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

Input:
Title: THE ASSOCIATION BETWEEN CHRONIC BULLYING VICTIMIZATION WITH WEIGHT STATUS AND BODY SELF-IMAGE: A CROSS-NATIONAL STUDY IN 39 COUNTRIES
Review round: 1
Reviewer: 2

Output:
Basic reporting: The present paper concerns a study aimed to investigate the association between bullying victimization with weight status and body self-image in 39 countries. Data set was drawn from HSBC.
Globally speaking the paper is clear, well written and there is a good coherence among objectives, hypotheses, methodology, analytic strategies and results.
The first impression is that the research does not add new knowledge to the literature: a large number of studies, without exceptions, prove the association between weight status and school victimization. Methodology and measures are not innovative.
Nevertheless, the study presents two important strengths: i) the large number of participants (213,595 adolescents) makes the results very robust; ii) the attention to the risk associated to underweight status generally less considered in studies focused on the relationship between overweight status and school victimization.
Abstract:
 the last sentence in the conclusions “Both actual weight and perceived weight are associated with chronic bullying victimization. Adolescents with underweight are also at higher risk of being chronically bullied” needs to be reformulated, that both overweight and underweight children are at risk of bullying victimization.
Introduction:
I believe that introduction should give more space to the literature related to bullying and weight status. In the first part of the introduction, the authors make a general introduction to bullying but in the second part they report the literature concerning the relationship between weight status and bullying very quickly. More specifically, they do not report some studies concerning the jointly association between weight status, self-image and bullying victimization that is the focus of the present study. They could cite:
Bacchini et al., Childhood Obesity, 2017;  13 (3), 242-249,
Reulbach et al., J Paediatr Child Health 2013; 49: 288-293;
Sutter et al, J Adolesc 2015;  43: 20-28;
Wilson et al.,. Int J Environ Res Public Health 2013; 10: 1763–1774
Who analyze the mechanisms that link weight status, body-image and bullying victimization.
Experimental design: Materials and methods
HBSC study is a well-known international survey but a reference where people can obtain more information is needed (also a website address).
Criteria for attributing a weight status to participants should be specified. The authors refers to Cole classification. An expert reader knows that BMI is not a sufficient parameter to classify weight status during the developing years because it needs to be standardized by age and sex.  Please specify.

Results
No information is given about the distribution of the variables.
Before running regression analyses, the authors should show (at least in the supplementary materials) correlations among variables.
Validity of the findings: I think that the major criticism concerns the way to interpret the results.
A first point concerns the high number of underweight children. It is well known that in western countries (especially in US and in some European countries such as Spain, Greece, and Italy) overweight and obesity is a widespread concern of public health whereas the underweight status is relatively rare. I think that the bias related to the self-evaluated measure should be more discussed (the authors make a point in the section on the limits of research). So they can not say “real weigth” vs “ perceived weight” but they should say  “perceived weight” vs “perceived body-image”
A second point concerns the lack of analyses concerning the differences among countries. I understand that this is not a point that the authors intend to investigate in the study but it sounds as a limit that the authors do not mention. How the variables are distributed across countries (at the least considering major groups such as north American vs European countries)? The normativeness of overweight in some countries could play a crucial role in determining the association between weight status and bullying victimization.
A third point concerns the lack of interaction analyses between weight status, body-image and victimization. For instance, the above cited studies showed that negative body self-image amplifies the association between a negative weight status (underweight od overweight).
The statement “bullying is less frequent in European countries than in US” has not correspondence in the literature.
Additional comments: References
It is unusual to cite the reference within the text after a point. Is a journal’s rule?
The two references of Olweus 1994a and 1994b are identical.


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

Input:
Title: THE ASSOCIATION BETWEEN CHRONIC BULLYING VICTIMIZATION WITH WEIGHT STATUS AND BODY SELF-IMAGE: A CROSS-NATIONAL STUDY IN 39 COUNTRIES
Review round: 2
Reviewer: 1

Output:
Basic reporting: The revised manuscript is much better and improved. It is clear and unambiguous, and professional English.

The literature review is still not satisfied. As I mentioned in the first round review, I insist that the literature review section should include a brief review of the influencing factors of being bullied (as the school climate, family structure, SES, demographic etc.). Without these reviews, the only concentration on BMI and overweighted could not provide enough information to readers. And, obviously, some of the important influencing factors were not included/controlled in the analysis because using a secondary data, and the authors should discuss these limitations in the discussions.

The methods and results are well-presented.
Experimental design: The experimental design is good and acceptable, research questions are clear, methods are appropriate.
Validity of the findings: The findings and analysis methods are validated, providing useful contributions.
Additional comments: The literature review should be improved, and some comments of the missed influencing factors should be acknowledged in the discussion section.


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

Input:
Title: THE ASSOCIATION BETWEEN CHRONIC BULLYING VICTIMIZATION WITH WEIGHT STATUS AND BODY SELF-IMAGE: A CROSS-NATIONAL STUDY IN 39 COUNTRIES
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors  responded satisfactorily to the concerns of the reviewers.

However, some issues remain to be solved:

-  Hypotheses sound too much descriptive. I think that the authors, on the basis of the previous literature, could advance more specific hypotheses. For instance: i) the association between BMI and victimization is consistent across countries (as using multilevel analysis they control for the country-effect); b) weight and body image are indipendent and unique predictor ecc.

Lexicon: reading the paper I was not sure if the authors were referred to bullying perpetration or bullying victmizaztion. For instance "bullying involvement" is an ambigous expression.
Experimental design: -  In the rebuttal letter, the authors say that in the supplementary they inserted correlation among variables but I was not able to find them. I remain curious to know whether perceived  body image and perceived BMI are two overlapping or indipendent measures. For instance, in a my contribution (Bacchini et al. 2017) BMI and Physyical were indipendent predictor of victimization. I think that in the discussion the difference and the contiguity between the two constructs should be discussed.
Validity of the findings: see above
Additional comments: No additional comments


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

Input:
Title: THE ASSOCIATION BETWEEN CHRONIC BULLYING VICTIMIZATION WITH WEIGHT STATUS AND BODY SELF-IMAGE: A CROSS-NATIONAL STUDY IN 39 COUNTRIES
Review round: 3
Reviewer: 1

Output:
Basic reporting: The language is professional, sufficient filed context provided. Article structure is professional. results are sound.
Experimental design: The research is well designed, and the research questions are well defined and generated, the data analysis method is appropriate.
Validity of the findings: The findings are meaningful, having some kinds of novelty.
Additional comments: NO


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

Input:
Title: THE ASSOCIATION BETWEEN CHRONIC BULLYING VICTIMIZATION WITH WEIGHT STATUS AND BODY SELF-IMAGE: A CROSS-NATIONAL STUDY IN 39 COUNTRIES
Review round: 3
Reviewer: 2

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: Paper has been significant improved. Responses' authors satisfied referees' requests.


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

Input:
Title: THE ROLE OF AWARENESS OF REPETITION DURING THE DEVELOPMENT OF AUTOMATICITY IN A DOT-COUNTING TASK
Review round: 1
Reviewer: 1

Output:
Basic reporting: The paper is well written, and the description of the design and presentation of the results is clear.  The review of the literature is relevant and clear, but does not include some literature that I think is relevant. In particular, the manipulation and assessment of awareness in learning has been a fraught topic for decades, and while it is true that these questions have not been studied in tasks closely resembling the present one, the large literature on implicit sequence learning that has developed over the last 20 years or so contains much relevant consideration of both theoretical and methodological issues.   I think that citing this literature would be useful, and might help to the authors’ choice of manipulation and assessment.
Experimental design: The research question is clearly identified, and the experimental design and procedure are straightforward.  However, the logic linking the reviewed theory to the design seems to have a gap. My understanding of Logan’s theory is that each instance is an independent memory representation. Therefore, it’s not clear that that the theory attention to, or awareness of, repetition per se should affect the rate of learning.  It seems to me that the manuscript does not clearly connect the statements in lines 120-139, which focus on attention to characteristics of the stimulus and response (i.e., pattern and numerosity) to the manipulation, which focuses on the relation between rather than within trials.  In fact, on my reading, Logan’s theory would not predict an effect of this particular manipulation of awareness (because each instance is a separate memory representation), though it may predict an effect of awareness of the relation between pattern and numerosity within instances.  In this sense, the null results may support the theory in a more specific way than described in the manuscript.  On the other hand, the authors may have an argument, left implicit in the present manuscript, that links awareness of repetition with attention to the task elements critical for forming an instance representation.

It would be helpful to better defend the specific manipulation of awareness in terms of the hypothesized effect on the development of automaticity, if that is possible, and to explicitly contrast the content of this awareness with other possible contents that might have different theoretical relations to the phenomena.
Validity of the findings: The data provide a robust replication of well-established effects important to instance theory (and to other theories of skill acquisition).  As the authors note, though, their important conclusions depend on accepting the null hypothesis concerning the effect of awareness of repetition. This may be theoretically significant in the present case, but no power analysis is presented.  It is true, as the authors say, that there was sufficient power to detect some effects – overall speedup and reduction in slope – but these are known to be very large effects.  However, I did not find this a convincing argument that there was sufficient power to detect important effects.  The number of participants is quite small for a between-subjects design intended to detect an effect of unknown size.  Moreover, as the authors also note, it is possible that awareness was not in fact successfully manipulated – the design included no manipulation check.  In fact, it’s not clear how participants would have used awareness of repetition on individual trials – by hypothesis, the retrieval of instances is automatic in the sense that the appearance of the stimulus is supposed to trigger recognition and thus instance retrieval.

