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: PATIENT-REPORTED OUTCOMES MEASURED WITH AND WITHOUT DIZZINESS ASSOCIATED WITH NON-SPECIFIC CHRONIC NECK PAIN: IMPLICATIONS FOR PRIMARY CARE
Review round: 1
Reviewer: 2

Output:
Basic reporting: The present study aimed to compare quality of life and disability among patients with chronic non-specific neck pain with or without associated cervicogenic dizziness. This study has an appropriate design but there are some aspects that should be addressed with the aim of improving the quality of the study. In spite of this, I believe that this is a useful study that provides interesting information in this field.
Experimental design: Sample Size (Line 138)
Is this calculation correct?
I think the authors should check it out.
Validity of the findings: No comment
Additional comments: This study was well described.
-General comments
Introduction section
Line 21-27: NSCNP definition should be reformulated. A brief description of why chronic neck pain is considered mostly nonspecific should be included (e.g: difficulty in establishing an accurate pathological diagnosis, the role of imaging test, multidimensional nature, etc.).
Line 37-41: A brief differentiation between cervicogenic dizziness and several clinical entities dealing with vestibular alterations should be included here (e.g. whiplash and associated disorders, BVVP vertigo (neurophysiological framework)).
Line 52-55: Objectives should be rewritten by please deleting parentheses.
Results section
My main concern in this section is the following:
Patients with NSCNP without cervicogenic dizziness had a mean of 55 months of chronicity (approx.). However, kinesiophobia scores showed a mean of 24 points. In addition, the NDI showed mild disability. I feel that these values are low compared to the chronicity time shown by these patients. This should be discussed.
In addition, Could the authors add the kinesiophobia score that the scientific literature reports as clinically relevant?
Finally, the comparison of the subscales (Harm and Activity avoidance subscales) between the two groups should also include (results section and also in Table 4).
Discussion section
The discussion should be deeply broadened.
Previously, the neurophysiological model has been mentioned. The hypothesis of this neurophysiological model resides in the fact that the alteration of a sensory input, specially provided by the neurophysiological pathways between the upper cervical region and the subsystems that form the postural control, could cause sensations of dizziness. It is therefore that this hypothesis resides in an integration incongruity between the aberrant cervical somatosensory input and the expected physiological sensory patterns. This was reported by Kristjansson and Treleaven a few years ago. Here, vestibular and somatosensory systems could compensate for alterations in balance by increasing the rigidity of the body, mainly in the cervical region, thus explaining the hyperactivity of the cervical musculature in patients with cervicogenic dizziness. However, Deborah Falla, Gwendolen Jull and colleagues (2004) found that patients with NSCNP presented an alteration in cervical neuro-sensorimotor control and conversely, these patients did not present dizziness. All of this should be discussed.
Apart from that, Ischebeck and colleagues (2017) recently have found that the cervico-ocular reflex is increased both, in patients with chronic non-traumatic neck pain without dizziness, and in patients with chronic traumatic neck pain due to whiplash with associated dizziness. The role of reflexes in comparing NSCNP patients with or without dizziness should also be addressed briefly.
In addition to this, a very relevant aspect (extremely important I would dare to say) in cervical dizziness is the cervical movement. I think that in the future, a hypothesis that goes towards the term "dizziness evoked by cervical movements" would be more appropriate than the current neurophysiological hypothesis “cervicogenic”. It has been reported that cervical dizziness may cause a reduction in cervical mobility in general terms, and specifically, cervical rotation movement.
Why was the active cervical range of motion not evaluated in the present study?
I can understand that the objectives were to assess disability and quality of life, but I think that range of motion can also play a relevant role in this variables, don't you think?
I feel this should be considered as a limitation.


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: PATIENT-REPORTED OUTCOMES MEASURED WITH AND WITHOUT DIZZINESS ASSOCIATED WITH NON-SPECIFIC CHRONIC NECK PAIN: IMPLICATIONS FOR PRIMARY CARE
Review round: 2
Reviewer: 1

Output:
Basic reporting: the article fulfills everything
Experimental design: the article fulfills everything
Validity of the findings: the article fulfills everything
Additional comments: I congratulate the authors for the great work of adapting to the manuscript according to the requests made previously. The manuscript is ready to be published.


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

Input:
Title: PATIENT-REPORTED OUTCOMES MEASURED WITH AND WITHOUT DIZZINESS ASSOCIATED WITH NON-SPECIFIC CHRONIC NECK PAIN: IMPLICATIONS FOR PRIMARY CARE
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: First of all, I would like to thank the authors for their efforts to address all the issues requested by both reviewers. I think the study is better now and would recommend acceptance for publication.
I would just like to add, although this is already of little relevance, that the cut (value) from which kinesiophobia is considered to be present or not was not added.
 Patients with neck pain without dizziness showed less than 28-29/44 points and therefore, we cannot consider them to have kinesiophobia. It would have been interesting to compare patients with neck pain without dizziness who did have high levels of kinesiophobia. It is not clear to me whether this can be considered a limitation, but I just wanted to let the authors know.


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: ACUTE NET STRESS OF YOUNG ADULT ZEBRAFISH (DANIO RERIO) IS NOT SUFFICIENT TO INCREASE ANXIETY-LIKE BEHAVIOR AND WHOLE-BODY CORTISOL
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript by Aponte and Petrunich-Rutherford investigates the effects of net acute stress on anxiety-like behavior and cortisol levels in young adult zebrafish. The data is clear, and the manuscript is nicely written. However, I have a few issues that need to be addressed and some concerns about the methods used.
Experimental design: I think that one of the major issues in the MS is the housing condition of the animals. In both works, the first one with adult animals (Tran and Gerlai, 2014; 2015) and this one with young adults, is that they do not discuss about the fact that zebrafish are isolated prior the stress and the behavioral task. Once that zebrafish is a social specie and that previously works showed that isolation changes zebrafish behavior (Parker et al., 2012), the authors should mention and discuss about this as an important variable and as a limitation of this work. In Tran works, they also moved the isolated the animals 7 days prior the test and, in this MS, animals were kept in this condition since 15 dpf. Further works aiming to analyze those differences should be very interesting, comparing about the differences in housing conditions vs stress reactivity, once that pair-house animals may have a different response compared to those that were kept isolated. Moreover, the authors should add more details about the housing condition in the Methods section (e.g. tank size, if animals had visual contact with the others).

(lines 134 – 136) Why the animals were fed once daily? I have some concerns that this could also affect zebrafish behavior, once that this is not part of standard zebrafish husbandry.

(lines 136 – 138) “On the day of the experiment, the individual tanks housing young adult fish (approximately 90 days post-fertilization) were removed from the system and moved to the experimental room”. The experimental room had the same temperature conditions from the fish room?

Although the authors mentioned that the light/dark task was used to assess zebrafish anxiety-like behavior, two recent papers have been showed that there are differences between the light/dark and white/dark test (Facciol et al., 2017; 2019). I think that is important the authors change the terminology used here and briefly comment about these differences in either Methods or Discussion section.

(lines 159 – 161) The authors said that one of the sides were covered with black and the other one was remained uncovered which is different from some protocols that cover the other side of the tank with white material to avoid contact with the external environment. This could also impact on zebrafish scototaxis responses.

(lines 189 – 192) “For the cortisol assays, a total of four samples (two samples from each treatment group) were removed from analyses due to issues with the extraction procedure. For the behavioral analyses, two samples from the non-stressed group were excluded from analyses due to incomplete video files”. However, in the excel files, the authors said that 3 animals were excluded for the cortisol analysis and 1 for the behavioral task, this only in the novel tank. For the light-dark more 1 animal was excluded from cortisol and other from behavioral analysis. Please, check the number of animals excluded and make the appropriate correction in the MS.

I noticed that the cortisol levels of the novel tank vs light-dark have a average difference of almost 10 ng/g. Where these animals from different batches? How many batches of fish the authors run?
Validity of the findings: The discussion section is well written and the conclusion well stated, however as previous mentioned, the authors should discuss about the limitations of their experiments, once that there are variations between this and previous experiments and is not only the age of the animals.
Additional comments: In the lines 67-78, should be interesting to mention about the cortisol production as one advantage of using zebrafish. Once that many others animal models do not produce this molecule.

(lines 82-85) The authors mentioned that in the novel tank animals usually spend half of the time in each zone, however previously works described the baseline behavior of zebrafish as initial increased preference for the bottom and then a gradually exploration for the top area (Cachat et al., 2010; Blaser and Rosemberg, 2012). Furthermore, in your data the animals showed in general a preference for the bottom of the tank, so I think that the authors could change this sentence and references to improve the rationale.

Please check the references, there are some places where the format is incorrect (e.g. Magno et al. (2015) - lines 102 and line 161).


