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: MANIFESTATIONS OF MUSCLE FATIGUE IN BASEBALL PITCHERS: A SYSTEMATIC REVIEW
Review round: 2
Reviewer: 1

Output:
Basic reporting: The changes made by the author have improved the focus and clarity of this paper.
Experimental design: The changes made by the author have improved the focus and clarity of this paper.
Validity of the findings: I think some further minor changes are needed before publication to ensure the interpretation matches the strength of the reviewed literature.
Additional comments: Thank you for responding to my previous feedback. The changes that you have made have greatly improved the manuscript. I have the following additional comments about the results and discussion that I think should be addressed before publication.

Line #334-335: that is only a 2.5 degree change in knee flexion angle which is a very small change. Please acknowledge effect magnitude not just direction i.e. “…small increase in knee flexion…” whenever you are discussing findings in the discussion section.

Table 3: please add the mean difference or pre- post- values for these significant changes so that the magnitude can be compared with the other studies. You have done this for all studies except this one.

Line #338-339 “A retrospective analysis study identified, during a game, the greatest muscle fatigue occurs in the extensor carpi radialis longus” this needs to be linked to kinematics which is the focus of the paragraph based on the subheading.

Line #345: the reference to Chalmers et al (2017) seems to be a narrative commentary on pitching mechanics and injury risk. Is there actually data to say that “slight deviations” cause injuries or is that just the author’s opinion? If the latter, please revise so that it is clear that “slight deviations” are thought to be important but such data does not exist. I’m assuming it does not exist otherwise it would have been formally included in your review.

Line #351-354: this section talks about forces but reports percentage data. What do the percentages indicate? You report one for the first and last innings so they don’t appear to indicate percentage change. Please make this clearer.

Line #396 “Fatigue linked to injury”: Only two studies (Lyman et al. 2001 and 2002) in Table 5 have findings that relate fatigue (self-reported fatigue or pitch count) to pain (none have done this with injury). Clearly there is a need for more work like this and for injuries not just pain to be considered. You need to acknowledge the lack of research into the relationship and emphasize the importance of this in the future.

Line #419: why is the Fleisig et al. (2009) study not included in your review if it reports injury incidence in injured pitchers?

Discussion: please add a limitations paragraph that highlights the lack of true fatigue and injury studies (i.e. only the Lyman et al. 2001 and 2002 studies have done this and they used pain not injury) and the fact that this review is based largely on data from junior or collegiate athletes rather than professional athletes. It is important to highlight these issues with past literature so that future studies can address them. Also explain why meta-analysis was not possible and what could be done to allow meta-analysis to be undertaken in the future. True Level 1 evidence should be the goal for this topic eventually.


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: MANIFESTATIONS OF MUSCLE FATIGUE IN BASEBALL PITCHERS: A SYSTEMATIC REVIEW
Review round: 2
Reviewer: 2

Output:
Basic reporting: Basic reporting for the revised manuscript has been substantially improved from the original submission.
Experimental design: The methodology in the revised manuscript has been improved. The manucript demonstrates methodological rigour associated with the eligibility criteria and the risk of bias assessment.
Validity of the findings: The findings presented in the revised mansucript presented clearly. Conclusions are supported by the findings.
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: MOBILE LEARNING IN DENTISTRY: USAGE HABITS, ATTITUDES AND PERCEPTIONS OF UNDERGRADUATE STUDENTS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The study has spelling that is not correct. Lines: 53...programmes, line 267...utilised. And others.
References cover the domains of content. they are well based.
Hypotheses are correct and valid, tables support them.
Experimental design: Well done
Validity of the findings: No comment
Additional comments: This is important information for educators as schools plan for future efficiencies in learning and success in life long learning models and vehicles.