I did think that the authors were admirably clear in pointing out the limits of their results and conclusion, and in considering alternative hypotheses.
Additional comments: I’m sympathetic to the aims of the study – examining the effects of awareness on a type of learning that has been important to theory in the field.  I wish, however, that the manipulation were more clearly linked to the theory, and the robustness of the null finding were easier to evaluate.


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

Input:
Title: THE ROLE OF AWARENESS OF REPETITION DURING THE DEVELOPMENT OF AUTOMATICITY IN A DOT-COUNTING TASK
Review round: 1
Reviewer: 2

Output:
Basic reporting: The English is clear, unambiguous, and professional.

The article structure is professional, and easily understood.

The article is self-contained.

The term "automatic" is not consistently defined across studies (see Logan, 1988, and more recently Reynolds & Besner, 2006). I think it's important that researchers in the area are explicit about what they mean by the term, and which features of automaticity they are concerned with. I believe a discussion of this problem is warranted.

Data is shared, but only in aggregate form making it impossible to replicate the analysis from scratch, or conduct alternative analyses with different decisions around outlier removal etc. I would prefer to see the original trial-level data included as well (anonymized, of course).

General Statement on Open Science
I think that open science principles are critical to improving research, and have adopted a policy of promoting them in my reviews. To this end, I would invite the authors to provide the data, materials, and analysis files/scripts in a public repository such as http://osf.io so that others can verify and re-examine claims, use the results to inform their own study designs, and ensure a more complete record of experiments to counteract publication bias in meta-analyses. Alternately, manuscripts should include a clear statement justifying the decision not to provide these materials. (See opennessinitiative.org for more on open science practices)
Experimental design: I leave it to the editor to determine if the article fits within the scope of the journal, though I see no reason why it shouldn't, given other articles that have appeared here.

The research question could be clarified with a clearer statement of how automaticity is defined and operationalized here.

The design is an extension of a paradigm used by Lassaline & Logan (1993). The authors test more subjects (17 per condition rather than 4) but with far fewer trials (540 vs. 6000+) per subject, and conducted in a single session rather than across 13 sessions. I think a discussion of these differences is warranted (it's worth noting that Speelman and Townsend (2015) used a similar design, and observed the same pattern of a reduction in the slopes that Lassaline and Logan observed).
Validity of the findings: This is the area of greatest concern to me.

1. If I understood correctly, the index of "time to automaticity" is the first block of 18 trials in which the subject achieves a slope of 100ms or less. However, it's quite clear from the data that in many cases the participants do not *remain* below 100ms from this point on, which raises the question of whether the task can really be considered to have become automated at that point, or if perhaps some index that takes into account the stability of that slope going forward is needed. The slope for each block is, after all, based on a maximum of 18 trials (3 for each number of dots). This is likely to be highly volatile.

2. Relatedly, the authors have adopted this 100ms/dot criterion as the index of automaticity. However, many of the subjects seem not to have achieved that measure consistently during Phase 5 (given the error bars there). How do the authors reconcile these with their conclusion that automaticity is achieved? Perhaps Speelman and Townsend's findings that not all subjects achieve automaticity in this task would be worth discussing here.

On reporting and interpreting Bayes factors:
3. I applaud the authors for using both Bayesian and traditional Null Hypothesis techniques to address the question, however the conclusions they draw from the BFs are misleading. Bayes factors around 1 are equivocal: providing little evidence in either direction. Statements such as "A similar conclusion is suggested by a a Bayes .... (Bayes Factor (.05) = 1.63)" (line 301-302) are thus misleading. The Bayes factor of 1.63 suggests no ability to draw conclusions of any kind.

4. The key hypothesis here was that subjects in the aware condition might achieve automaticity faster than subjects in the control condition. This would be indicated by the presence of a group x phase interaction for the slopes such that the aware group's slopes dropped faster than the control group. The ANOVA analysis considers this interaction directly, but the Bayes Factors are reported only for the main effect of group. Why are Bayes Factors for the critical interaction not considered?

5. I'm unfamiliar with the notation "Bayes Factor (.05)". What is the "(.05)" here? Similarly, no indication is given of which model the Bayes factor favours. BF=1.63 (line 302) may indicate slightly more evidence for the null, or for the alternative. (Based on the t statistic, I think the BF likely favours the null here.) This ambiguity needs clarification throughout.

6. The BFs all being around 1 also speaks to the statements in lines 326-330. BFs in that range should be taken to imply that more data is needed to discriminate the two hypothetical models (null, vs. alternative). They certainly can not be taken to support the view that since some effects are significant, there likely isn't a power problem. However,
 I reiterate that the BFs reported do not appear to directly address the critical interaction.
 (It's also worth noting that the significant effects are all main effects of phase, while the critical, non-significant results are interactions - ANOVA is more powerful for main effects, so the presence of those effects does not offer much defence against power concerns.)
Additional comments: The article raises an interesting question (can awareness of relevant features of stimuli speed the process of moving from "slow and effortful" to "fast and automatic"). The design of the study is a simple extension of one used before, however I wonder at whether the design is well-enough powered to conclude there is no effect. The use of Bayesian analysis is great, but as applied here does not help to address the most important feature of the data.

There is a significant benefit to the decision to use Bayes Factors. If the BFs for the interaction are equivocal as they are for the main effects of group, the the authors are welcome to simply collect more data until the Bayes Factor is able to discriminate the base model (main effects of Phase and Group) from a model with the addition of the interaction.

I also worry about the way that "achieving automaticity" is operationalized here since subjects seem to go from the "automatic" 100ms/dot to "effortful" 101+ms/dot from block to block (though it's hard to be sure how much this occurs, since the data provided are aggregated into phases of 6 blocks.) This suggests to me that there is a high level of volatility in the measure as an index of automaticity.

I'm not sure how difficult it will be to address my concerns, but it seems to me that it isn't impossible so I am currently recommending "Major Revisions."


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

Input:
Title: THE ROLE OF AWARENESS OF REPETITION DURING THE DEVELOPMENT OF AUTOMATICITY IN A DOT-COUNTING TASK
Review round: 2
Reviewer: 1

Output:
Basic reporting: The paper now cites work on the role of attention in the literature on implicit learning, as I suggested.
Experimental design: The authors have clarified the rationale for their predictions and how they follow from instance theory.  Making this explicit is a major improvement.
Validity of the findings: The authors have provided a more appropriate and robust statistical approach to supporting the robustness of the finding of no interaction.
Additional comments: The authors have responded effectively to my earlier concerns, so I have limited this re-review to noting that.  I think the revision greatly clarifies the contribution of this work.


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

Input:
Title: THE ROLE OF AWARENESS OF REPETITION DURING THE DEVELOPMENT OF AUTOMATICITY IN A DOT-COUNTING TASK
Review round: 2
Reviewer: 2

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


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: EVIDENCE FOR WIDESPREAD DYSREGULATION OF CIRCADIAN CLOCK PROGRESSION IN HUMAN CANCER
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: See bellow
Additional comments: The authors used a comprehensive bioinformatics pipeline, including a newly created metric-delta clock correlation distance, to screen publically available data on healthy tissues for mouse, and to subsequently generate a signature of clock gene co-expression. Using their methodology, the authors concluded that clock dysregulation is a common treat in human cancer which is in agreement with other published studies.

The bioinformatics and statistical analysis presented in this study is solid and supports the conclusions of the paper. The established of a web tool will be useful and will contribute to facilitate the application of the methodology presented. The manuscript is of broad interest, the same sort of pipeline can be applied also to address other research questions, beyond the cancer vs healthy comparison.
I would therefore recommend publication in Peer J.

 Nevertheless, a few points should be improved and/or better explained before publication, as listed bellow:
1 - A more detailed scheme of the pipeline used would facilitate the understanding of the methodology. In Figure 4A the authors have such a pipeline which could be completed by indicating the different levels/types of biological samples use. This would be particularly relevant since the authors use mouse samples to generate a signature for clock genes, this is later validated in human “healthy” samples, and in a next step used to analyse human cancer samples.  This procedure should also be included in the scheme of Figure 4A.

2 - The authors include the cell line U2OS, a human osteosarcoma cell line, in the group of healthy human tissues – why? This sample should not have been included in this group. The authors must justify why they have decided to do so, and to which extent does this sample influence the overall results.

3 – The authors should better explain how their tool can be used to distinguish a phase shift from an expression change, between samples, for the core-clock genes.

4 – In line 348 the authors mention that tumour samples have a “weaker pattern”, this should be better explained since it is not clear what “weak” means, in this context.

5 - Figure 1A and Figure 2A show the expression of clock genes in human and mouse. The plots should be at least partially comparable, however Figure 1A uses a normalization in the range (1,-1), but Figure 2A uses a normalization in the range (2,-2; 3, -2). There is no apparent reason to do this and the same normalization should be used. Figure 2A is very difficult to interpret because the clustering is not well visible, it seems to be quite poor.

6 - Figure 3A should be revised, the samples have very different sizes and this makes interpretation difficult.

7 – The authors calculate a new score of delta-CCD, but have only one figure (Figure 4B) with this score. However, the figure is not very good at supporting the conclusion that the dysregulation of the circadian clock is a common mean by which human cancers achieve unrestrained growth and division. If the dysregulation would be so common for cancers, shouldn’t there be many high scores and a few low scores or at least a clear prevalence of high scores?