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: ACUTE NET STRESS OF YOUNG ADULT ZEBRAFISH (DANIO RERIO) IS NOT SUFFICIENT TO INCREASE ANXIETY-LIKE BEHAVIOR AND WHOLE-BODY CORTISOL
Review round: 1
Reviewer: 2

Output:
Basic reporting: Graphs displaying some behaviours would helpful.
A schematic showing the behavioural testing designs and outlined behavioural zones (i.e. top) would be useful.
Experimental design: Is it possible that sample sizes may be too small? We normally consider n = 20 as appropriate for statistical power in behavioural zebrafish studies (this is specific to our lab but I could not find a simple article to support this claim). Could more samples be added?
Is euthanasia by clove oil too slow a method to accurately capture cortisol levels immediately after death? Why were fish not decapitated instead (as was the protocol in Tran et al. 2014)?
Could distance to top, instead of distance traveled in top zone, be a better measure? This may be a better behavioral measure of "bottom dwelling" (a well characterized anxiety-related behavior).
Validity of the findings: Why was an ANOVA not used to investigate a main effect of net stressor? It also would have been interesting to see the effect of the net stressor over time (repeated measures).
Additional comments: I do not think it is necessary to include how long the net stressor was every time it is mentioned. Simply explain in the methods that it was 30 seconds then just call it an acute net stressor. Continuously saying "acute (30 second) net stressor" makes the read a little hard at times, especially in the results.
Line 112 – “despite the evidence that zebrafish are demonstrate anxiety-like behaviors” please remove the “are”
Line 271 - “the lack of significant findings in the behavioural tests MAY be attributable...” please add "may" or similar word to clarify this sentence.


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: ACUTE NET STRESS OF YOUNG ADULT ZEBRAFISH (DANIO RERIO) IS NOT SUFFICIENT TO INCREASE ANXIETY-LIKE BEHAVIOR AND WHOLE-BODY CORTISOL
Review round: 2
Reviewer: 1

Output:
Basic reporting: English is very clear and the MS is well-written.
Experimental design: Research question is well defined, and the methods described had sufficient information to replicate.
Validity of the findings: The findings of this MS are very interesting and very important, especially from those scientists that works with zebrafish models.
Additional comments: The MS from Aponte and Petrunich-Rutherford was significantly improved, the new version has much more details about the methodology used and they have now addressed all the Reviewer concerns. The English is very clear, and the MS is well-written. Overall, I recommend this MS to be accepted in PeerJ.


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: ACUTE NET STRESS OF YOUNG ADULT ZEBRAFISH (DANIO RERIO) IS NOT SUFFICIENT TO INCREASE ANXIETY-LIKE BEHAVIOR AND WHOLE-BODY CORTISOL
Review round: 2
Reviewer: 2

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: All previous comments have been addressed appropriately. I have reviewed the new manuscript and have 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: DEVELOPMENT OF GENOMIC SIMPLE SEQUENCE REPEAT MARKERS FOR GLYCYRRHIZA LEPIDOTA AND CROSS-AMPLIFICATION OF OTHER  GLYCYRRHIZA SPECIES
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: The method was not enough and not clearly descripted for SSR identification.
Validity of the findings: The development of SSR markers is one of the most important part for the manuscript. However, there was nothing mentioned for the identified SSR number, the frequency and distribution of different SSR types in the genome.
Additional comments: In this manuscript entitled "Development of genomic simple sequence repeat markers for Glycyrrhiza lepidota and cross-amplification of other Glycyrrhiza species" to describe the development of SSR markers in Glycyrrhiza lepidota, and use some of these markers to assess the genetic diversity. In general, this paper is good writing, but miss several important parts in methods and results. The overall research content was not enough for published on Peer J. Certain specific comments are addressed below:
Major comments
1. The development of SSR markers from the genomic sequence is an important component of the entire manuscript, while the detail on SSR analysis in the method and result was missed. For example, approximately 28,000 SSRs were identified in G. lepidota. What is the criteria used for identification of different SSR motif? What are the frequency, distribution and characterization of different SSR motif?  These important information were not mentioned in the manuscript.
2. The Author Contributions was also missed in 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: DEVELOPMENT OF GENOMIC SIMPLE SEQUENCE REPEAT MARKERS FOR GLYCYRRHIZA LEPIDOTA AND CROSS-AMPLIFICATION OF OTHER  GLYCYRRHIZA SPECIES
Review round: 1
Reviewer: 2

Output:
Basic reporting: This is a very well done work and it is very well presented. The authors have informed about a new set of markers very useful for different studies regarding this species.
In general, writing is very clear and the objectives are very well faced.
There are some minor things that I suggested to check before accepting this manuscript.
Experimental design: The experimental design and analysis follow the standard protocols for this kind of studies.
I feel that this work is original enough to be published here.
This a very well designed and performed work.
Validity of the findings: The authors have informed about the first set of SSR markers for this particular species. It is unique and hard to perform work.
Their findings, despite limited to this species or their related ones, will have an impact on the upcoming studies in different areas.
Additional comments: You have done a very intense and hard work. There are only some minor comments that I suggest you to consider.
You will find them in the PDF draft.


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: DEVELOPMENT OF GENOMIC SIMPLE SEQUENCE REPEAT MARKERS FOR GLYCYRRHIZA LEPIDOTA AND CROSS-AMPLIFICATION OF OTHER  GLYCYRRHIZA SPECIES
Review round: 2
Reviewer: 1

Output:
Basic reporting: This the second revision of this manuscript. The authors made all the recommended changes and I suggest to accept the manuscript as it is now.
Experimental design: Minor changes recommended before were include.
Validity of the findings: No additional comments
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: PANGENOMIC TYPE III EFFECTOR DATABASE OF THE PLANT PATHOGENIC RALSTONIA SPP.
Review round: 1
Reviewer: 1

Output:
Basic reporting: The language is clear and well written. The authors use of active voice is a nice change.
Experimental design: The rationale behind the analysis is clearly laid out. Treating frameshifts as fall calls seems questionable but the database makes the call clear and checking each frameshift is beyond the scope of this study, although systematic verification of frameshifts and pseudogene status really should be on the priority list.

The T3SS secretion assays are lacking a cytoplasmic leakage control.
Validity of the findings: The authors are clear as to why they have made their analysis choices and openly acknowledge any limitations.
Additional comments: This is a truly valuable and important resource for the community of MPMI researchers.


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: PANGENOMIC TYPE III EFFECTOR DATABASE OF THE PLANT PATHOGENIC RALSTONIA SPP.
Review round: 1
Reviewer: 2

Output:
Basic reporting: The authors improved a resource that is extremely valuable to the Ralstonia solanacearum community. I do not see any issues with this manuscript besides English language and grammar. In several instances, sentence structure, punctuation, singular/plural, and past/present tense need to be corrected. In some cases, these mistakes make it difficult to understand the meaning of sentences. I corrected some of these mistakes myself (see attached pdf) but I think it would be best if the authors still employed a professional editing service to do so in order to ensure a high quality manuscript.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: Impressive work! I am looking forward to seeing this published.


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

Input:
Title: PANGENOMIC TYPE III EFFECTOR DATABASE OF THE PLANT PATHOGENIC RALSTONIA SPP.
Review round: 1
Reviewer: 3

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: In general, I found this manuscript to be well written and timely. The goal of this work is to update a previous iteration of the Ralstonia effector database and provide increased usability in terms of the functions and strains included. As such, it's a pretty straightforward descriptive work that *could* be useful to a variety of different research groups.

I really have no major qualms with the manuscript, the database and data are what they are and the authors do a good job of highlighting strengths and weaknesses in their approach.

The authors were careful in evaluating genomes for quality before inclusion, but I would suggest one more metric (since even complete genomes can be polluted with a bunch of frameshifts due to errors in long reads). Mick Watson came up with a handy way to at least quantify how often these frameshifts might be present in the genome (http://www.opiniomics.org/a-simple-test-for-uncorrected-insertions-and-deletions-indels-in-bacterial-genomes/) and I think it might be good to use a metric for the Ralstonia genomes. I'm guessing it won't change much, but something to think about as a platform for comparison of genome quality across sets.

Otherwise, some slight critiques:

L22: "causing the vascular" better as "causing vascular"

L52-53: "This bacterium is also particularly studied as it" could be rewritten a bit for clarity

L56: "bacterium in interaction" better as "bacterium during interaction"

L62: Legionella should be capitalized?

L78: "accommodating" better as "enabling"

L81: "This was performed taking into" better as "This analysis took into account:

L89: please define more clearly what you mean by "complete" genomes

L106-108: please include more details about why these genomes were of insufficient quality

L141: better as "and nine only contain"

L159-160: please rewrite this first sentence for clarity

L182: please include details of the PCR reaction conditions

L210 (and throughout): please evaluate all instances using the word "homology". Homology implies the exact same function. With effectors it's difficult, but for where you are just talking about sequence similarity, please replace the word "homology" with "similarity"

L269: ",the least Rips are conserved" better as "the lower the number of conserved Rips"

Please make sure that the words within the figures are legible (perhaps larger and a different font), I'm not sure that they are in this iteration.