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: MOBILE LEARNING IN DENTISTRY: USAGE HABITS, ATTITUDES AND PERCEPTIONS OF UNDERGRADUATE STUDENTS
Review round: 1
Reviewer: 2

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: Please describe the independent groups compared and the study population (see description below in comments).
Additional comments: This article presents an interesting study to assess mobile learning in undergraduate dental students. Please address the following comments to improve this manuscript:
•	IRB approval-Add reference number
•	In the abstract, under Materials and Methods, it is listed that total scores of two independent groups were compared with Mann Whitney test and of three independent groups with Kruskall-Wallis test. These respective independent 2 and 3 groups are not described in the manuscript. Rather, the Materials and Methods section describes that no sampling method was used. Please describe how these groups were selected.
•	Under Introduction, paragraph 2 “In addition, whilst it is possible for students…..”, change ‘effectively’ to ‘effective’ as it is intended to be used as an adjective and not an adverb
•	In Materials and Methods, it is mentioned that “the study was designed as a cross-sectional survey (2014)” while the study is conducted in 2017-18. Please clarify if the survey was designed in 2014, if so, was it published then and add a reference to that publication.
•	The study population, undergraduate dental students, should be described in detail, specifically if these were 1st, 2nd, 3rd or 4th year students as from the quoted responses, it seems the survey was taken by the junior dental students only.
•	It should also be discussed if the dental curriculum in the study institution has an online educational system or curriculum management system. The conclusion that the students use internet primarily for shopping, social networks and communication, and secondary for educational purposes might not be accurate if they are not introduced to an internet-based educational system in the first place.


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: MOBILE LEARNING IN DENTISTRY: USAGE HABITS, ATTITUDES AND PERCEPTIONS OF UNDERGRADUATE STUDENTS
Review round: 1
Reviewer: 3

Output:
Basic reporting: Overall, this is an interesting study and it seems like it offers something new to what is already known about m-learning in dental education.  I believe it would benefit greatly from attention to several points that I have outlined in my comments.

1.  There are multiple areas where the language needs to be corrected or the read-ability of a sentence could be improved.  I have noted specific instances of these and offered suggestions below.

2.  The review of the literature is generally well done, however the introduction would benefit from the addition of a paragraph outlining what is currently known to be associated with attitudes towards m-learning.

3.  The paper has been structured well, and the flow of materials is logical.

4.  The results reported fit well with the stated study aims.

5.  I have the following comments for your introduction:
-line 38 – write out “IT” in full as this is the first time you use this abbreviation
-line 41 – I find the use of the word “solutions” ambiguous.  Consider rephrasing to read as “…educational technologies have taken advantage of this opportunity.” (if I am understanding your intent with this sentence correctly).
-line 47 – “It is emphasized …”  I would suggest making the source of the emphasis explicit in this sentence and re-phrasing it to read something like this:  “It is emphasized in the m-learning literature that …” or “Results of m-learning research emphasize that …”
-line 50 – I would suggest qualifying your description of courses as “intensive” – do you mean they are time-intensive, knowledge-intensive, or in a different way intensive?  I think that specifying this will help strengthen your argument for why m-learning is so important.
-line 53-54 – Consider re-wording this sentence to improve clarity and grammar.  For example, “In addition, whilst it is possible for students to withstand intensive course content delivery over a short period of time, prolonged instruction at this intensity may make meaningful learning more difficult (reference).”

-paragraph starting at line 50:  Consider adding an opening sentence and several linking words to summarize the content of this paragraph.  For example, “There are multiple advantages that m-learning brings to education.  First …  Second …. Third ….”  This would make the content in this paragraph easier for the reader to follow.
Experimental design: 1.  This paper appears to fit with the stated aims and scope of this journal.