8 – The data presented does point to a dysregulation of the clock in tumours, but this bioinformatics analysis provides no evidence for a dysregulated clock as a means of achieving “unrestrained growth and division” (line 533). The authors should better justify this statement, eventually reformulate it.


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: EVIDENCE FOR WIDESPREAD DYSREGULATION OF CIRCADIAN CLOCK PROGRESSION IN HUMAN CANCER
Review round: 1
Reviewer: 2

Output:
Basic reporting: Overall, the manuscript from Shilts and colleagues is well written and a well‐designed study with some major and minor cause for revision.
-	Line 87: “In addition, although CYCLOPS can be used to infer rhythmicity in the expression of individual genes, it is not designed to quantify the differences in circadian clock function as a whole between multiple conditions.”. This sentence is unclear to me. What are differences in clock functions?
-	Figure 1A shows Zeitgeber time, but in the description it is stated that samples were not labelled with the time of day (line 273). Are these predicted/estimated ZTs?
-	Fig 1A is redundant with Fig. S3.
-	Line 317: “The patterns of clock gene co-expression in human tissues and cells were similar to the patterns in mice (Fig. 2 and Fig. S4),..” The description of comparison of patterns should be made more clear. Therefore it would be helpful to state which Figures to compare with what, since in this case Figure 2 and S4 are not the ones to be compared to understand the descriptions of the results, rather Figure 1 and Fig. 2. This also applies to other comparisons.
-	Line 322: “The strong pattern in human skin was due to clock gene co-expression both between the three time-points (9:30 am, 2:30 pm, and 7:30 pm) and between individuals at a given time-point (Fig. S6).” The Figure is labelled 14:30 and 19:30 instead of 2:30 or 7:30.
-	Line 322: The description order of Fig. S6 needs to be the same as illustrated in the Figure.
-	Description of Figure 2 are not in line with the test in the results. Fig 2 and Fig 2C have been described, but not Figure 2A, B.
-	What is the difference between Fig 2 and S7? (ZTs?) Where is the description of lung and liver samples in Fig. 2?
-	Line 391: “To better understand the nature of clock dysregulation in tumors, we compared clock gene expression in human cancer and in knockouts of clock genes in mice.”. Describe what tissue was used.
-	Line 426: “In contrast, human tumor samples tended to show similar or somewhat higher MAD compared to non-tumor samples.”. Please comment on the fact that higher MAD was found in tumor tissues, which may indicate enhanced rather than reduced rhythmicity in tumor tissues compared to non-tumor tissues.
-	Line 428: Taken together with the differential expression analysis, these results imply that clock dysregulation in human cancer is not due solely to loss of activity of one or more core clock genes. “. This sentence or the conclusion is unclear to me.
-	Line 451: Remarkably, we observed a consistent pattern, in which genes with an acrophase near tended π to show the strongest reductions in expression in tumor  samples (Fig. 6C, P ≤ 0.001 of non-zero amplitude by permutation testing for each dataset). Using Phase Set Enrichment Analysis (Zhang et al., 2016), we found that these genes were particularly enriched for involvement in G protein-coupled receptor signaling (Table S4).” This sentence is unclear to me. Which results support this?

Figure legends:
-	abbreviations need to be xexplained even in figs
-	 S6: “Pattern of clock gene expression in human skin is due to both time-of-day variation and inter-individual variation.”. This sentence does not make sense to me.
-	S14: The header is incorrect: Cry1/Cry2 KO are in the bottom panel not the top as indicated on the right. I suggest to write the specific genes in the header and the WT or KO on the right.
Experimental design: Experiments have been designed carefully and are mostly well described.
-	Line 155: A brief description of ComBat would have been helpful to understand the procedure to reduce batch effects.
-	Line 162: It would be important to know for which data sets the “strength of circadian rhythmicity” has been calculated.
-	Line 232: A short explanation on what “gene modules” are would be helpful for the reader?
Validity of the findings: 1.	The strength of the described work lays in the quantification of similarities between gene expressions patterns of control and tumor tissues. However this technique has not been applied to all parts of the study. It puzzles me that the technique is only introduced at the last third of the manuscript, but not from the beginning on. While reading the manuscript my major criticism was the lack of quantification for the entire first half of the results, quantification was missing although the technique was available.
A statement such as “weak or absent” cannot be concluded from these results, since no quantification has been done. Similar descriptions of results have been made in the entire results section. Sentences in the results section such as “In non-tumor samples from most cancer types, we observed a similar pattern of clock gene co-expression as in the healthy mouse and human datasets (Fig. 3A-B and Fig. S8A) or “In contrast, in tumor samples from each cancer type, the pattern was weak or absent.” (line 348) need to be rewritten.
The introduction of the quantification for the very first dataset and then for all datasets following would strongly improve the manuscript and support/validate statements like “we observe the same trend of expression patter”  or “we confirm our findings in additional human organs” such as in line 328 or
- line 317: “The patterns of clock gene co-expression in human tissues and cells were similar to the patterns in mice (Fig. 2 and Fig. S4),..”
- line 319-326: ” The pattern was less distinct in human blood (Fig. S5), likely because several clock genes show weak or no rhythmicity in expression in blood cells (Hughey & Butte, 2016). The strong pattern in human skin was due to clock gene co-expression both between the three time-points (9:30 am, 2:30 pm, and 7:30 pm) and between individuals at a given time-point (Fig. S6). Compared to co-expression patterns in mouse organs and human skin, those in human brain were somewhat weaker, which is consistent with the weaker circadian rhythmicity for clock genes in those two brain-specific datasets (Hughey & Butte, 2016).”
-	Line 303: “In each dataset, the correlation pattern was preserved in both daytime and nighttime samples (Fig. S3).” This needs to be quantified before such a conclusion can be made.
-

2.	Describing a similarity between species is impossible without quantification.
-	Line 317: “The patterns of clock gene co-expression in human tissues and cells were similar to the patterns in mice (Fig. 2 and Fig. S4), consistent with our previous findings of similar relative phasing of clock gene expression in mice and humans (Hughey & Butte, 2016). The pattern was less distinct in human blood (Fig. S5), likely because several clock genes show weak or no rhythmicity in expression in blood cells (Hughey & Butte, 2016). The strong pattern in human skin was due to clock gene co-expression both between the three time-points (9:30 am, 2:30 pm, and 7:30 pm) and between individuals at a given time-point (Fig. S6). Compared to co- expression patterns in mouse organs and human skin, those in human brain were somewhat weaker, which is consistent with the weaker circadian rhythmicity for clock genes in those two brain-specific datasets (Hughey & Butte, 2016).”. There is not enough evidence presented that human samples are similar to mouse samples. Mouse patterns are rather strong, while human co-expression patterns are rather weak. This is not restricted to only blood samples as indicated by the authors, rather applies to all samples excluding from skin. The clock in humans and thereby gene expression amplitudes are not generally weaker. How do the author’s explain the differences in the expression profile strength between mice and humans? Saying they are similar as stated in line 317 is incorrect, since there are major differences in the contrast between both species. A quantification would give room for better phrasing, however to argue something is more similar to another can only be made, when a negative controls exist.
-	Line 346: “In non-tumor samples from most cancer types, we observed a similar pattern of clock gene co-expression as in the healthy mouse and human datasets (Fig. 3A-B and Fig. S8A). In contrast, in tumor samples from each cancer type, the pattern was weaker or absent. We observed the same trend when we restricted our analysis to only matched samples, i.e., samples from 350 patients from whom both non-tumor and tumor samples were collected (Fig. S9).”  A well defined quantification (including positive and negative control) is needed to state such a conclusion. For example (line 354) the described perturbation in Fig. 8B is not obvious to me. I would need more evidence including a quantification.

3.	Another major point addresses the validation of conclusion made for the functionality of the circadian clock. As the author’s state in the introduction and in the discussion that the method introduced does not rely on time points, a conclusion about the functionality or rhythmicity of the clock is impossible to make. The results in the manuscript describe a specific clock gene pattern at a specific time point, not throughout the circadian cycle. Therefore sentences describing the rhythm or the circadian aspect of the clock have to be re-phrased. There are many sentences in the entire manuscript. Some examples are:
-	the title of the manuscript “Widespread dysregulation of the circadian clock in human Cancer”. No statement can be made about the circadian clock status since no circadian/rhythm analysis have been performed. I rather suggest “altered co-expression profiles of clock genes”.
-	Line 28: “Furthermore, by analyzing a large set of genes previously inferred to be rhythmic in healthy human lung, we observed systematic changes in patterns of circadian gene expression in lung tumors.” Instead of “circadian gene expression” I suggest rather “clock gene expression”.
-	Line 272: ” Such a pattern could be used to infer the activity of the clock, even in datasets
-	273 in which samples are not labeled with time of day (Fig. 1A).”.What does even the “The activity of the clock” means? The status the expression level of specific clock genes?
-	line 304: “These results indicate that our approach can detect an active circadian clock in groups of ….:). I would rather suggest something like “These results indicate that our approach can detect a co-expression pattern at a specific time points, similar to the one detectable in data from tissues with a functional circadian clock.”
-	Line 337: “To investigate circadian clock function in human cancer, we applied our approach to…”. Circadian clock function cannot be investigated with these kind of analysis.
-	needs to be changed  accordingly
-	line 378: ” Overall, these results suggest that the circadian clock is dysregulated in a range of human cancers.”. The presented data do not justify a conclusion like that, because the circadian aspect is irrelevant for the applied method.
-	Line 417: “In the clock gene knockouts, rhythmic expression of most clock genes was reduced or lost (Fig. S16).” No rhythm analysis has been performed, so no conclusion about it can be drawn here. Same holds true for the legend of Fig. S16!
-	Line 455: “Taken together, these findings suggest that dysregulation of the circadian clock in human lung cancer is accompanied by systematic changes in broader circadian gene expression.”
-	Line 469: “Our approach for detecting a functional clock relies on three principles.”. The functionality of the circadian clock cannot be detected by the applied approach, rather a “normal” phase-relationship between clock genes, whereby “normal” means compared to a previously defined control pattern.
-	Line 480: “Altogether, these principles enable our method to detect dysregulation of the clock in groups of samples whose times of acquisition are unknown and whose coverage of the 24-h cycle is incomplete.”.
-	The conclusion needs to be re-phrased accordingly.
Additional comments: Apart from the changes I have suggested for the quantification, I enjoyed reading the manscuript and the idea behind the introduced analysis is well-thought-out and of importance for the cancer field.