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

Input:
Title: MOLECULAR EVIDENCE THAT THE CHANNEL ISLANDS POPULATIONS OF THE ORANGE-CROWNED WARBLER (OREOTHLYPIS CELATA; AVES: PASSERIFORMES: PARULIDAE) REPRESENT A DISTINCT EVOLUTIONARY LINEAGE
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: I really enjoyed reading Hanna et al. The paper is very well written and is an important contribution to the phylogeographic and ornithological literature. I especially appreciated the detailed and thorough descriptions of the methods, because the genetic data did not have clear phylogeographic breaks in the form of clades the authors were forced to examine several populations groupings. This required detailed descriptions of how the samples were grouped and the authors should be proud that these descriptions were clear and concise. I found them very easy to follow and made the paper pleasant to read.

I believe the paper is acceptable for publication in PeerJ. There is little to critique with the manuscript overall. The figures and tables are all necessary and easy to read. The methods clear and the data generated done so following proper animal care and use guidelines and plans made to make datasets etc publically available. My only overall comment concerns the discussion. There are a few confusing comments made about incomplete lineage sorting and gene flow. This is the only part of the paper where I believe the authors get tripped up. It appears at the beginning of the discussion they make the argument that lineage sorting is responsible for shared variation among populations but later on they contradict this statement and suggest it is geneflow. I just think these ideas need to be reconciled and it wouldn’t require a major revision to do so. Below I point to the specific instances of discord in the text.

Line 421-423: I am not convinced that the lack of structure and high levels of shared alleles is solely due to incomplete lineage sorting. Seems to me that there is just as good of a chance that there is high gene flow that flows through adjacent populations which is why you see a pretty strong signal of IBD (the most distant populations being the most diverged). I would just be cautious with this statement.

Line 432-435: I understand your reasoning that the low diversity might be indicative of a founder event, bottleneck or strong selection, but your mismatch and D and F stats don’t appear to back up this conclusion. So what makes you lean towards such events. Low diversity on islands can also come from persistent stable but small populations.

Line476-477: This statement contradicts you statement in the opening paragraph that suggest incomplete lineage sorting is the process behind shared variation.

Minor recommended edits:

Line 40: delete “and” at the end of the sentence.


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

Input:
Title: MOLECULAR EVIDENCE THAT THE CHANNEL ISLANDS POPULATIONS OF THE ORANGE-CROWNED WARBLER (OREOTHLYPIS CELATA; AVES: PASSERIFORMES: PARULIDAE) REPRESENT A DISTINCT EVOLUTIONARY LINEAGE
Review round: 1
Reviewer: 2

Output:
Basic reporting: The paper is well-written, though as in all such papers the molecular laboratory methodology and bioinformatics analyses are essentially impossible to present in a fashion readable and intelligible to the non-specialist (note that this is not a generic problem, not a failure of this particular contribution).   The general topics, results and interpretations are presented clearly.

In setting up the study (lines 26-28), it might be worth mentioning that there is geographical variation in songs of Oreothlypis celata, and the slightly slower and more patterned songs of sordida (in contrast to more rapid, less patterned songs of the nearest populations of lutescens) have been commented upon by some authors.  In the absence, however, of well-designed, quantitative studies of song variation, perhaps there is nothing to site here beyond some qualitative mention in popular literature (Dunn and Garrett 1997). Perhaps also worth mentioning that sordida is the only subspecies that regularly nests well above ground level (see BNA account).  The habitat term "riparian chaparral" (line 31) is a bit odd, but certainly lutescens does breed commonly in the chaparral/riparian ecotone. Line 39 (breeding range of orestera) might better say "and in some mountain ranges within the western deserts of North America" so as not to imply they nest in desert habitats; also, that sentence seems to be truncated in my version.
Experimental design: One very minor comment: Regarding population sampling (lines 66-74), bear in mind that some southern California coastal breeding Orange-crowned Warblers can disperse well away from breeding areas by mid to late May (e.g. up into the higher mountains or even onto the deserts), so you might point out how you took care to be certain your samples came from breeding sites and did not include dispersing post-breeders.
Validity of the findings: Regarding your population #7: It is unfortunate that there were no available specimens and no realistic collecting opportunities in places like the Santa Monica Mtns. in Los Angeles/Ventura Counties where lutescens nests in close proximity to mainland populations of sordida (the latter well-known from the Palos Verdes Peninsula but probably locally a bit farther up the coast, and in several populations down the coast in Orange and San Diego Counties). You might look at Philip Unitt's comments in the San Diego Bird Atlas on the status of sordida in San Diego County, the southward range expansion of lutescens there, and the possibility that lutescens if "swamping out" sordida in costal San Diego.
Additional comments: A good contribution that does not exactly argue that current subspecies delineation is flawed (insofar as the subspecies concept is informative, as I believe it to be), but does demonstrate that the underlying genetics are far more complex.  Your conclusions of the distinctiveness of sordida will hopefully spur more work on the ecology, behavior and phenotypic distinctness of this taxon, as well as a deeper investigation of the relationship between coastal mainland breeding populations of lutescens from Santa Barbara and Ventura Counties south to San Diego County and the localized mainland breeding populations of sordida.


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

Input:
Title: MOLECULAR EVIDENCE THAT THE CHANNEL ISLANDS POPULATIONS OF THE ORANGE-CROWNED WARBLER (OREOTHLYPIS CELATA; AVES: PASSERIFORMES: PARULIDAE) REPRESENT A DISTINCT EVOLUTIONARY LINEAGE
Review round: 1
Reviewer: 3

Output:
Basic reporting: Mostly fine.
Experimental design: This is good -- strong geographic sampling in western N America; mtDNA and nuDNA.
Validity of the findings: I thing IBD is overemphasized given the overall results; more suggestions below.
Additional comments: PeerJ #31580. Hanna et al. on orange-crowned warbler population genetics.

The authors used mtDNA and 10 microsatellite loci to assess genetic properties of the orange-crowned warbler across part of its range (focusing on western North America), including all four recognized subspecies and 192 individuals. The sampling represents a considerable degree of field effort over many years. Overall, there were low levels of divergence, attributed in the abstract to IBD (though I would change; see below). Channel Islands populations are focused on as being most distinct.

Overall, the dataset, analyses, and writing are fairly sound. I do have some suggestions for improvements, however. With the infusion of some arbitrariness in making geographic assignments, the geographic relationships and differences are enhanced. This results I think in needless de-emphasis of insular isolation and phenotypic attributes (subspecies), which seem to be much stronger influences on patterns in the data.

The paper focuses rather heavily on the second of four arrangements of geographic analyses (Figs. 1, 2b[needs to be labeled that], and 3). This arrangement partitions the samples (incompletely) into 8 populations (Fig. 1, lines 75-78). Designation of some of these populations and omissions of some of the sampled localities (Fig. 1, NW Canada) seems to have a great degree of subjectivity. The omission of 7 disparate points in NW Canada and the island-mainland separation of populations 7 & 8 would bias the subsequent analysis. You can see the bias this would introduce to enhance different signal in the data: eliminating samples in NW Canada that are similar across great distances and emphasizing the islands (dissimilar over short distances) as separate populations despite proximity. Ultimately, there were four groupings considered, but the first, with only locality and with “weak” evidence (line 387) for geographic distance being important is unduly de-emphasized. I am not objecting to multiple approaches, because it is useful to explore what factors best explain the distribution of genetic variation across this space, but a key result remains “these initial analyses did not reveal spatial structure in the genetic data” (line76), and the paragraph beginnng on line 384. The overall approach used can appear as being “let’s throw things at the wall until something sticks,” and in Methods at least it might be delivered better. Currently it reads like “Well, straight-up raw analyses didn’t show anything interesting, so we tried these other things...”

I am a skeptic of using locality priors in Structure analyses. Its effect is to impose some structure where it might not exist on a genetic basis alone. That is problematic in organisms with large dispersal distances. With these animals being seasonal migrants with imperfect site fidelity and probably long dispersal distances, pure analyses of the underlying genetic properties are more informative. So Figure 3 should not be the Structure result that takes priority. The first Structure analysis that includes all samples and no locality information (line 391, K = 1) should be at a minimum 3a. That is much more comparable to most published Structure analyses in the literature. The legend also needs to include what data were used and whether priors were used.

Despite there being a *lot* of statistical tests in Tables 1-4, S2-S4, there is little evidence of multiple test corrections (line 334 mentions Bonferroni corrections outside tabulated results). This has become less acceptable, and it is where the penalty is usually paid for a “let’s see what sticks” approach. Careful, methodical planning between rounds of tests is usually evident to balance these tradeoffs. This should be addressed. It is partly dealt with in language in the text looking for consensus patterns among analyses, but more is needed.