2.  I think your research aim could be better structured.  Consider stating the study’s general purpose and then following that up with specific study objectives.  For example, “Therefore, the aim of the current study was to determine the mobile technology usage habits of 81 undergraduate dental students and their attitudes and opinions towards m-learning” [study purpose].  Specifically, the study objectives were as follows:  (1)  to describe usage of mobile technology in dental students, (2) to explore students attitudes towards m-learning, and (3) to explore the role of the students’ demographic information and the purpose and habits of using mobile technologies as examined by previous research 90 studies (Deshpande, Kalaskar, & Chahande, 2016; Khatoon et al., 2014; Rung, Warnke, & 91 Mattheos, 2014).  Or, state the study objectives using the breakdown detailed in lines 153-159.

3.  You have included mention of ethical review as requested by the journal.

I think your methods require greater detail and some of your decisions require support as outline below:
-lines 94-95 – You have repeated the study’s purpose here.  Consider revising to read as follows “To address the study’s aim, a cross-sectional survey design was utilized.”
-ethical approval is included

-line 99 – “The study consists of 81 volunteer participating undergraduate students … would read better as “The sample consisted of 81 undergraduate students who volunteered to participate …”

M-learning Attitude Scale
-line 104:  Omit “with” and change to past tense (consists to consisted)
-line 108 – to help the reader, specify what a higher score indicates (ie. a better or worse attitude towards m-learning)
-please specify the scales here

Student Information Form
–More detail on this item or an example of an item would benefit the reader.  For example, can an example of an item that captured current usage be included?  Was a student asked about usage that day or in that term or in the past 2 weeks?

Opinions about mobile learning
–include the 5 questions here

Statistical analysis
-line 143 – This is the first mention of two independent groups.  This needs to be described earlier in the methods, because the reader does not know what criteria was used to divide the 81 students into these two groups.
-it is unclear why you decided to only include reasons for internet usage in your regression analysis – why not include sociodemographic information?
Validity of the findings: 1.  You have provided your dataset as requested by the journal.

2.  I have the following comments for your results section:
-Line 186 – “students” should be “students’ ”
-line 229 – Interpretation of the r square would help readers understand your results.  For example, 27% of the variation in … was explained by ….(r2=0.268).”
-line 238 – Consider making this sentence flow better by re-wording as follows “The students were asked about positive aspects of courses with m-learning,…”

3.  I have the following comments for your tables:
Table 1
-specify that “M” stands for the median as your tables should be able to stand alone from your discussion of statistical methods
-including the possible range of scores would also help provide context for the data you present here

Table 2
-because usage habits are not described in the methods, I am unclear as to what the total score refers

Table 3
-a note about the different factors here would be helpful as otherwise it is unclear as to what “F1” refers without having to go back and check your methods

4.  I have the following comments for your discussion:
-lines277-278 – “…are very much into …”  Consider revising to more professional language such as “…are willing and eager to use …”
-lines 282-283 – Please specify the findings of this reference – did the other authors also identify the same trend of personal use first and educational use second?
-line 292 – Change “reported” to “reporting”
-line 293-295 has multiple grammatical issues.  Consider revising to “The results of a study by Khatoon et al. (2014) suggest that smartphone usage may be greater than that of computers due to the latter being more difficult and impractical to carry.”
-line 305  “Contributes” should be “contribute”
-line 317 – Select one of “suitable” or “appropriate” but use of both words here is redundant

-a discussion of the limitations of the study is missing and needs to be added

-consider including an implications paragraph
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: MOBILE LEARNING IN DENTISTRY: USAGE HABITS, ATTITUDES AND PERCEPTIONS OF UNDERGRADUATE STUDENTS
Review round: 1
Reviewer: 4

Output:
Basic reporting: Please see comments below.
Experimental design: Please see comments below.
Validity of the findings: Please see comments below.
Additional comments: General comments
=============
This manuscript aims to evaluate attitudes and perceptions towards mobile learning
(m-learning), as well as to identify variables related to those attitudes amongst undergraduate dental
students.. I would like to congratulate the authors for their efforts in planning the research and writing this manuscript.