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: EVIDENCE FOR WIDESPREAD DYSREGULATION OF CIRCADIAN CLOCK PROGRESSION IN HUMAN CANCER
Review round: 2
Reviewer: 1

Output:
Basic reporting: The article  meets the necessary standards.
Experimental design: See general comments.
Validity of the findings: See general comments.
Additional comments: In this revised form of the manuscript the authors have addressed most of my comments, as well as the comments from the other reviewer. I find this study very well-thought, of overall interest, and only have a few remaining minor comments, which can be easily addressed before publication of the manuscript.

My main worry goes back to my previous comment regarding the usage of U2OS (human osteosarcoma) as “normal clock” and the subsequent statement by the authors in the rebuttal letter “we believe stronger rhythms in tumour samples are unlikely”.
The authors consider that the clock in U2OS cells is “strong” (I would agree). Furthermore, other studies (Relogio et al Plos Genetics 2014, cited by the authors in this manuscript) also show the existence of different clock phenotypes (including strong clocks) within the same cancer type. Such strong rhythms observed in cancer cells contradict the statement “we believe stronger rhythms in tumour samples are unlikely”.
Altogether, it is not trivial to associate the “quality of the clock” with cancer, even though compelling evidence points to a strong clock-cancer correlation with which I would also tend to agree. As such, I would suggest that the authors are more critical regarding the interpretation of the data, and rewrite a few sentences in the discussion.
1-For e.g. in the first paragraph of the discussion the authors write “…we discovered that clock progression may be perturbed in multiple types of human cancer” what do the authors mean by clock progression?
2-On pg 13 of the discussion the authors write “…the dysregulation of clock progression in cancer maybe readily reversible”. Again, this is a rather strong statement based on the fact that some cancer cells lines have a strong clock, maybe these cells have always kept a “healthy clock”.
3-Finally, to help reinforcing the message of the paper regarding the dysregulation of the circadian clock in cancer, the authors could add the following (very recent) references to the discussion, which strengthens the conclusions and perspectives presented:
The circadian clock as a tumour suppressor:
a) Ticking time bombs: connections between circadian clocks and cancer. Lamia KA. F1000Res. 2017
b) The Ink4a/Arf locus operates as a regulator of the circadian clock modulating RAS activity. El-Athman R, et al. PLoS Biol. 2017
c) Circadian disruption promotes tumor growth by anabolic host metabolism; experimental evidence in a rat model. Guerrero-Vargas et al BMC Cancer. 2017
The circadian clock in treatment:
d) The tumor circadian clock: a new target for cancer therapy? Kiessling S, Cermakian N. Future Oncol. 2017
e) Dosing time dependent in vitro pharmacodynamics of Everolimus despite a defective circadian clock. Zhang Y, et al Cell Cycle. 2017


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: EVIDENCE FOR WIDESPREAD DYSREGULATION OF CIRCADIAN CLOCK PROGRESSION IN HUMAN CANCER
Review round: 2
Reviewer: 2

Output:
Basic reporting: Overall, the manuscript from Shilts and colleagues has improved significantly since the last submission. It is well written and a well‐designed study with some minor cause for revision.

Results
-	In the results section the Supplemental Figure citations (from Fig S6, S7, S8) are not in order. Fig. S6A, B are described first, then Fig. S7 and S8 and finally Fig. S6C, D. Either Fig. S6C, D becomes the new Figure S9, or its decription needs to be reorganized.

Figure legends:
-	abbreviations need to be explained even in figs, such as ZT = Zeitgeber time
-	statistical values such as p<0.005 for the p-values such as in Fig. 2 are not standard. p<0.05, P<0.01, P<0.01 and P<0.001 is the standard.
Experimental design: no comment
Validity of the findings: In the new version of the manuscript the authors have included my suggestion and added analysis to the relevant sections and rewrote the conclusion about the functionality of the clock to my satisfaction.  Although I have some minor comments:
-	Line 365- ongoing: I am puzzled by the fact that the human tumor and non-tumor data have always been compared with the reference data set obtained on mouse tissue, although human datasets have been available and even more with indication about the Zeitgeber time. As indicated in the results Fig. 1, human data show rather similar pattern as results obtained from mice, but as indicated elsewhere co-expression pattern in 2 out of 6 human data sets are not significantly similar to the ones obtained from mice. Shouldn’t this facts have lead the authors to generate a new reference set using the available human data set with time information to be used for all the following comparisons between human g non-tumor and tumor data?
-	Using tissues from clock knockout mice with only loss of one clock gene (excluding Bmal1), such as Clock or Reverba should not be pooled with data sets obtained from mice with either deletion of two essential clock genes or Bmal1, which cause arrhythmicity. Complete circadian disruption is only accomplished by genetic disruption of Bmal1 or two essential clock components such as Cry1/2-/-, whereas RevErba-/- mice and Clock-/- mice are still rhythmic, but with a different tau! Thus, in their own circadian day length the phase relationship of clock genes to each other collected from samples during the 24 day is likely changed. This may explain the differences in Fig. S13 and S14 within the knockout cohort and also the results described in line 401-406 regarding fig. 3A rather than the small sample size as indicated by the authors. I would rather recommend under this circumstances to keep it simple and stick to completely arrhythmic mice or organs.
-	Line 408-416: The relevance to compare human cancer and clock knockout data sets is not intriguing, since human cancer tissues usually show disrupted clock gene expression, but usually not a complete loss of any of the core clock components. The difference between clock knockout and suppression or upregulation in tumor tissues which results in arrhythmicity of the expression, is a completely different situation and not comparable. Literature reports strong up or down-regulation of essential clock genes, which then influences the status of interconnected clock genes, however, I am not aware of reports indicating complete loss of clock genes due to mutation in human tumor cells. Therefore this comparison is invalid or irrelevant and very artificial to me and the conclusion e.g. indicated in line 106: ” Our findings suggest that dysregulation of circadian clock progression is present in a wide range of human cancers, is not caused solely by the loss of core clock genes, and is accompanied by systematic changes in broader circadian gene expression.“  seems a wrong interpretation and misleading.
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: EVALUATING THE ADAPTIVE EVOLUTIONARY CONVERGENCE OF CARNIVOROUS PLANT TAXA THROUGH FUNCTIONAL GENOMICS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript is clearly written with clear English. Enough background and references are provided. Figures and tables are well described. Although many raw data were provided as supplementary materials, I would suggest the authors to include their custom-made ORF models and GO annotations to ensure the reproducibility.
Experimental design: The question and hypothesis were described well. I found no problem on technical and ethical standard.
Validity of the findings: The current version of the manuscript lacks a statement on their implicit assumption on phylogenetic relationships. Despite of the author’s clear recognition as seen in Fig. 2, the authors did not take into account phylogenetic relationships in their analysis. Their approach implicitly assumes a star tree for analyzed species. In other words, all species were treated as equally distant to each other, without discriminating carnivorous origins (e.g., Utricularia and Genlisea were implicitly treated to have different origins as carnivore). I would suggest the authors to explicitly mention on this problem as a source of potential bias.
Additional comments: The authors reported, by bioinformatics analysis of published genomes, a signature of convergent gene ontology enrichments in separately evolved carnivorous plants. The manuscript may benefit from the following comments:
	L94: In the commentary you cited, I could not find the claim on 16 independent origins. Their description is: “…, these species represent at least nine independent origins of the carnivorous habit per se…”. You might have misunderstood their Table 1, in which genera are labelled with their trap strategies. Some of them share the common carnivorous ancestry even if they use different trap strategies (e.g., three genera in Lentibulariaceae), and therefore carnivorous origins do not total sixteen.
	L95: “Lower plants” might not be appropriate as a terminology. I would suggest “bryophytes” or “non-vascular plants” instead.
	“Neofunctionalization” is not limited to duplicated homeotic regulatory functions. Please rephrase or remove it.
	L157: To my knowledge, GO-based approaches cannot usually identify lineage-specific repurposed genes in genomes which lack experiment-based annotations. Such novel genes would be annotated with GOs identical to their parent genes by the annotation pipelines based on sequence similarity (such as BLAST2GO used in this study). In such cases, they appear to be non-differentiated in function. Indeed, this manuscript did not gain any insights into neofunctionalization at all.
	L371: I failed to understand why you try to associate the GO terms only to digestion rather than to carnivorous syndrome in general.
	L405: In carnivorous plant genomes, not all of enzymes serve as digestive enzymes, so there is no “clear roles in direct digestive function”, at least only by GO enrichment analyses. The same applies to L415.
	Please clearly distinguish GO terms from the rest (e.g., consistently using quotations for GO terms). For example, the statement starting from L417 is quite confusing. It sounds that you actually measured water channel activity, but what you did was the enrichment analysis of the GO term “water channel activity”.