The gene flow analyses seem incomplete. I have not used IMa2p with mtDNA and microsatellies, but most analyses on this topic provide an actual rate estimate of individuals per generation and effective population size estimates, not just that gene flow is asymmetrical and that population sizes differ in the expected direction. Traditional analyses (which the authors were not shy to use elsewhere in the study) should be used to analyze mtDNA and microsatellite data separately to provide effective populations size and gene flow estimates (which can then be directly compared to a vast literature on these topics in population genetics). Using more traditional IM analysis on mtDNA could also provide a decent divergence estimate and improve the authors’ suggestion in the Discussion (lines 453-459) that divergence is more recent than previously suggested.

The ending seems really limp to me, and actually misdirecting from the strongest takeaway results. IBD was not supported with mtDNA and it was remarkably weak with microsatellites. On the other hand, insular isolation on the Channel Islands produced the strongest signal of population divergence, and the significant differences among subspecies (Table S2) were also quite remarkable. How is this consistent with IBD “having generated the genetic distances and clusters we observed”? This overemphasizes the IBD signal and underemphasizes other aspects of the data. To me, the results indicate that life history (lower migration) plus island isolation and then, among other subspecies, ecological/life history aspects not considered are more important drivers of the distribution of genetic variation in this species across this space. In fact, an a priori prediction would have probably ranked island isolation, subspecies, and distance in that order as the likely best explanatory variables. If the authors think not, a more convincing case should be made for the aproach they’ve taken to emphasize distance alone. For a start, I’d move Fig. S3 and Table S2 into the main body of the paper, then provide a more robust consideration of all the factors to explain the distribution of genetic varaition in the data. (And did I miss an AMOVA explanation of the amount of variation explained by subspecies?).

Other notes made:

Line 302: “...we recovered significant isolation by distance in the microsatellite data” is contrary to lines 76-77 (“did not reveal spatial structure”). In fact, the raw microsatellite results in showing such a poor relationship (lines 386-390) corroborate the SAMOVA result here: IBD is not an important component of these populations. I think this needs emphasis.

Line 422: Why focus only on ILS? Gene flow is probably far more important for the “high level of shared alleles.”

The supplementary tables need legends in the files, including what the values are, what the data are, and the singificance levels of the asterisks.


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

Input:
Title: MOLECULAR EVIDENCE THAT THE CHANNEL ISLANDS POPULATIONS OF THE ORANGE-CROWNED WARBLER (OREOTHLYPIS CELATA; AVES: PASSERIFORMES: PARULIDAE) REPRESENT A DISTINCT EVOLUTIONARY LINEAGE
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: I have no further comments. The authors have fully addressed my previous comments and concerns. Nice work.


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

Input:
Title: MOLECULAR EVIDENCE THAT THE CHANNEL ISLANDS POPULATIONS OF THE ORANGE-CROWNED WARBLER (OREOTHLYPIS CELATA; AVES: PASSERIFORMES: PARULIDAE) REPRESENT A DISTINCT EVOLUTIONARY LINEAGE
Review round: 2
Reviewer: 2

Output:
Basic reporting: All of the minor concerns I expressed in the initial round of review have been adequately addressed by the authors.
Experimental design: No comment
Validity of the findings: no comment
Additional comments: Again, all of the minor concerns I expressed in the initial round of review have been adequately addressed by the authors. These concern some details on breeding seasonality, potential issues with early post-breeding dispersal, possible vocal differentiation of sordida, and areas where sordida and lutescens breed in close proximity on the mainland.  I reiterate that I am not qualified to assess the techniques and conclusions of the molecular work.


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

Input:
Title: MOLECULAR EVIDENCE THAT THE CHANNEL ISLANDS POPULATIONS OF THE ORANGE-CROWNED WARBLER (OREOTHLYPIS CELATA; AVES: PASSERIFORMES: PARULIDAE) REPRESENT A DISTINCT EVOLUTIONARY LINEAGE
Review round: 2
Reviewer: 3

Output:
Basic reporting: See all comments in comments to the authors.
Experimental design: See all comments in comments to the authors.
Validity of the findings: See all comments in comments to the authors.
Additional comments: Hanna et al. 2019 PeerJ population genetics of orange-crowned warbler

I saw this paper before and see many improvements following from my previous comments. It is a good study, and publishable. But there are still two places where I think the authors could improve the overall impacts of their paper.

The first response does not address my point. Reviewer 1 commented on the clarity of the grouping treatments, not on the appropriateness of those groupings nor on how the analyses were subsequently treated. I did not critique their explanation, but rather their subsequent treatment (and a stylistic approach I would not choose myself). De-emphasizing the unpartitioned results (i.e., “did not reveal spatial structure” line 104) was I thought somewhat misleading. “We recovered low levels of divergence...” in the Abstract I think minimizes the results summarized in line 104 and overemphasizes the post-partitioning results, which masks an important result of your study. Using geography alone, no significant structure was found. However, with geographic partitioning that took into account, e.g., “ecologically significant” landmarks (line 112), and subspecies (line 119), significant levels of structure/divergence were recovered in your data. Styllistically, I would have made the paritioning seem less subjective and more directly in pursuit of factors likely to cause genetic structure, but differences there are valid choices.

Thank you for pointing out the multiple-test corrections. It looks like I did not look carefully at the table legends given on separate pages from the tables themselves per an unfortunate journal style (that should be changed, please, editors).

I also appreciate minimizing issues of IBD and ILS. That helps. The new Table 4 does, too.

In the response, I am surprised by this statement:“...we were unable to effectively calculate migration rates scaled by population size...” It needs to be clear that you have *chosen* not to effectively calculate these by not doing analyses that have been routine in mtDNA and microsatellite studies.

Contrary to your opinion, IM is well suited to analyzing mtDNA for population genetics. In fact, Jody Hey et al’s program IM has been used in hundreds of studies doing exactly what I suggested you do (see, e.g., Hey 2005 PLoS Biology and the citations of it). MtDNA is nonrecombining, which is a strength for this analysis, and the results apply only to females (e.g., effective population size estimates). Your results would not only go directly to answering some key questions in your study (date of split, effective population sizes, rates of gene flow), they would be directly comparable to these hundreds of similar studies. Unlike IMa2, it does not require a supercomputer.

Similarly, your microsatellite data provide a genomically independent, complementary basis for evaluating gene flow. Arguably the best direct approach would be to use Beerli & Felsentein’s 1999 (software updated through 2012) program migrate.

So I still see an easily filled gap in your objectives given your data to provide estimates not of relative rates, but of actual individuals per generation (using two great datasets!). Because you are right: “Furthermore, comparisons of island taxa and their mainland counterparts are fundamental to assessing the taxonomic status of island endemics, many of which are of conservation concern (Wilson et al., 2009).” (First paragraph of Introduction). No ddRAD data are needed to do this, and it’s been routinely done in literally hundreds of other studies like yours. Fringe benefits include actual estimates of effective population sizes and divergence dates for the Channel Islands population.

I think it is a good study. But it would be fairly easy to make it a great study, given the data you already have in hand.


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: LARGE PELVIC TUBERCLE IN ORANGUTANS RELATES TO THE ADDUCTOR LONGUS MUSCLE
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: The presented manuscript provides valuable information in the field of comparative and evolutionary anatomy that is related to the presence and the great development of the iliopectineal tubercle in orangutans, as well as its relationship with the tendon of insertion of the adductor longus muscle. The manuscript is well written and its methodology conforms to the professional standards required in this type of research work. In my opinion, I believe that the publication of the manuscript after a minor revision can be considered.
	- In the Materials & Methods section, as well as in Tables 1 and 2, it should be specified if the osteological sample used belongs to individuals raised in the wild or in captivity. In the case of having individuals from both sources it would be interesting to analyze, especially in Pongo, if there are differences between them in the presence and/or in the development of the iliopectineal tubercle. Do the authors think that the development of different locomotion patterns in primates raised in the wild and in captivity can affect the presence and/or development of the iliopectineal tubercle? Also, no information is presented on the number of males and females in the osteological sample analyzed. I think it would be interesting to add this information and analyze the existence of possible differences between males and females to see how the iliopectineal tubercle behaves in highly dimorphic genera such as Pongo and Gorilla.
	- I also think it would be interesting to add quantitative information regarding the adductor longus muscle. During the dissections carried out, have the authors collected quantitative muscle information, such as the absolute weight of the muscles, the length of the muscle fascicles and the angle of pennation? With this information you can obtain interesting quantitative parameters such as the absolute and relative weight of the adductor longus with respect to the other muscles of the thigh or with respect to body weight, or calculate the physiological cross-sectional area. With this information, it can be analyzed if there is a relationship between the values of these parameters and the presence and/or development of the iliopectineal tubercle comparing the different dissected species among them. Although the number of dissected individuals is low for some genera (Gorilla), the fact of having dissected 4 Pongo, 3 Pan and 3 Hylobates can provide information on the relationship between the iliopectineal tubercle and the anatomical and functional characteristics of the adductor longus.
	- In Figure 4 the muscle designated as psoas major in B and D corresponds at this level to the iliopsoas muscle. In the legend of the image 4c it should be indicated that the pectineus muscle has also been eliminated.
	- In Table 3, the value of C2 = 1 in Gorilla should be 32?
	- In Table 4, the value of C2 = 0 in Pan should be 0?