Specific comments
=============

Major comments
---------------------
1. I understand that the study population consisted of volunteers (n = 81) and the data collection was carried out through a virtual survey. Have you considered the fact that these students (due to the fact of voluntariness), have already had some ease in handling social media and other internet-related technologies? This could explain why the authors found, in general, positive attitudes. I have not seen this in the Discussion section (especially in the limitations).
2. Results section: Some data presented in the tables are unnecessarily repeated in the text. For example, lines 172-173, 175-177. Please correct.

Minor comments
---------------------
1. Line 38: Please write the full term of "IT", and then use the abbreviation in parentheses.
2. Line 88 ("These variables are...") to line 92: This text is probably unnecessary as it is already presented in the Methods section
3. Lines 279-280 have the same information that lines 284-286.
4. Line 291: Why the sencence starts with "although"?


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: MOBILE LEARNING IN DENTISTRY: USAGE HABITS, ATTITUDES AND PERCEPTIONS OF UNDERGRADUATE STUDENTS
Review round: 2
Reviewer: 1

Output:
Basic reporting: Please see comments below.
Experimental design: Please see comments below.
Validity of the findings: Please see comments below.
Additional comments: The authors have addressed all the suggestions. I believe the manuscript is now suitable for its publication.


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

Input:
Title: THE DISAPPEARING HAND: VESTIBULAR STIMULATION DOES NOT IMPROVE HAND LOCALISATION
Review round: 1
Reviewer: 1

Output:
Basic reporting: I find the manuscript clearly written. Sufficient background and reference to previous literature is provided. I have especially appreciated the fact that three competing scenarios are presented to the reader: each is properly detailed, supported by references, and maps unequivocally to specific outcomes of the main task. The structure of the paper is adequate, and does not depart from what is customary in the field. The raw data is provided in the form of an excel file, which I could assess, with appropriately labeled headers; replication of the analyses is thus in principle possible. All the relevant results from the experiment, as far as I can tell, have been described in the main text/supplementary materials.

1) I only have a minor suggestion concerning the introduction: lines 71-76 appear rather vague. For the benefit of readers unfamiliar with the DHT, the authors may consider rephrasing this passage along the lines used in the methods (e.g. 177-180). Moving Figure 1 about here may also help.

2) Figures are appropriate (however, Figure 2 uses cm as units, whereas data in Table 1 is given in mm).
Experimental design: The research falls within the scope of the journal. The research question is important and well identified. The methods are sound. Statements concerning ethics approval are given.

Only a few details in the methods are missing, and in my opinion should be reported:

3) Line 127: power analysis. Justification for choosing an a-priori effect size of 0.3 should be given, for example by referring to previous similar research.

4) I commend the authors for using a complementary Bayesian approach. This approach is particularly interesting in order to provide support for H0 (instead of a mere failed rejection). However, please consider the following points:

a) Bayes factors are appropriately introduced in lines 258-262 and reported for the main tests (270-278). However, they are not interpreted. That Bayes Factors generally support H0 should be, instead, mentioned in the results section, and perhaps rehearsed in the discussion (e.g. around line 408, when discussing the importance of a-priori well powered studies capable of supporting H0). Ultimately, these indices strengthen the idea that H0 may indeed be more likely than H1, arguing against a falsely negative finding.

b) Because all the BFs favor H0, the authors may consider switching to Base 1 BFs (obtained as the reciprocal to 1 of BF10). This would make more intuitive assessing the degree of support for H0. For example, a BF10 of 0.123 would become 1/0.123= 8.13, indicating that the probability of obtaining the data would be about 8 times more likely under H0 with respect to H1.

c) Bayesian analyses are, to some extent, prior-dependent. Thus, the prior distribution used should be described. I believe JASP (which, by the way, should be referenced: JASP Team (2018). JASP (Version 0.8.6)[Computer software].) uses “default” Cauchy-distributed objective priors, centered on 0, and with a scale parameter of 0.707 (i.e. 50% of possible standardized effect size values are expected to fall between -0.707 and + 0.707).
Validity of the findings: The conclusions capitalize on negative results; however, such null findings can be interpreted considered the a-priori power analysis and the Bayesian statistical approach. Raw data is provided for anyone to replicate/expand these findings. Several possible limits of the study have been identified and discussed. Conclusions are consequent to the results, well stated, and when speculative appropriately identified as such.