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: EVALUATING THE ADAPTIVE EVOLUTIONARY CONVERGENCE OF CARNIVOROUS PLANT TAXA THROUGH FUNCTIONAL GENOMICS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript #21378 "Evaluating the adaptive evolutionary convergence of carnivorous plant taxa through functional genomics" by Gregory L. Wheeler and Bryan C. Carstens. The presented study shows interesting and important findings which are related with expectations about evolutionary convergence in carnivorous plants. The manuscript is well written in a proper and high-quality language. Also, the authors provide enough references to support the ideas which are addressed in the manuscript. Figures are relevant, high quality, well labeled and well described.
Experimental design: The originality of the research is within aims and scope of the journal. The research question is well defined, relevant and meaningful. Also, methods are well described and the author provides enough information to replicate all analyses performed.
Validity of the findings: The data analysis part of this manuscript has been conducted very thoroughly, even the annotation process from some available genomes was made using a single methodology. This, in my opinion, it's a success because can considerably be reduced the bias that surely exists when genomes which are compared were annotated using distinct methodologies. A Huge effort has also been made by adding statistical support to the analysis.
I conclude saying that I will support this manuscript once that authors address some issues which I summarize in the next section (general comments for the author).
Additional comments: 1. To my knowledge, de novo transcriptome assembly in highly heterozygous species typically yields a higher number of contigs than the actual number of genes expressed, thus rendering a redundant and fragmented reference transcriptome. Considering this possibility is not possible to say how many homologs (or orthologs) genes are present in the whole genome using transcriptome data. This is something that the authors do not consider when they referred to some previous publications, e.g. Lines 141 and L 142. "Utricularia gibba L., which has a minute genome only 80 megabases in size, contains at least 77 distinct genes for nitrogen transport functions". This need to be paraphrased or probably change it by something like: "the high number of unigenes (at least 77 distinct transcripts) annotated like nitrogen transporters in de novo transcriptome of Utricularia gibba L. strongly suggest that in the minute genome of this carnivorous plant species, the nitrogen transporters families are expanded in comparison with other plant species.

2. Even when is possible that differences in some gene families could be related with tandem duplication or gene transposition duplication, it is well known that gene duplication and subsequent gene retention or loss (fractionation) are often attributed to recent and/or ancient whole-genome polyploidy events. None mention about these possibilities is presented in this manuscript and in my opinion, this need to be included at least in the discussion section considering that whole-genome duplication and selective contraction, resulting in enormous genetic diversification. It is necessary to discuss if the divergent sub/neo-functionalization events, might have occurred after the whole genome duplication events (which are different for some of the taxon’s which were analyzed). This probably could strengthen the conclusion of the authors in which they suggest that plant carnivory can evolve using multiple independent metabolic pathways.

3. Considering that both U. gibba and G. aurea have significant differences in terms of the genome size and they are members of Lentibulariaceae family, I consider necessary that authors provide an explanation (hypothesis) about why they have substantial differences in terms of the portion of carnivory-associated functions showing strong signals of genomic overrepresentation. This is relevant considering that even when they show different trap system, both carnivorous plant species are considered lack roots and the prey capture is the main source for uptake some nutritional compounds.


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: EVALUATING THE ADAPTIVE EVOLUTIONARY CONVERGENCE OF CARNIVOROUS PLANT TAXA THROUGH FUNCTIONAL GENOMICS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The new manuscript is improved and answered most of my comments raised earlier. The only problem I found was their new addition of the statement on the phylogenetic dependency.

In their response letter, the authors replied as:
“We attempted to account for potential phylogenetic bias with our sampling design; however, this is never explicitly stated in the text. This was an oversight on our part, and an explanation of our intention with this design has been included to the “Sampling” portion of the Methods.”

This reply and their addition to the main text is less convincing to me. If you attempted to account for the phylogenetic relationships by sampling design without a phylogenetic comparative method, one carnivorous species and one non-carnivore in each lineage should have been sampled so that fair pairwise comparisons can be made (this is a classical workaround to homogenize different phylogenetic distances). Without validation or supporting references for your sampling strategy, one should refrain from stating that the bias was accounted, while the caution of the bias should be clearly stated.

Minor comments:
L106, “such at” might be “such as”.
L177, “functione” might be “function”.
L564, “detected..” -> detected.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: no comment


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: EVALUATING THE ADAPTIVE EVOLUTIONARY CONVERGENCE OF CARNIVOROUS PLANT TAXA THROUGH FUNCTIONAL GENOMICS
Review round: 2
Reviewer: 2

Output:
Basic reporting: As I said before, the presented study shows interesting and important findings which provide an interesting study with an original approach to testing for genomic convergence. The manuscript is well written in a proper and high-quality language. Figures are relevant, high quality, well labeled and well described. After one round of review, the author attends the reviewers' comments and provides new references to support the ideas which are addressed in the manuscript.
Experimental design: The hypothesis and aim were clearly described while methods are deep-described with sufficient detail and information to replica the whole analysis.
Validity of the findings: The experimental design provides a novel approach to testing for genomic convergence and the conclusions come from data analysis with enough statistical support.
Additional comments: The authors provide additional information in response to the reviewers' comments. Mostly, all comments were attended and the manuscript was significantly improved in comparison with the previous version. I consider that the manuscript, in the actual format, provides an interesting study with an original approach to testing for genomic convergence. I also believe that the manuscript will be of general interest to the community, mainly for those which employ genomic approaches to understanding problems in evolutionary biology. In my opinion, this manuscript has merits publication 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: DETOURS IN LONG-DISTANCE MIGRATION ACROSS THE QINGHAI-TIBETAN PLATEAU: INDIVIDUAL CONSISTENCY AND HABITAT ASSOCIATIONS
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comment in addition to review below
Experimental design: No comment in addition to review below
Validity of the findings: No comment in addition to review below
Additional comments: Major comments:
The manuscript presents some exciting data showing how Pallas gulls may move around a major ecological barrier during their annual migrations, and present the surprising finding that the gulls move up to twice as far as necessary to reach their wintering grounds, making sideways detours of considerable distance. The data appear to have been analysed appropriately (but see my comments about pseudo-replication, which need dealing with) and the results are of interest to the wider community. The basic reporting is good, the experimental design is fine and the validity of the findings are adequate, but see below for some thoughts that I think need addressing:

•	I am not sure that “extreme detours” is accurate. Yes, the birds are making a detour, but it’s double the distance, not 10 times, which might be more clearly “extreme”. Granted, it is difficult to set a cut-off, but perhaps this aspect just needs to be moderated by deleting “extreme” from the title and text.
•	“Habitat availability” (e.g. abstract line 16, methods line 144, etc). The proximity to water bodies that may or may not be suitable for gulls to use during migration is not the same thing as habitat suitability. Indeed, there is a whole Niche modelling field that uses that term and that is therefore implied here, when you have made no such analysis. I would be honest here and call it what it is – proximity to water bodies. You don’t know that these water bodies are suitable (they could be occupied by people (line 299), polluted, dried up etc). so just keep it simple.
•	Reverse migration – I am not sure what you mean by this? You give a definition on line 131 but in bird migration it has another meaning. I would suggest instead saying something like “in case the bird doubled back on itself during the migration”
•	As I see it, springtime, northward migrating gulls don’t exhibit much deviation from the shortest route. This seems sensible if the gulls were attempting to get to optimal breeding sites before competitors each year. This also could explain why the arrival date is consistent too (line 231). Autumn, southward migrating gulls, however, seem to be the ones making the detours. These can be considerable and one wonders what they might be doing there. It would seem sensible to use Google Earth to at least describe the land that they are deviating to, is it agricultural? Water bodies? You essentially hint at this on line 261 and 279 but I think it could and should be more explicitly discussed in a dedicated paragraph. You introduce several reasons for detours nicely in the introduction, I would expect to have seen each hypothesis individually discussed.
•	Navigation – This comes in at the end of the discussion and I am not sure that it is warranted. The study is sufficient without including discussion of putative navigation strategies. Certainly there are not robust data with which to test this. I would suggest removing it from the manuscript entirely.

Minor comments:
Abstract:
Line 8 – change “the” to ‘their’
Line 13 – I think it would be more honest here to say that you tracked four gulls, but for repeated years. The 22 migration seasons instead makes it sound like there is more data than there is.
Line 16 – see comment above about habitat availability.
Line 26 – see comment above about navigation.

Introduction:
Line 45 – “detoured with a short cut” is an oxymoron – can you rewrite this to whatever you mean?
Line 49 – also insert reference to Hawkes et al. (2012) Proc Roy Soc B 108, 9516 who showed that bar-headed geese detour considerably from the shortest route to avoid strong winds.
Line 54 – change “on extreme plateau” to ‘at high altitude’. Likewise for line 57.
Line 75 – needs a reference for this statement.
Line 77 – you should also cite Pennycuick (2008) Modelling the Flying Bird, Academic Press.