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: LARGE PELVIC TUBERCLE IN ORANGUTANS RELATES TO THE ADDUCTOR LONGUS MUSCLE
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript is very good and meets all the relevant criteria for this section. It is clear and well-written. The introduction and background provide plenty of historical context regarding the interpretation of the iliopectineal tubercle, and clearly communicate why this study is of interest. The figures, especially 2, 3, and 4, are very good. I suppose that space isn’t an issue so it is by no means necessary, but it seems to me that Figures 2 and 3 could be combined into a single figure with no loss of clarity. However, I understand if the authors wish to keep them separate.

I did notice that Fick 1895a is referenced once in the text but is missing from the list of Works Cited.
Experimental design: This research fills a small but important gap in our knowledge about the prevalence and expression of what the authors have termed the iliopectineal tubercle. The research questions are pointed and well-defined. My only comment about the design as it currently stands is that Homo is not included in the osteological sample. If one is going to assess whether the iliopectineal tubercle is unique among primates and within Hominoidea, it seems that Homo should be included, even if we are very confident that no iliopectineal tubercle will be found. If I had to guess why Homo was not included, it would be because one of the stated aims of the study is to determine if the iliopectineal tubercule is homologous to the pubic tubercle in humans and thus there would be an element of circularity in determining the character state in Homo. One solution to this is to “reverse” the research questions as stated in lines 135-137 (and as reported in the results and discussion).  First, it is assessed which soft tissue structure the iliopectineal tubercle is related to, then assess whether the tubercle is unique among primates/hominoids. Since it had been determined by the soft tissues that the iliopectineal tubercle is NOT homologous to the pubic tubercle in humans (as was suspected), the authors are then free to score the character in humans (presumably as zero) without interference. Now, I could be completely missing the mark here, and this is by no means an insistence on including modern humans in the osteological sample. But I did wonder why they were not included and it might be worth addressing why you chose not to.
Validity of the findings: The authors find that the iliopectineal tubercule is expressed in all adult and some subadult osteological specimens of Pongo. The authors find the iliopectineal tubercle variably expressed in other adult hominoids (with the exception of Homo), and not at all in other subadults. Dissection shows that, in Pongo, the iliopectineal tubercule is associated with the proximal attachment of the adductor longus and is not homologous with the pubic tubercle of Homo. These findings seem valid and are well supported by the results of their study.
Additional comments: I enjoyed reading the paper, nice work!


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

Input:
Title: LARGE PELVIC TUBERCLE IN ORANGUTANS RELATES TO THE ADDUCTOR LONGUS MUSCLE
Review round: 1
Reviewer: 3

Output:
Basic reporting: The article is well-written and clear, and provides sufficient introduction and background to demonstrate how the work fits into the broader literature. The figures are relevant and clearly illustrated.
Experimental design: The submission clearly defines the research questions in lines 134-137 and places them within a functional and evolutionary context. The methods are clearly described, and the sample is of an appropriate size and composition to address the research questions. The authors note that a larger cadaver sample and one encompassing a broader age range would be useful in future investigations.
Validity of the findings: The data are robust (n = 294, 28 primate genera) and included in the tables and supplemental information. The conclusions are appropriately stated and connected to the original research questions, and supported by the results.
Additional comments: My only general comment has to do with lines 190-192, in which the authors describe the location of the tubercle as medial to the anterior superior iliac spine (“An incipient...or small iliopectineal tubercle is present on the iliopectineal eminence medial to the anterior superior iliac spine and lateral to the pectineal line...”). Given the shape of hominoid ilia, most features on the iliopubic ramus are medial to the ASIS. Can the tubercle's description be clarified with reference to another feature?


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: MIRDRN—MIRNA DISEASE REGULATORY NETWORK: A TOOL FOR EXPLORING DISEASE AND TISSUE-SPECIFIC MICRORNA REGULATORY NETWORKS
Review round: 1
Reviewer: 1

Output:
Basic reporting: No Comment
Experimental design: No comment
Validity of the findings: Regarding the use of databases for miRNA targets, the authors relied on the HMMD database that provides data about experimentally validated miRNA targets. The web-based tool described by the authors can include a filter that allow users to exclude miRNA targets based on the experimental method used to validate those miRNA targets. (For example excluding targets validated by methods that used mRNA expression analysis by QPCR and include only targets validated on the protein level, or based on the knockout/overexpression methods for functional analysis of those miRNAs…etc). This is due to the fact that validating miRNA targets biologically can be challenging.
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: MIRDRN—MIRNA DISEASE REGULATORY NETWORK: A TOOL FOR EXPLORING DISEASE AND TISSUE-SPECIFIC MICRORNA REGULATORY NETWORKS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript by Liu et al. describes a database, miRDRN, that compiles associations between miRNA, target genes/proteins, and diseases. The database considers multiple levels of data  and computes correlation between miRNA, genes and diseases by using principles of the set theory. The data used to build miRDRN was obtained from other databases that mostly rely on experimentally-validated sources, however, the authors do not provide any comparison to previously published alike databases or even to the ones from which they obtained the data for building miRDRN (e.g. HMDD, TarBase).
Experimental design: -- line 111: how did the authors integrate the different data types ? or did they mean 'compiled' ?
-- the definition of Jaccard score for detecting the overlap between two sets of genes as explained by the authors is very informative and very well-suited to GO. However, for pathways and since the authors account for primary and secondary interactions, it will be worth weighing the genes/proteins in a pathway depending on how near/far they are from the target genes (i.e. order of steps from/to target in a pathway)
-- line 161: step 3 mentions that targets of a specific miRNA are collected from two sources (HMDD and TarBase). Does the authors take the overlapping targets from the two sources (intersection) or union ?
-- It is not clear to me how the authors integrated the miRNA-disease association data. They have elaborated on how miRNA-target associations were computed but not for the disease association. Also, it is not clear how they define co-morbidity from a computational point of view.
Validity of the findings: -- lines 292-293: Are the 26 targets that have literature support well-validated targets ? if yes, I would suggest that the authors curate a list of positive and negative association 'gold standard' targets and then use this set to quantitatively evaluate the performance of their tool.
-- The authors should provide a comparison to existing similar tools and highlight the advantages of using their tool in particular. I am not up-to-date on microRNA databases but a quick search shows there are several against which the authors should mention (e.g. HMDD, miRwayDB). Also several of these databases have experimentally validated targets, so I strongly suggest that the authors provide quantitative performance evaluation measures (AUROC, F1 score, FPR ... etc)
-- the authors should clearly hint that the findings form their tool are exploratory in nature with the goal of prioritizing miRNA-target-disease associations.
Additional comments: -- the discussion on potential failure of the anti-AD drug (case #3) is very interesting and demonstrates the utility of the database in full. I would strongly suggest that the authors follow the same for the other two case studies (cases #1, #2).  The authors could also hint to potential applications of building similar databases in the introduction and highlight that their databases is very comprehensive in guiding associations across several types of data that could potentially lead to identifying new drug targets, understanding drugs mode of action ...etc.

minor comments:
-- the names of the diseases in the 'disease' drop box need to be revised, for instance 'acute', 'chronic' and 'hepatocellular' are not proper diseases names.
-- what is the default p value cutoff ?
-- line 132: the intersection and union symbols are reversed.


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: ULTRAVIOLET DISINFECTION IMPACTS THE MICROBIAL COMMUNITY COMPOSITION AND FUNCTION OF TREATED WASTEWATER EFFLUENT AND THE RECEIVING URBAN RIVER
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript “ Ultraviolet disinfection impacts…” by Kauser et al. is well written. Intro and background are informative and rationale clearly stated. Literature reference appear to be valid and relevant throughout the manuscript. Reference section appears to be flawless which is rare these days. Structure of the manuscript confirms to PeerJ standards. No issues with fig 2—5, but it is hard to understand fig 1 without the details in the method or caption: two replicates are plotted for each time point?
Experimental design: Research is original and within Aims and Scope of PeerJ, but more detail is needed.
Lane 144-150. It was just one experiment run simultaneously? How many replicates where sacrificed at each time point? Two? (30 samples total?)
Lane 184. Only the forward reads were used?
Lane 197 DeSeq2…I’m not familiar with this program, what was it used for?
Lane 218. As SybrGreen based assay was used, melting curve analyses was not conducted?
I’m not sure if base and stormflow lab-setup (just based on the %) and conclusions will hold and reflect bacterial community dynamics in real-world situations.
Validity of the findings: Data and replication is limited but appear statistically sound
Conclusions are limited but valid based on the results
Additional comments: Authors have put quite a bit effort into writing this manuscript and clearly have a good knowledge of background research/literature It is just unfortunate that the paper appears to be based on the analyses of one wastewater effluent sample part of which was UV treated, both of which where then diluted  or undiluted in river water and analyzed at 0, 24 and 48h (30 samples?).
Somewhat ambitious goal to assess potential ecosystem-level impact (lane 92-93), not sure if this can be deduced from a small lab experiment which involved mixing river water with sewage.
Concern is that only one experiment with limited data points (three), but authors do acknowledge weaknesses in the conclusions. Conclusions are limited but valid.