5) The control (sham) condition is appropriate though far less unpleasant than the active CVS condition; this can be hardly avoided, as distress is to some extent inherent to a vestibular perturbation. However, one may wonder how effective the blinding procedures (line 205) would be under such strikingly different after-effects. Can the authors elaborate about this?

6) I must (partially) disagree on the authors’ assessment in lines 454-457 – “we did not lodge and lock our experimental protocol (…) failure to do so is a shortcoming of this work”.
I am myself a fierce defender of open practices, including pre-registration. Pre-register and possibly peer-review the protocol before data collection (i.e. registered report article format) is, I find, an effective way to improve methodological practices in one field, and counter-act publication bias by favoring methodologically sound studies regardless of their outcome. This is also possible and it has been done recently with vestibular stimulation protocols.
However, while such formats may inspire further confidence in the results, I don’t think that any study that is not formally pre-registered is necessarily inferior, or has shortcomings. This would be a radical opinion and I don’t think it has been ever stated this way. Instead, shortcomings arise in case of undisclosed flexibility in data acquisition or statistical approaches. For example, false positives are inflated for data peeking that leads to multiple sequential analyses and thus affects the sampling plan (i.e. stop data collection as soon as one test is significant, or continue data collection if one test is *almost* significant). Other degrees of freedom of the researcher – removing outliers, change statistical approach, etc. – also yield increased risk of false positives. All research is characterized by the presence of several arbitrary steps, and this is not eliminated by pre-registration; what pre-registration tackles is the transparency in which a researcher is forced to report all these choices, which are in this context undertaken independently of the results.
In this study: a sampling plan was established a-priori following power analysis; one participant was, indeed, discarded, but this has been clearly disclosed and motivated (and her/his data reported in the supplementary materials); analyses seem to be rather customary for the task. Ultimately, I suggest the authors to re-consider this point.
Additional comments: This study is important and timely. I am positive it will provide food for thoughts for many researchers in the 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: THE DISAPPEARING HAND: VESTIBULAR STIMULATION DOES NOT IMPROVE HAND LOCALISATION
Review round: 1
Reviewer: 2

Output:
Basic reporting: This experimental work exploits a perceptual illusion that gives participants conflicting information about their hands position (proprioceptively- vs. visually-encoded). The authors’ hypothesis is that caloric vestibular stimulation would improve hand localization enhancing the proprioceptive location over the visual one. The underlying aim of the study is to expand the understanding of the role of vestibular information in bodily self-consciousness. The literature review informs the basis of the research itself, since contrasting findings exist regarding the effects of vestibular stimulation on self-location mechanisms and body-ownership feelings.

Overall, I think that this work makes an interesting contribution to the field. For the improvement of the paper I have the following suggestions:

The introduction section needs to be improved including a straightforward presentation of your research question and experimental work.

Lines 71-80: While the rubber hand illusion procedure is well known, the disappearing hand trick is far less known. I think it would help the reader to spend a few more words on how the mirage box practically works. Also, being the drift one of your measurements, its description needs to be expanded.

Lines 91-94: in this section it seems that you’re making predictions about the possible effects of CVS without having explicitly introduced your experiment and measurements. Also, mentioning slope and intercept here (as well as in lines 104-106) is confusing.

Lines 107-111: This paragraph is vague. If you believe that CSV induce spatial attention effects, then you should clarify in which direction these effects are expected to affect your results, and discuss this prediction in the results and discussion section.

The information that you’re testing both hands is missing. Do you have any specific prediction about the effects of CVS on the initial localization of left and right hand/drift?