Methods:
Line 98 – what does “complete” mean?
Line 129 – need to very clearly describe these as randomly generated longitudes and observed bird longitudes throughout.
Line 132 – This is a bit long winded at present. How about instead: “If the observed bird longitudes fell below the 2.5th percentile, or above the 97.5th percentile of the 1,000 randomly generated longitudes, the observed bird longitude was considered to significantly deviate from the migratory route that might have been expected.”
Line 137 – the linear distance between
Line 139 to 143 – this isn’t clear to me, I don’t understand what other metric you are collecting here and how it differs from line 137?
Line 144 – see comment above about ‘habitat suitability’
Line 146 – needs a reference for this point
Line 155 - In order to compare between northward (spring) and southward (autumn) migrations statistically as you outlined here, you need to account for individuals (as you show on line 228). Line 81 states “from two to four years” suggesting that three of the four birds may have contributed more data to the analyses and plots than the one that only lasted two years. Essentially, data presented in Figure 3, for example, may be pseudo-replicated. I would suggest something more sophisticated than a t-test should be used, at least something like a GLM where you can include individual as a random factor. For plotting, you should ensure that the same number of trips per animal go in, i.e. if one gull was only tracked for two trips, only two from the other gulls should go into the plot as well and the other trips excluded.

Results:
Line 175 – Give the n per individual (with only four birds seems little point in giving a mean +/- s.d.). Can you also give the duration over which the birds were each tracked as well?
Line 178 – is “two-four” a typo?
Lines 183, 184, 186, 187, 190,  – there is a statement on each of these lines (e.g. “larger”) that needs to be supported by a statistical result. From this point onwards, the stats have been reported well.
To me, line 184 is the major finding of the whole study and needs to be pulled out to be more prominent, e.g. by start the paragraph at line 205 with it, and calling that paragraph “Temporal patterns of detours” so it marries well with the previous paragraph heading.
Line 189 – add (n=x per gull). The 75% here is unnecessary, just say three of the four gulls (same for line 193).
Line 198 – how many per individual? I am hoping it is three each!
Line 200 – I would delete “In contrast…barriers after detours” and start the sentence at “Gulls covered”
Line 205 – then you need to give the effect size here as well, it would be in km.
Line 206 – a loop migration is something different to what you have observed.

Discussion:
Line 249 – this sentence is muddled – you can’t detour with a shortcut? I suggest rewriting it to whatever you mean.
Line 251 – likewise muddled
Line 253 – what does “inhospitable at ground” mean?
Line 254 – I would argue that the availability of stop-over sites doesn’t necessarily mitigate the altitudinal climb required for the gulls to get onto the QTP from Bangladesh.
Line 263 – some typos here, I think you mean “might not be the only...”
Line 266 – do you mean significantly lower altitudes?
Line 269 – but are gulls a species that undertakes considerable fuel loading prior to migration? If they stop lots of times they may not need to.
Line 270 – this should be rewritten as something more like: “Additionally, the energetic costs of locomotion are reduced at lower altitudes”
Line 302 – this sentence is a little confusing (“trans-barrier” comes up for the first time) – what do you actually mean here? Perhaps that one might imagine that once a gull had climbed onto the QTP it wouldn’t fly back off it again any sooner than it absolutely had to? I suggest changing this sentence.
Line 304 – I disagree and think the most interesting is the extent of the detour in northwards versus southwards migration.

Figures and Tables
Figure 1 – does this need to be in colour? Given that much of the world prints to black and white printers, it would be better to produce the figure in black and white if possible. The land could have a black outline and needs some country labels. The tracks could be solid dark grey for northwards and dashed dark grey for southwards migrations (the 3,000 metre polygon would still be visible and clearly distinguishable). I think you should plot the shortest route on the map as well so we can see the differences – this could be a thick black line. I would also reduce the number of tick marks and labels to just 4-6 on each axis. The legend should also start “Map showing…”

Figure 2 – I think the legend could more clearly explain the plot, it’s not clear enough right now and doesn’t mention the randomisation routines. I believe something like this might be more helpful: “Bar plot showing the mean longitudinal deviation from the shortest route in latitudinal bins of three degrees, for northward (blue) and southward (red) migrations. Error bars show standard deviation, pie charts show proportion of routes that deviated significantly from 1000 randomised routes starting and ending at the same point, as measured using a XX test and p<0.05.” Again, this plot could be in black and white instead of colour.

Figure 3 – I would shade the boxes in grey, which makes the plot easier to read. I would also consider adding notches to denote significant differences. I am not sure what “Turkey style” whiskers are? Given the sample size, I am surprised the data met a normality test to use a t-test and there is no mention of a normality test in the methods. Can you check they are normally distributed and if not, use a Mann-Whitney? The legend should also start “Boxplots showing…”. It is also not clear how pseudo-replicated the boxplots might be, since I imagine there is more than one trip per gull in there (otherwise there are only four datapoints making each box). You should use the same number of trips per gull and explicitly state this in the legend.

Figure 4 – See my earlier comment. Calling this “suitable habitat” may be misleading, or rather, it would be more accurate just to say “proximity to water bodies” as that is what you have actually measured. Again, this plot could be in black and white instead of colour. The legend should also start “Line plots showing…”

Figure 5 – I feel like there should be better way to show these data, or perhaps just to mention it in the text instead. If you do keep the figure, I would change the legend to “satellite derived ground surface temperatures”, which I believe is what you actually used, although I don’t think there is any mention of where these data came from in the methods? I am also not sure that “overshot” is accurate (did they overshoot or did they mean to go there? Not possible to know). The legend should also start “Line plots showing…”.


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: DETOURS IN LONG-DISTANCE MIGRATION ACROSS THE QINGHAI-TIBETAN PLATEAU: INDIVIDUAL CONSISTENCY AND HABITAT ASSOCIATIONS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The English could be improved throughout. A few citations could be changed/added as well. (See General Comments.)
Experimental design: I believe that the migratory detours need to be calculated in relation to a great circle route as opposed to a straight line route. (See General Comments.)
Validity of the findings: I have some minor quibbles related to the Discussion section. (See General Comments.)
Additional comments: General Comments
I like this paper a lot. I think that it details an interesting dataset and offers some novel insights into the drivers of migratory route choice in long-distance migratory birds. I have only one ‘major’ concern: I think the migratory deviations should be calculated in relation to the great circle distance between non-breeding and breeding sites, as opposed to the straight distance. Otherwise, my only other general suggestion is that the writing needs to be tightened up throughout the manuscript and especially at the spots I note below. All in all, well done!

Specific Comments
Lines 13-14: I do not fully understand the second clause of this sentence.

Lines 45-47: I do not entirely understand this sentence.

Lines 66-70: I am not sure if this large vs. medium-sized bird dichotomy really exists any more in terms of tracking studies. Tons of tracking studies of all sorts have focused on ‘less-than-large species’ recently, including a number from the shorebird literature related to within and between individual repeatability (e.g., Conklin et al. 2013, PLoS One). I think that you can probably delete these two sentences if you would like.

Line 77: See also Parr et al. 2017 in Journal of Avian Biology for another recent trans-Himalayan migration example.

Line 89: I presume that the gulls were breeding at Qinghai Lake?

Line 107: What were the duty cycles on the PTTs?

Lines 111-112: Shouldn’t you calculate the great circle distance instead of a straightness index? Birds have frequently been shown to adhere to great circle routes.

Lines 118-119: Do you present these data in the results?

Lines 145-146: Citation?

Lines 243-246: See comments below about moult migration.

Lines 247-248: I am unsure of how your study provides any information on flight range capacity. Given that your gulls were stopping over along their route, these detours were not part of non-stop flights and thus were not extending our knowledge of truly long-distance flights. What your data do shed light on is how expensive migratory detours actually are in terms of the annual energy budget of an individual, but that is not their flight range capacity, per se.

Lines 266-270: I think that you need to mention the literature on moult migration here. See, for instance, Mosbech et al. 2012 in Polar Biology for a discussion of this behaviour in Little Auks (Alle alle). Even if this is not a plausible explanation for the northward fall movements that you observed in Pallas’ Gulls, numerous waterfowl and seabirds exhibit large detours as part of their moult migrations and these should at least be noted.

Lines 272-274: Nilsson and Sjöberg 2015 were generally unable to correlate observed reverse migrations over Falsterbo with any weather conditions. I think that Senner et al. 2015 in Journal of Animal Ecology is a stronger citation here.

Lines 278-279: I do not think that faster springer migrations necessarily mean that a species is a time-minimizer. For one thing, wind regimes could simply be more favorable in spring for migration, enabling individuals to migrate faster than in fall, but saying nothing about the relative use of time- vs. energy-minimization strategies.

Lines 281-283: Citation?


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: DETOURS IN LONG-DISTANCE MIGRATION ACROSS THE QINGHAI-TIBETAN PLATEAU: INDIVIDUAL CONSISTENCY AND HABITAT ASSOCIATIONS
Review round: 2
Reviewer: 1

Output:
Basic reporting: See general comments below.
Experimental design: See below
Validity of the findings: See below
Additional comments: General Comments:

The authors adequately addressed all of my concerns from the first draft of the manuscript — well done! However, I noticed one other statistical issue — regarding the relationship between repeatability and consistency — that I missed in the first draft (my apologies) and which I think needs to be addressed. Otherwise, I just have suggestions about ‘word-smithing.’