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: ULTRAVIOLET DISINFECTION IMPACTS THE MICROBIAL COMMUNITY COMPOSITION AND FUNCTION OF TREATED WASTEWATER EFFLUENT AND THE RECEIVING URBAN RIVER
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript is well written, in an unambiguous, professional English.
The literature references is well documented with many recent publications. However, the introduction could better describe the state of the art in that particular field, and emphasize what has been done and what is missing. That would help the reader to understand what gap this paper is filling.
Some figures could be improved (see general comment).
Experimental design: Research questions are well defined, relevant and meaningful. As stated above, the introduction could be re-structured to be able to identify right away how this research fills an identified knowledge gap.
The experimental design seems appropriate to answer the question. However, details about the experimental design are missing (see general comments).
Validity of the findings: Duplicate of experiments was done. RT-PCR where run in triplicates.
Data seems robust. A minority of statistical tests are irrelevant (comparison of 2 vs 2 values or 3 vs 3 values), but that issue can be easily addressed (see the general comments for more details).
Conclusions are well stated, linked to the original research question and limited to supporting results.
Additional comments: General comment

In the present study, the authors performed a lab experiment to evaluate the impact of UV disinfection on the bacterial community composition and activity in the wastewater effluent and receiving waters. An RNA-based approach was used to avoid the amplification of free or damaged DNA, which is particularly relevant after wastewater treatment processes. Active bacterial community structure was assessed targeting the V1-V3 region of the 16S rRNA gene. Bacterial activity was monitored using quantitative RT-PCR of the bacterial ammonia monooxygenase (amoA), while resistance gene was assessed by targeting the tetracycline resistance gene (tetW).
The authors collected effluents from a wastewater treatment plant (WWTP) in Chicago (that did not undergo a tertiary treatment) and river water samples located 1 km downstream from the WWTP discharge point. Three conditions were tested: (1) to evaluate the influence of UV-disinfection on wastewater effluent, effluent samples were UV-treated or not. (2) To assess environmental implications, treated or untreated effluent samples were added to river samples to mimic stormwater conditions. (3) River sample no spiked with effluent was also monitored to mimic baseflow conditions.
The authors observed that while some organisms are inhibited by UV disinfection (alpha diversity decreases following treatment), many organisms not only survive, but remain active. When effluent were combined with river water, some organisms could persist in the environment and likely enhance microbial functions such as nitrification and antibiotic resistance.

Overall, the paper is well written and easy to read. Figures are appropriate and supplementary data provides useful information and data. I only have three concerns. First, in the Introduction, the authors should better describe the literature and the gaps that remain in the field (more RNA-based approach, etc.). Second, the Method section could be more precise. For example, details about the microcosm incubation conditions are missing (e.g., agitation speed, light, etc.). Moreover, the authors have to describe the statistical procedure they followed to perform the random forest regression analysis (package, number of trees, etc.). The version of usearch should be provided. Does the default LDA threshold of 2 is relevant in the context, a threshold set at 3 would be more stringent. Finally, although the results are congruent with in situ observations (Chaudhary et al. 2018), I would highlight the fact that results are originated from a laboratory experiment that might not entirely reflect what can be observed in situ.

Specific comments:

Abstract
Line 22: the authors already introduced “WWTP” in the text, so “urban wastewater treatment plants” could be deleted.
Lines 24-25 (and lines 61-62): that is true for combined systems.
Line 28: since a lot of studies used “16S rRNA” to describe DNA amplicons, I would write in the abstract the word “RNA” alone (somewhere) to make it clear that the authors targeted only RNA and not DNA (to highlight that specificity).

Introduction
Lines 48-50: redundant with the 2nd sentence.
Line 63: it is true for the US, but the monitoring of FIB to evaluate the effluent quality is not the case in all countries of the world.
Paragraph 66-85: I would reorganize this paragraph. The first sentence is not completely true: in the presence of a secondary and tertiary treatment, both steps must drastically shape the bacterial community composition (BCC) and activity (BCA) (and not only the final treatment). With all the studies made on this particular topic (see for example the next part of the paragraph or the beginning of the discussion), the authors can’t state that “the effect of disinfection on microbial community composition and functional potential in receiving waters is unknown”. If it is actually true, they have to better explain what have been done in the field, and what is missing (what they partially did in the next part of the paragraph).

Methods
The versions of the R packages or commands that the authors used should be mentioned. Some citations are missing in the “statistical analysis” section.
I understand that the authors don’t want to describe in details the Picrust analysis (for only three lines in the Results section), but this analysis should be introduced in the Method section.
Line 143: Does “Chicago 2011” refers to “Illinois Department of Resources 2011” or “Metropolitan Water Reclamation District of Greater Chicago 2011”?

Results
Please consider to not cite references in the Results sections when it is redundant with the Methods section.
Lines 237-243: Did the authors statistically tested two points against two points? I would just mention “the alpha diversity is lower or higher”, not much.
Moreover, I would specify that despite the difference visible in Figure 1, the alpha diversity is about the same between the five treatments (around 3.5).
Line 246: Does “all timepoints” include T0?
Lines 284-285: Something is wrong with the sentence, “identified that could be” should be deleted?
Line 286: How Arcobacter can be both an indicator bacteria and a genus identified to be associated with the indicator bacteria?
Line 289: Something is missing after “with”?
Line 290: Consider “OTUs” instead of “OTUS”
Lines 300-302: The parenthesis can be deleted without loose information since it has been clearly described previously.
Lines 315-317: Overall in that part, the authors keep explaining the treatment 2 (effluent + river samples = stormwater conditions) and treatment 3 (river sample = baseflow conditions). That makes the text hard to follow. By removing these repeats, the text would be more clear and concise.

Discussion
Lines 404-415: The authors highlighted the fact that some taxa predominant in effluent are also associated in the literature with natural habitats. If publicly databases are available (V1-V3), the authors could verify if the taxa they recovered are mainly associated with human fecal sources, sewage infrastructures or environmental sources.
Line 412: The authors mention that some OTUs couldn’t be taxonomically assigned to the genus. In that specific example, the use of the out-of-date Greengenes database can be challenged. The use of more recent databases could solve the issue.
Lines 424-433: In addition to Arcobacter spp., Acinetobacter spp., are also among the predominant bacterial taxa in sewage influent (see references already cited by the authors in the paper).
Line 438: Did the authors observed OTUs assigned to the acI lineage? I’m surprised to observe them in rivers since acI are mainly inhabitant of lacustrine environments (I can be wrong – but Chicago river is fueled by Michigan Lake?).
Line 448: Please consider “published” instead of “done”.
Line 449-450: I wouldn’t mention that detail in the manuscript.
Line 454: Please consider “significantly” instead of “signficantly”
Line 474: I would delete “HGT” since the authors never use the abbreviation after that.
Line 493: Does the word “significantly” refers to a statistical test comparing 3 points (24h) against 3 points (48h)? I would just say that the expression drastically dropped after 24h but reach the “control” level expression after 48h, as the authors did in the Results section.

Figures
Figure 1:
I would delete the sentence “Effluent samples are effluent only”.
I am not sure that boxplots are the most appropriate way to plot the data considering that there are only 6 points per box. Maybe a jitter plot or a pirate plot would be more appropriate.
I can’t see 6 points for each boxplot. Are they overlapping with other points or did the authors exclude some samples from the analysis/sequencing?
Figure 5:
The number of replicates should be mentioned in the legend. Moreover, I'm still not sure if a histogram is the most appropriate way to plot these data (see Figure 1).

Supplementary data
Standard curves are missing to fully interpret the RT-qPCR 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: ULTRAVIOLET DISINFECTION IMPACTS THE MICROBIAL COMMUNITY COMPOSITION AND FUNCTION OF TREATED WASTEWATER EFFLUENT AND THE RECEIVING URBAN RIVER
Review round: 2
Reviewer: 1

Output:
Basic reporting: The manuscript is well written. Introduction and background are sufficient and informative. The rationale is clearly stated. Literature reference appear to be valid and relevant throughout the manuscript. Structure of the manuscript confirms to PeerJ standards. The concerns have been addressed.
Experimental design: Research is original and within Aims and Scope of PeerJ. Sufficient information has been provided in the methods section. The concerns have been addressed
Validity of the findings: Data and replications is limited but appears statistically sound. Data has been provided. The concerns have been addressed.
Additional comments: The concerns have been addressed.


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

Input:
Title: ULTRAVIOLET DISINFECTION IMPACTS THE MICROBIAL COMMUNITY COMPOSITION AND FUNCTION OF TREATED WASTEWATER EFFLUENT AND THE RECEIVING URBAN RIVER
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: I really appreciated the effort the authors made to respond to our remarks and questions.
The authors took into account the suggestions I made. If not, they justified their position (I was not expecting such vivid argumentation, especially regarding the greengene database).

