10 quadrats; fence 2011, 12 quadrats), and 10 outside."
L134-135: "vegetation ~ grazing." Omit.
L151 cumulative "logit" mixed model?

Discussion
L274-279; These sentences are too descriptive and unnecessary. They don't include anything found from this study.

Figure2; What does "+" indicate?
Table1, Table2; Show the z-value, the probability (P value) and the statistical significance in each parameter.


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: SOIL BACTERIAL BIODIVERSITY IS DRIVEN BY LONG-TERM PASTURE MANAGEMENT, POULTRY LITTER, AND CATTLE MANURE INPUTS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Please see my general comments below
Experimental design: Please see my general comments below
Validity of the findings: Please see my general comments below
Additional comments: In this manuscript, Yang et al described their study on the impact of long-term conservation pasture management on soil bacterial diversity using 16S rRNA gene amplicon sequencing. This is a very interesting studies. The manuscript is well written.

My comments are given below

1) The authors started with soil health. Based on the text, I can see microbiome diversity is an important factors. However, more detailed information on key factors affecting soil health is necessary for both introduction and discussion.

2) The raw data processing is quite ambiguous  to this reviewers.  It seems all analysis are at the phylum level. There is no mentioning of the basic unit OTU or ASV (amplicon sequence variant). Additionally, 16S data is often compared at family or genus level where functions are more specific or meaningful. Please explain here

3) Are there similar studies on soil microbiome using similar technology? In any case, it is important to start with summary of the overall community profiles at the different taxonomy levels before performing any comparison. This can serve as reference for future studies, for instance, a table or taxonomy trees describing the abundance of key taxonomies at different levels

4) Figure 5 LEFSe results is also strange. It should all more taxa with some of them are significant. Showing only two taxa is really unusual

Minor comments:

1) One important findings are the impact of poultry litter and cattle manure on microbiome diversity. This is clearly of agricultural interest by themselves. Are there studies on the microbiome of poultry litter or cattle manure? It will be very interesting to compare or at least comment on this matter

2) Figure 2 and 3 are a bit repetitive, could be merged to be more informative.

3) Suggest to remove: L324-336 and Supplementary materials are not very interesting as the predicting of microbiome functional potential are mainly for human gut microbiome, PiCRUST performance is very poor for soil micrcrobiome

4) L232-233 Please explain "these results suggest the main bacterial functions remained structural stable" the reviewer don't feel this is logical conclusion from the previous text


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: SOIL BACTERIAL BIODIVERSITY IS DRIVEN BY LONG-TERM PASTURE MANAGEMENT, POULTRY LITTER, AND CATTLE MANURE INPUTS
Review round: 1
Reviewer: 2

Output:
Basic reporting: This paper meets the requirements for language, background, structure, and hypothesis.
Experimental design: The experiment consists of an appropriate, long-term trial and its research questions and methods are appropriate.
Validity of the findings: The paper suffers from only a need for minor corrections to clarify some findings.
Additional comments: In this paper, Yang et al. present their findings on the effects of a thirteen-year pasture management experiment on the soil microbiome. The authors show that land management and manure amendments created a gradient of soil bacterial richness which was lowest in sites that received no manure inputs and highest in continually-grazed cattle pastures. The authors suggest that increased grazing pressure may promote belowground biodiversity. The manuscript is generally clearly written, though there are a few minor points that need clarification (see below). My only major concern is the paragraph discussing PCoA based on Bray-Curtis dissimilarities across treatments. I feel this figure is being under utilized. PERMANOVA could also be applied to this section to determine if the clustering of the pasture management is meaningful. Otherwise, there are a few changes that I think are important for clarity, especially for readers coming from outside of the livestock/agronomy sphere.

Specific comments:

L40-Throughout: There is inconsistency in the use of broiler litter and poultry litter throughout the manuscript. I think it would be better to stick to poultry litter throughout.

L46-53: This summary is awkward as it alludes to the negative effects of grazing on plants, though this is not explored. Also these sentences are somewhat difficult to follow. The use of albeit to link disjointed statements does not help

L65-Throughout: As previously, there is inconsistency across the manuscript using H for hayed e.e. L117.

L75: Is penetration resistance referring to water or physical intrusion?

L89-90: Citation needed.

L96-97: This statement could benefit from further citation.
L103-104: Which citation goes with which statement?

L129-130: A citation for these soil classifications (i.e. FAO classification? USA classification?) is needed.

L132-149: A more clear description of the control treatment should go here.

L162-166: Were samples homogenized?

L180-183: I can’t tell if this is just a fancy way of saying rarefied?

L186: R is software, not a package.

L250-252: This discussion on Verrucomicrobia should be expanded. There is growing interest of its role in soils Navarrete et al 2015 (doi: 10.1007/s10482-015-0530-3), Bergmann et al., 2011 (doi: 10.1016/j.soilbio.2011.03.012), Cabello-Yeves et al., 2017 (doi: 10.3389/fmicb.2017.02131)

L256-throughout: The authors consistently remind the reader that they are using the Chao index. Personally I do not think this is needed, but the authors may disagree.

L314: Is microflora an appropriate term? Would microbiome be better?

Fig 5: I think this figure should be moved to the Supplementary Material as it does not really add anything to the paper.

Fig 7: Purely an aesthetic suggestion, but I think that the dots should be coloured by treatment as well to make the distribution of data clearer to