Specific Comments:

Abstract: I think that the Abstract could be shortened significantly (e.g., Lines 25-29 could be deleted).

Line 9: I think that the use of the word ‘adaptive’ here is odd.

Lines 10-12: This is a bit of a run-on sentence.

Lines 28-30: Do you have evidence to support the first part of this sentence? If not, you should use the word ‘may’ instead of ‘can.’

Line 39: Should be ‘energetic’ instead of ‘energy’

Lines 41-43: This is an awkward sentence.

Line 44: What is a ‘notably’ heterogeneous environment? This sentence, in general, is unintelligible to me.

Lines 51-53: I think that you want to cite Gill et al. 2009 (Proceedings of the Royal Society B) here as a counterpoint, since large-fuel loads can enable birds to avoid stopping in dangerous areas, etc.

Line 64: Yes, low PPO2 is omnipresent over high plateaus, but plateaus present other barriers as well (depending on the species) and may offer fewer stopover areas than other regions.

Line 66: You are referring to phenotypic flexibility here, not phenotypic plasticity (sensu Piersma and Drent 2003, Trends in Ecology and Evolution).

Lines 258-269: I apologize for not catching this in the first draft of the paper, but there is an important distinction between ‘consistent’ and ‘repeatable’ in statistical terms. Repeatability — as a statistical measure — is always comparing an individual’s behavior to the expected amount of variation exhibited by the entire population. This means that low amounts of among-individual variation can lead to left-skewed measures of repeatability, while high amounts of among-individual variation can lead to right-skewed measures of repeatability. (See Conklin et al. 2013 (PLoS One) for an extensive discussion of this issue.) In this paper, there are two potential issues: (1) Four individuals do not necessarily provide a representative sample of the amount of population-level variation existing among Pallas’ Gulls, and thus your measures of repeatability may be right-skewed. (2) Regardless of whether or not the repeatability measures are skewed by the amount of population-level variation, declaring within-individual variation in arrival dates of ±14 days ‘consistent’ seems a bit odd. Many migratory birds vary their arrival dates by only ±3-5 days across years (again, see Conklin et al. 2013), and thus arrival dates that vary ±14 days do not appear especially consistent. I would therefore suggest reporting both measures of repeatability and within-individual variation in Table 1 (e.g., Arrival date for an individual ± variation across years for that individual).

Lines 285-287: “and the detours under these circumstances are even more difficult to explain” — this does not make sense since you go on to explain these detours in the rest of the Discussion.

Line 303: ‘Detoured’ is not the proper word here.

Lines 308-311: This is a run-on sentence.

Line 312: “might be one but not the only driver” — you need a comma between ‘one’ and ‘but.’

Lines 315-327: This addition seems tangential to your core focus and relies on data that you don’t fully present in this manuscript. I would leave it out.

Lines 355-359: See my above comments about repeatability and consistency.


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

Input:
Title: THE FIRST REPORT OF THE PREVALENCE OF NOSEMA CERANAE IN BULGARIA
Review round: 1
Reviewer: 1

Output:
Basic reporting: In this study, Hristov et al. aimed to analyze the presence of Nosema apis and Nosema ceranae in samples taken from different regions of Bulgaria. The information provided is interesting. As it is mentioned in the General comments, I recommend performing some analysis in order to complement the manuscript.
The structure of the article is acceptable. I recommend some modifications in the introduction and to remove the figure.
Experimental design: More details of the experimental design should be provided.
L85-87- Which was the criteria of the number of samples taken from each region? Was it according with the number of colonies from each region? Please, provide details of the experimental design.
L91-92- Why were the samples taken from the entrance or from the frames away from the brood nest? Was any bias in the method of sample collection according with the region?
L95- Which cell lysis buffer? Please, provide information and references.
L125- Did the authors include positive controls? Did the authors verify the identity of at least one of the PCR fragments of each Nosema species? According to Martín-Hernández et al., 2007 the annealing of these primers is 61.8ºC and not 50ºC.
Validity of the findings: The findings are not novel but are valid. The conclusion should be revised.
L37- Please, remove “that confirms the worldwide dissemination and prevalence of Nosema ceranae”. The main conclusion is that N. ceranae is the main Nosema spp. in Bulgaria. As there are regions of the world where there is no information on the prevalence and distribution of this pathogen, it is not possible to say that these results confirm the worldwide dissemination. Also, I do not understand what the authors mean with that “confirms the prevalence of Nosema ceranae”.
L186-191- This information is not the conclusion. Please, remove it.
Additional comments: L25- Please, correct “the objective of the present study was to”
L28- I think that the authors should re-organize the information, mentioning first that only N. ceranae was detected and not N. apis and then the percentage of infection from each region.
L29-30- “rate of infection”, not of invasion
L37- Please, remove “that confirms the worldwide dissemination and prevalence of Nosema ceranae”. The main conclusion is that N. ceranae is the main Nosema spp. in Bulgaria. As there are regions of the world where there is no information on the prevalence and distribution of this pathogen, it is not possible to say that these results confirm the worldwide dissemination. Also, I do not understand what the authors mean with that “confirms the prevalence of Nosema ceranae”.
L42-48- Please, remove this paragraph. This is a general background of the beekeeping activity in Bulgaria and it is not directly related to the purpose of the manuscript. I recommend starting the introduction section highlighting the importance of bees in pollination and for the ecosystem maintenance and then introduce the sanitary problems that the bees have to face. Authors can also mention that there have been reports of colony losses in different countries and that Nosema is one of the main threats that affect honeybees.
L49- rewrite these sentences in order to not repeat the words
L53- Please, provide references. Also, replace Nosematosis for Nosemosis and correct this in the whole manuscript.
L53-54- Nosema apis and Nosema ceranae are the only two microsporidia so far reported to infect honeybees. However, there are other Nosema spp. Please, correct this sentence and provide references.
L68-70- The difference in morphology between both Nosema species is only distinguishable by very expertise technicians. Authors can mention the morphological difference between both types of spores and the advantages of using molecular techniques to distinguish between both species.
L72- Evans et al., 2013 reviewed different techniques to be used in Apis mellifera research but not restricted to Nosema. Please, provide correct references.
L80- Please substitute “spreading” for “distribution”
L80-82. Please, remove this sentence. Nosemosis is usually used to the describe the disease and in this study, the authors analyzed the presence of N. apis and N. ceranae but this was not related with symptomatology. I would recommend restricting this word to sentences in this context. Also, I agree that the information regarding the presence and distribution of these pathogens is important and contribute to gain insights and compare it with other countries. However, this sentence is too ambitious.
L85-87- Which was the criteria of the number of samples taken from each region? Was it according to the number of colonies from each region? Please, provide details of the experimental design.
L91-92- Why were the samples taken from the entrance or from the frames away from the brood nest? Was any bias in the method of sample collection according with the region?
L95- Which cell lysis buffer? Please, provide information and references.
L125- Did the authors include positive controls? Did the authors verify the identity of at least one of the PCR fragments of each Nosema species? According to Martín-Hernández et al., 2007 the annealing of these primers is 61.8ºC and not 50ºC.
L128-remove specific
L135. Please, remove this sentence. It is already mentioned in L88
L137- Provide the percentage in order to compare with the infection level of the other two regions.
L141- Please, substitute “invasion” for “infection”. Also, “in comparison”
L153-Does the authors has historical information about the presence of N. apis and N. ceranae in the country?
L161- It has been also reported that there are bees tolerant and resistant to Nosema (Fontbonne et al., 2013) which is in agreement with your hypothesis. Authors may include this study in the discussion.
L162- I would recommend reorganizing the information in this paragraph. From the previous one, the authors point out that there might be bees more susceptible to Nosema than others and then argued that this may be the case in Bulgaria. But the first sentence only says that there are differences in the prevalence of Nosema in the flat and mountains areas. So, for a better understanding, it is important to say directly that these results might explain the differences in the prevalence observed in the different areas since different subspecies of bees are reared in each area.
L175-L177- Does the author has analyzed the genetic origin of the bees? If the authors still have the samples which were used for analyzing Nosema, it would be interesting to analyze their genetic origin and prove (or not) the hypothesis that they are proposing. I think that this analysis would contribute to making the article much more interesting.
L178- honey bee colonies
L182- Is this the case of the different regions in Bulgaria? Please, expand this idea.
L185- As I mentioned before, the authors analyzed the presence of N. apis and N. ceranae, so I would recommend using the word nosemosis only when talking about the disease.
L186-191- This information is not the conclusion. Please, remove it.
Table I- Please, re-write the title of the table. “Differential diagnostic investigations” is not appropriate.
Table I- “co-infection” instead of “co-invasion”
Fig. 1- I recommend to remove this figure.


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

Input:
Title: THE FIRST REPORT OF THE PREVALENCE OF NOSEMA CERANAE IN BULGARIA
Review round: 1
Reviewer: 2

Output:
Basic reporting: This manuscript reports on the presence of N. ceranae, an emerging exotic pathogen of the honey bee, in populations of honey bees in Bulgaria. The introduction and background top the study are well laid out, with some interesting references to the country’s beekeeping. Results are clear: N. ceranae is widespread, but the native congener Nosema apis was not detected.

The language of the ms is generally good throughout. I make some suggested changes in the section ‘general comments’ below, plus a suggestion for the inclusion of an additional reference. The structure of the ms is fine. Table 1 is clear, Figure 1 is of good quality but country labels should be added.
Experimental design: This ms clearly presents original research: results from PCR testing of honey bees from Bulgaria. The question is clearly posed, and the methods have been described in sufficient detail to allow replication of the study. On the technical side, the methods employed are generally robust. Below I list three additional points.