I have some minor comments that should not need to be reviewed before publishing the manuscript. I made a comment regarding the line 28 in the abstract. (1) Because of this comment, the sentence is off now. Sorry, the first version was better. (2) Add "RT-" before qPCR on line 32 (it was likely what I wanted to say the first time). (3) One space is missing before the parenthesis line 172. (4) There is a display problem line 262.


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: TAXONOMIC REASSESSMENT OF THE GENUS DICHOTOMIUS (COLEOPTERA: SCARABAEINAE) THROUGH INTEGRATIVE TAXONOMY
Review round: 1
Reviewer: 1

Output:
Basic reporting: The authors do a nice job of outlining the reason why this phylogenetic study was done. They have sufficiently cited past studies, and have justified their choice of data and taxon selection.
Experimental design: 1. When introducing the 16S marker in the materials and methods, please indicate the number of species for which this marker was available.
2. They should be very explicit and clear in the methods to explain why they ran haplotype networks on the 3' and 5' COI ( line 213/214) separately.
3. Symbols seem to be missing in line 225.
4. The figure 2 should include an image of the genitalia measured.
5. The phylogenetic data is presented in a very clear and concise way.
Validity of the findings: This phylogeny is an excellent first step in evaluating this genus. The data is robust, and uses several independent sources that are largely congruent.
Additional comments: 1. Some words are italicized that need not be, line 29 on page 1, " richest within the New World Scarabaeinae". Line 93/94 has extra set of parathenses: "(although this result is based on a small number of
2. 93 species of Dichotomius and only one individual per species, Bocak et al. 2014; Monaghan et al.
94 2007)."
3. Lastly, the references are not uniform in the application of the "-" and "en dash" between page numbers.


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: TAXONOMIC REASSESSMENT OF THE GENUS DICHOTOMIUS (COLEOPTERA: SCARABAEINAE) THROUGH INTEGRATIVE TAXONOMY
Review round: 1
Reviewer: 2

Output:
Basic reporting: My review doesn't exactly cover these items, but I see no issues in this regard.
Experimental design: Largely good. My detailed comments (in the 'General Comments' box) describe concerns about how some of the data were parsed, analyzed, and interpreted.
Validity of the findings: Overall I believe the conclusions are largely justified. However, some of their results could be presented more clearly, and some recommended reanalyses might alter the conclusions in small ways. More comments below.
Additional comments: This paper evaluating (very preliminarily) the phylogeny of the large Neotropical genus Dichotomius has a strong introduction. The authors do a good job of creating a good story from a group of beetles that has received relatively little press. The combination of nuclear and mitochondrial molecular data and morphometric data on genitalia allows for some generally well executed and synthetic analyses addressing species level phylogeny, some phylogeographic questions, and species delimitation. So I think most of the ingredients are in place for an excellent paper. However, I do have a number of concerns, mainly with the presentation, which can be hard to follow in places, and with their overpartitioning their data, treating two contiguous fragments of COI as if they were independent markers. The authors also seem to misinterpret the purpose and results of 'species delimitation' analysis, since most species included aren't (apparently) represented by multiple exemplars. So I would recommend some reanalyses, a fair bit of figure reformatting, and considerable text revision to address these concerns, detailed further below. In addition, my annotated copy of the pdf includes a number of additional minor edits, comments, and questions that should be addressed.

There's no justification (nor any presented) for treating the two halves of COI separately in the quantitative and phylogenetic analyses. These sequences should be concatenated and treated as coherent haplotypes (albeit with gaps at the primer junction). Furthermore, I don't understand why so much of the COI data was excluded for higher level phylogenetic analyses. Were the authors concerned about missing data? They don't state this as a their justification, and shouldn't assume that it would be a problem without attempting some analyses. I would think that having the largest numbers of individuals included that are available for the best represented marker would be very valuable for obtaining clearer resolution among the species.

Through the figures and text, it's difficult to keep track of how many specimens represent each species in each analysis, and it's therefore difficult to interpret various statements about the monophyly and coherence of species. 79 of 95 individuals represent D. satanas, and another 52 came from GenBank. Without going into the supplementary tables one can't easily tell how these other specimens are distributed across the tree. So when the authors claim that iBPP was done for 'all species and D. satanas', we can't really tell how good a test of these other species coherence this represents.

The species delimitation figure places posterior probability boxes on branches below multispecies clades. How are these to be interpreted? Not as species probabilities, but as clade probabilities? If this is intended as a true species delimitation analysis, these should only be applicable to terminal clades with species represented by more than a single individual for molecular data. It is not clear to me that this is true for any species other than D. satanas.

Other miscellaneous points:

The figures use elaborate color schemes to convey information on classification, distribution, and natural history. These seem overly complicated, and are very hard to interpret, as many colors are very similar, and the significance of some of the habitat distinctions is unclear/questionable.

The phylogeographic results for satanas requires a map to put it into some kind of explicitly geographic context. It means relatively little any reader unfamiliar with Colombian geography.

Rooting the satanas tree with D. agenor seems a strange choice, and may result in a spurious rooting. Why not use one or more closer relatives?

The discussion goes into quite a bit of detail with regard to internal relationships and classification. But with only 16 of 171 species represented by DNA data here, it's hard to take these very seriously. Probably a lot of that could be compressed and made a bit more tentative.


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: TAXONOMIC REASSESSMENT OF THE GENUS DICHOTOMIUS (COLEOPTERA: SCARABAEINAE) THROUGH INTEGRATIVE TAXONOMY
Review round: 2
Reviewer: 1

Output:
Basic reporting: See general comments..
Experimental design: No comment
Validity of the findings: No comment
Additional comments: While the changes the authors have made to their analyses represent a significant improvement, the paper is still rough in interpretation and presentation. Please see my submitted, tracked-changes version for more (not exhaustive) details. Some of the main issues have to do with discussion of groups (subgenera and others) found to be non-monophyletic. The authors don't explain the findings very clearly, and seem almost to contradict their results in a couple places. Most of my other comments are quite minor, but for a second revision, this is still a little rough.


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: TAXONOMIC REASSESSMENT OF THE GENUS DICHOTOMIUS (COLEOPTERA: SCARABAEINAE) THROUGH INTEGRATIVE TAXONOMY
Review round: 3
Reviewer: 1

Output:
Basic reporting: see general comments
Experimental design: see general comments
Validity of the findings: see general comments
Additional comments: This version looks clearer. I note just a couple spelling changes in the attached (one a verb tense mismatch, one just a misspelling).


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: SEX DIFFERENCES IN THE RESPONSE TO ANGIOTENSIN II RECEPTOR BLOCKADE IN A RAT MODEL OF ECCENTRIC CARDIAC HYPERTROPHY
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript is written in clear unambiguous professional English language. The references used are relevant and adequate and the authors provide enough background to show context.

Raw data are shared. However, the reporting of the gene expression could be improved. The data from the sham groups are missing. Additionally, the identification of the animals is also missing and the units of the data are missing in the table.
Experimental design: Walsh-Wilkinson et al study sex dimorphism and differential response to chronic treatment with valsartan in male and female rats with cardiac volume overload due to aortic valve regurgitation. Sex dimorphism in response to treatment in rodent models of volume overload has not been previously studied. The authors show that females show left ventricular wall thickening and that this effected is at least partially prevented by treatment with valsartan.

In general terms the study is well designed, and the results and analysis are correct. However, some aspects should be considered for improvement.

It would be helpful to present together, in the same tables and figures, the results in the sham group and in the aortic regurgitation (AR) groups and to include the statistical comparison between the sham untreated group and the AR untreated and treated groups. This would allow to see the effect of AR and to what extent valsartan prevented that effect. In its current for it is difficult to compare AR with Sham. Maybe males and females can be separated in different tables.

On the other hand, all the analysis related to cardiac weight are focused on raw cardiac and left ventricle weights. It would be interesting to index the weight either by body weight or by tibia length. Additionally, it would be useful to include the echocardiographically calculated left ventricular mass index.

From a methodological point of view, regarding mRNA analysis it is not stated which house-keeping gene was used for normalization.

A statement on the ethical approval should be included in the Methods section.

Minor comments:
- Lung weight data are not presented in the results.
- LOX is not included in the description of methods.
Validity of the findings: Regarding the statistical analysis, All the the analyses has been performed assuming a parametric distribution of the data but according to the methods this has not been checked. If possible data should be presented as dot plots instead of bar graphs.

As the authors state in the introduction they have previously shown in male rats that blockade of the renin-angiotensin system (Arsenault et al. 2013; Plante et al. 2009), had a beneficial effect in a similar experimental procedure reducing left ventricular hypertrophy and dilation. The lack of beneficial effect with valsartan in comparison to those previous studies should be discussed in depth. Why did valsartan have no effect on left ventricular dilation neither in males or females or on hypertrophy in males?

Overall, the conclusion is supported by the results.