1. Were positive controls employed in PCRS to avoid the issue of false negatives? Duplicate PCRs might help in clarifying this issue if positive controls were not available.

2. Statistical testing needs to be clarified.

3. (line 99, and more a comment for future reference) Do not use EDTA in re-suspension buffer when dissolving DNA as it inhibits PCR (it chelates Mg ions of the PCR buffer). Use Tris alone.

4. (line 91) Were all 5 bees per colony analysed individually or as a single DNA extract?
Validity of the findings: In large part, the findings seem clear-cut and robust and sound.

1. (line 91) Why only 5 bees collected per colony when it is standard (Fries et al. 2013) to analyse 30 honey bees per colony?

2. Lines 132-142: Provide results of statistical tests to support statements

3. Lines 178-182 The authors hypothesise that warmer climates may favour N. ceranae over N. apis and vice versa. One paper explicitly exploring this issue is:
Natsopoulou et al. (2015) Interspecific competition in honeybee intracellular gut parasites is asymmetric and favours the spread of an emerging infectious disease. Proceedings of the Royal Society of London B: Biological Sciences 282:20141896.
Additional comments: Suggested changes to the text (where relevant, by line number):

Abstract and elsewhere: Do not write about ‘Bulgarian honey bees’ as though they are different in some meaningful way from those in surrounding countries; rather, use ‘honey bees in Bulgaria’.
29-30 highest rate of invasion → highest prevalence
43 Give a date for the First Bulgarian Empire
48 Delete ‘etc’
49 various specific → numerous
53-4 Two main species of Nosema have been identified “in honey bees” (text in “” is needed)
66 replace ‘, but’ with a semicolon
69 invasion → infection
80 spreading → distribution
92 at entrance’ → at the entrance
94 add ‘a’ before ‘single bee’
119 add ‘a’ before ‘Little Genius’
122 add ‘a’ before ‘2%’
141 have demonstrated a higher level of invasion → had a higher prevalence of N. ceranae
145-7 Information should be transferred to the Introduction
165 infection → prevalence


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

Input:
Title: THE FIRST REPORT OF THE PREVALENCE OF NOSEMA CERANAE IN BULGARIA
Review round: 1
Reviewer: 3

Output:
Basic reporting: i recommended this article to published in Peer J
Experimental design: Experimental design is good, well defined
Validity of the findings: as this is the first report from Bulgaria, so i approved this manuscript to published
Additional comments: In the submitted manuscript the authors present a study on the prevalence of Nosema ceranae and Nosema apis in honey bees in Bulgaria. However, I have several issues with the manuscript in its current form.
Since N. ceranae was first detected in 2005 in Apis mellifera colonies in Spain, many publications came up which confirm the presence of N.ceranae in many countries all over the world. It is now accepted that N.ceranae is distributed worldwide and it is assumed that it is replacing N.apis in several regions for so far not known reasons. It is thought that warmer climate conditions forward N. ceranae infections. Therefore, it is not stunning that N. ceranae can be detected in a Apis mellifera subspecies in Saudi-Arabia.
The submitted manuscript is yet another study on the prevalence of N. ceranae in yet another country and this is why the study is of low scientific interest.

my some minor comments are.

1. some latest references should be added in the text for instance "Ansari, M.J., Al-Ghamdi, A., Nuru, A., Khan, K.A. and Alattal, Y., 2017. Geographical distribution and molecular detection of Nosema ceranae from indigenous honey bees of Saudi Arabia. Saudi Journal of Biological Sciences.Volume 24, Issue 5, July 2017, Pages 983-991"

2. it will be more appropriate if author can add microscopic studies of nosema spores in the text

3. discussion should be elaborated in context to the prevalence of nosema in the adjacent countries of Bulgaria


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

Input:
Title: THE FIRST REPORT OF THE PREVALENCE OF NOSEMA CERANAE IN BULGARIA
Review round: 2
Reviewer: 1

Output:
Basic reporting: In this study, Hristov et al. aimed to analyze the presence of Nosema apis and Nosema ceranae in samples taken from different regions of Bulgaria. The manuscript improved a lot since the last revision. I consider that it should be published now.
Experimental design: The experimental design is clear now and the required information was provided by the authors.
Validity of the findings: No comment
Additional comments: Specific comments:
L39-46- Please, provide references.
L43- “On the other hand, honey bees are…”
L44- Remove “are”
L143-144- Please, submit the sequences to NCBI and provide their accession number


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 AN RNA INTERFERENCE (RNAI) GENE KNOCKDOWN PROTOCOL IN THE ANAEROBIC GUT FUNGUS PECORAMYCES RUMINANTIUM STRAIN C1A
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript is clearly written in the professional language and article structure with sufficient supporting tables and figures. The manuscript showed relevant results to answer hypothesis in RNAi Pecoramyces ruminantium strain C1A.
Experimental design: Generally, this primary research tried to fill in the knowledge gap on the development of RNAi in Pecoramyces ruminantium strain C1A that has not been demonstrated in anaerobic fungi. Although more rigorous investigation must be required to prove the deep mechanism of RNAi in Pecoramyces ruminantium, the methods described in this study is sufficient to know if siRNA delivery can work well in Pecoramyces ruminantium.

However, there is the lack of statistical approach to indicate the efficient and inefficient difference in table 1 and table 2. In material and methods, the authors should compare genes encoding Dicer, Ago, QIP, QDE3, RdRp in Pecoramyces ruminantium with those that were reported in filamentous fungi such as N.crassa, M.oryzae, etc.
Validity of the findings: 1. RNAi machinery in the Neocallimastigomycota
Although the authors have identified at least 2 Dicer protein, one protein for each Ago, QDE2, QIP protein and no protein homologs of RdRp in Pecoramyces ruminantium, It is not clear that which Dicer protein involves in RNAi machinery as described in filamentous fungi. In addition, the phylogenetic relationship should be shown between those proteins and RNAi components in Ascomycete group.

2. In the figure S1A, the author should explain how to evaluate the rates of fungal growth and fungal biomass. They should add more result on the rate of fungal growth in this case.

3. In the line 250-251, the author mentioned the rates of the germinating spores that picked up the siRNA, were 80-90%. Do you have any evidence supporting this conclusion because I think that those rates play important role in silencing efficiency? Are you sure that all experiments showed the same percentage if siRNA intake?

4. In table 1, could you please explain why the fungal biomass yield treated with 150 nM siRNA was lower than that with 100 nM siRNA that was considered as the most silencing efficiency (4 folds decrease of ldhD transcriptional level)?

5. I found that the results of 100-150 nm siRNA treatments were not identical in table 1 and table 2. Herein, silencing efficiency of LDHD gene was observed at 100 nM siRNA with 4 folds decrease of ldhD transcriptional level compared with untreated control and unrelated siRNA (table ). However, I found that D-LDH specific activity when treated with 150 nM siRNA, was lowest resulting the lowest fungal biomass yield in comparison with the treatment of 100 nM siRNA. Please explain clearly about this in the discussion part.

6. In figure 3, the authors should explain why they do not evaluate the correlations between time intervals of fungal culturing and silencing efficiency of the target gene. Due to artificial siRNA mediated gene silencing, the efficiency could be decreased by time.
Additional comments: Generally, the authors have tried to prove that RNAi could work in the anaerobic gut fungus, Pecoramyces ruminantium and provide RNA gene knockdown protocol for this purpose. I think the author should make clear some comments in this manuscript that should be improved upon before acceptance.


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 AN RNA INTERFERENCE (RNAI) GENE KNOCKDOWN PROTOCOL IN THE ANAEROBIC GUT FUNGUS PECORAMYCES RUMINANTIUM STRAIN C1A
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript is very well-written, clear, and concise. Appropriate references are included and the figures are generally acceptable. One suggestion would be to include a higher magnification of the spore siRNA uptake panels in Figure S1, and the inclusion of perhaps an outline of an individual spore at higher mag to give an clear indication of the boundary the is encompassing the fluorescence signal.
Experimental design: The experimental design is made clear, and appropriate controls are included. The methods are described clearly and a strong rationale for the experimental plan is presented. The RNA-seq data presented demonstrate some potential off-target or secondary effects. Are there any processes or functions that are enriched in the RNA-seq data that could tell you if these are related processes impacted by the siRNA or if they are truly random/off target effects? GO analysis perhaps?
Validity of the findings: The authors demonstrate clearly by a number of corroborating experiments that the siRNA is indeed being taken into the spores and is exerting a repressive effect on the target mRNA and the activity encoded by the target mRNA. Taken together with the RNA-seq data, there is compelling evidence that RNAi-mediated silencing is functional and may be a useful tool in expoloring further the biology of Pecoramyces ruminantium. Appropriate statistics are provided, and the data are presented both in such a way that any manipulation or normalization is apparent.
Additional comments: Summary of minor suggestions from above:
1.	Include a higher magnification of the spore siRNA uptake panels in Figure S1, and the inclusion of perhaps an outline of an individual spore at higher mag to give an clear indication of the boundary the is encompassing the fluorescence signal.
2.	Are there any processes or functions that are enriched in the RNA-seq data that could tell you if these are related processes impacted by the siRNA or if they are truly random/off target effects? GO analysis perhaps?