As a minor issue, in the discussion an effect on systolic blood pressure is mentioned, but these results are not presented in the current study.
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: SEX DIFFERENCES IN THE RESPONSE TO ANGIOTENSIN II RECEPTOR BLOCKADE IN A RAT MODEL OF ECCENTRIC CARDIAC HYPERTROPHY
Review round: 1
Reviewer: 2

Output:
Basic reporting: -	The abstract lacks contextual information. Please adapt in order for the abstract to include introductory information/background information in the beginning to place it into context and the perspective/importance/filling gap at the end. On the other hand, the results section in the abstract is too detailed and long.
-	The reference format in the text may not be correct and the way in which references are presented now makes reading the text difficult. Please place references between brackets in the sentences.
-	The start of the introduction lacks some flow, and it is difficult to understand the connections between the sentences. (lines 32-34)
-	The introduction is very short, which makes it difficult to follow the argumentation for the study. Please add more background information on for example cardiac hypertrophy, volume overload and the RAAS system. Also,.
-	The rationale and hypothesis of the study are unclear, and therefore the study design is difficult to assess. Why did you choose for a short study of 2 months instead of a chronic study? And why do you start treatment 1 week before surgery/AR induction, because this will not resemble a clinical situation (as medication will not be given before the problem arises)?
Experimental design: -	The age of the rats is not stated in the methods section. From the weights in table 2&3 and the growth chart from Charles River, it seems that the female rats are younger than the male rats. If this is the case, female and male results cannot be directly compared, so then it will be better to show the results in different tables and discuss them separately in the context of responses to AR.
-	What is the difference between the ‘complete echo exam’ (line 71-73) that is performed before AR induction and at the end of the protocol, and the ‘echocardiographic exam’ (line 75-76) that is performed 2 weeks after surgery and at the end of the protocol?
-	In the methods, please add RNA isolation details.
-	In the statistical analysis it is stated that student’s t-test is used when only 2 groups are compared (line 85). In the results, it is unclear when only 2 groups are compared and this test was used. How do you correct for multiple testing?
Validity of the findings: -	Figure and table locations are not logic. Table 2&3 can be combined and 4&5 as well, because now it is difficult to compare the values of sham and AR rats. Figure 3 and 4 have to be placed in the text instead of at the end.
-	Lung weight is an important factor to add to table 2/3, as it helps to assess whether rats are decompensating/have heart failure. Also, the baseline body weights will be informative, and if they changed due to surgery.
-	The ShMV group has a high SEM for most measurements. Was the group normally distributed or were there any outliers?
-	Please rewrite line 96-100 in a logical order taking into account the text before and the text from Figure 1.
-	In Figure 1, there seem to be differences for some conditions but they are almost all not significant. It will be insightful to present the results in a scatter plot including the mean/median and error bars, so that the distribution of the samples can be seen.
-	The legends to Figure 3 contain no information about subfigures A, B and C, so this has to be added. Also, please add ‘vs sham’ to the title of the y-axis behind fold change in figures 3&4, as this was also added in the other figures.
-	Line 124 states that KLF15 is ‘upregulated’ by LVH in males, but figure 3B shows this must be ‘downregulated’.
-	Line 126-127 seems contradictory to 127-129, because first you state that there are no important modulations and then significant changes are described. Please reformulate and replace ‘extracellular markers studied’ by mentioning the specific markers.
-	Why did you choose to study the specific extracellular matrix genes? In the discussion, you state that extracellular matrix remodelling is low in the early phase of disease (line 211-213), so it is expected that there are no differences. Maybe it would be better to test for early matrix response genes like Thrombospondin-2, Intergrin β1 or CTGF, because collagen responses are later in the disease? In addition, other gene expression markers of cardiac remodelling exist that better reflect the earlier remodelling process, including markers for cardiomyocyte metabolism (e.g. angiopoietin-like 4) and for inflammation (e.g. Interleukin-6, TNF alpha).
-	Line 162 misses a reference; to which side is ADME changed? (would you expect more or less effect of valsartan in females?)

Minor points:
-	Line 12: ‘The aim of the study’ instead of ‘aim of study’.
-	Line 24: ‘normalized’ or ‘helped normalizing’ instead of ‘help normalizing’.
-	Line 37: HF has to be between brackets.
-	Line 39: LV has to be written full, because this is the first time you use it in the main text.
-	Line 43: ‘increase in’ instead of ‘increase’.
-	Line 49: ‘as observed in…’ instead of ‘observed in …’, because it is a more general statement.
-	Line 53: specify treatment; what kind of treatment?
-	Line 81: why did you not use all animals, maybe better for significance/power?
-	Line 96: ‘every parameter’ or ‘all parameters’ instead of ‘every parameters’.
-	Table 4 legends: PW abbreviation description is missing.
-	Line 120-121: This sentence can be interpreted like the increased expression of Bnp was higher as Anp, which is not the case, so include ‘compared to male’.
-	Line 134: two times ‘relatively/relative’ in 1 sentence, so replace one.
-	Figure 1 caption: ‘LV hypertrophy’ has to be ‘hypertrophy’, because not only LV is measured.
-	Line 148-149: rephrase sentence
-	Line 164: rephrase sentence or leave ‘since’ out.
-	Line 190-191: Is there a study that used a higher dose of Valsartan?
Additional comments: this is a nice, well-written and timely paper. Addressing the comments will improve it further.


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 DIETARY CONCENTRATE TO FORAGE RATIO ON GROWTH PERFORMANCE, RUMEN FERMENTATION AND BACTERIAL DIVERSITY OF TIBETAN SHEEP UNDER BARN FEEDING ON THE QINGHAI-TIBETAN PLATEAU
Review round: 1
Reviewer: 1

Output:
Basic reporting: Overview this paper is clear, well wrote, all tables and figures were adequate and literature references are enough.
Experimental design: The experimental design  is correct, but the statistical analysis more appropriated  on different levels of concentrate in diet is regression.
Validity of the findings: On discussion about rumen pH, the time of collection of the ruminal liquid after the supply of the concentrate and or diet is fundamental to verify the change in pH. The decrease in pH is associated with a high starch on diet and acid lactic production. The discussion on protein content seems to be inadequate. Because  the bacteria need 7% of protein on diet, then only in the first treatment (HS1) the protein content was less than 7%. Regard to volatile fatty acids the term most accepted today would be short chain fatty acids. In addition, for the term of comparison between treatments the correct would be to present the data in proportion from the total and not in concentration. Necessary correct ml to mL on text
Additional comments: Considering the complex microbioma ruminal, this work is very important and contributes with new information about the interactions of the diet and microbiota


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 DIETARY CONCENTRATE TO FORAGE RATIO ON GROWTH PERFORMANCE, RUMEN FERMENTATION AND BACTERIAL DIVERSITY OF TIBETAN SHEEP UNDER BARN FEEDING ON THE QINGHAI-TIBETAN PLATEAU
Review round: 1
Reviewer: 2

Output:
Basic reporting: This study describes the potential effect of the diets varying in the ratio of concentrate to forage on the rumen fermentation, bacterial diversity and growth performance in Tibetan sheep. The study is interesting, and provides valuable insights to the ruminant nutrition community.

The English language should be improved. There are grammar errors throughout the manuscript.
Experimental design: 1. need more explanation and power analysis on why 20 animals, with 4 in each treatment groups is sufficient.

2. lacks information on how the DNA concentration was measured.
Validity of the findings: findings are reasonable

1. more information is needed on the number of reads mapped to each OTU.
2. the authors stated in the discussion that the ruminal pH values in this study did not show significant differences, though previous study by Kennelly (1999) did observe a significantly decreased pH values. Not sure why the protein level was proposed as a possible explanation. Please clarify.
Additional comments: Major comments:
1.	Line 76-77, is 20 animals total sufficient for this study? Please provide a power analysis.
2.	Lines 120-121, How the DNA concentration was determined, aside from using the 2% agarose gel? As the correct measure of DNA concentration is critical to downstream sequencing library preparation and analysis
3.	Line 187, What is the number of raw reads mapped to each OUT?
4.	The measurement of ruminal fluid pH, the authors stated in the discussion that the ruminal pH values in this study did not show significant differences, though previous study by Kennelly (1999) did observe a significantly decreased pH values. Not sure why the protein level was proposed as a possible explanation. Please clarify.
5.	The English language should be improved. Below are some examples (though not exhaustive)
a.	Line 152, please use the sign “”
b.	Line 268, please consider to remove the word “exist”
c.	Line 269, please consider to remove “because of our”
d.	Line 270, please rephrase the sentence “leading the protein level in diets were lower”
e.	Line 281, please rephrase the sentence “the expected increase the molar proportion…”
f.	Line 286, please rephrase the sentence “no difference among each treatments…”
g.	Line 316, please consider to change “have a habit of” into “has a habit of”

Minor:
1.	Line 251, please elaborate on what does “positively jointly” mean?
2.	Lines 320-321, “the abundances increased”, is this significant? What is the p-value?