Line 68: missing reference for the evidence that proprioceptive drift is correlated with the strength of the illusion (e.g., Tsakiris and Haggard 2005).
Line 75; 80; 373: Bellan et al. 2017 a or b?

In lines 82 and 299 you mention sections that seem not to be present in the manuscript.

Please, make the symbols in Table 2 uniform.

The whole conclusions section repeats lines 458-464!
Experimental design: Material and methods are well described, but in the experimental procedure section some details should be restructured. For example, initially it is not clear whether you’re testing only one or both hands. Also, in lines 193-194 the sentence “The direction of movements of the arrow does not affect localization judgments” doesn’t help with clarity and appears to be out of place.  This statement needs to be moved in a different position and expanded, for clarity, or deleted.
Validity of the findings: The conclusions are appropriate given the results.
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 DISAPPEARING HAND: VESTIBULAR STIMULATION DOES NOT IMPROVE HAND LOCALISATION
Review round: 1
Reviewer: 3

Output:
Basic reporting: The article is written in professional English and in a clear and unambiguous manner. Literature review is extensive and includes the most relevant studies on the topic. The article is self contained with results addressing the stated hypothesis. Raw data are shared and the file is clear and easily readable.
Table 1 could be improved by explaining in the table caption why "mm/trial" is indicated only  for the slope (the reader not very familiar with the task would have otherwise to go back to statistical analysis of the manuscript and infer why).
Table 1 uses mm but Figure 2 uses cm. Also, I am not sure both figure 2 and table 1 are needed: they are describing very similar parameters.
A similar reasoning on possible duplication applies to Figure 3 and Table 2. I wonder if the number of tables and figures could be reduced without impacting the quality of the manuscript.
Experimental design: The research question is well defined, and the authors did a particularly good job in stating why the research question is important (i.e. informing clinical studies).
The methods are well described and definitely allows replication of the study. Ethic standards are respected.
The chosen method is a valid and well recognized one.

There is 1 concerns about the methods reported in this study, for the CVS in particular.
The authors state that "during the actual experiment, the caloric or sham stimulation was performed as described above and, once nystagmus subsided, the participant walked to another, adjacent room where the localisation task was performed by a third experimenter, blinded to the condition (CVS or sham).". Walking after a vestibular stimulation actually disturbs the effect of the CVS itself. The different head position and the stimulation due to head movements of the vestibular receptors likely restored the system  functioning. I am wondering if the authors can back up their choice and/or can discuss the impact of this experimental choice.
Validity of the findings: Data analysis is sound. However, for the post hoc comparisons, authors did not apply any multiple comparison correction. This could lead to an increase of false positives. It is worth considering applying a Bonferroni correction to the comparisons reported and see if they survive the corrected threshold. This would impact how the authors interpret their results on the subjective report and laterality.

In general, the results of this study are of interest to better understand the contribution of the vestibular system to the development of a sense of the body.
However, given the issue with the experimental design explained above, if walking disrupted the effect of CVS, then I am afraid the reported results are simply due to the fact that individuals were not under the effect of the stimulation anymore when carrying out the task.
Until this is ruled out, I am concerned about considering the conclusions valid or not.
Additional comments: The paper is well written, clear and with an attention to details that clearly indicated an effort towards allowing replication and having a reliable results, which overall is commendable given all the discussion on good practices in psychology which e are witnessing nowadays.
I think the authors need to carefully consider the effects of walking between the stimulation and the execution of the experimental task, and secondly they should consider protecting themselves from false positives in multiple comparisons.
Once these 2 points are cleared, i think the paper would provide a much stronger contribution to the literature.


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 DISAPPEARING HAND: VESTIBULAR STIMULATION DOES NOT IMPROVE HAND LOCALISATION
Review round: 2
Reviewer: 1

Output:
Basic reporting: -
Experimental design: -
Validity of the findings: -
Additional comments: The authors addressed all my points in a satisfactory way.


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 DISAPPEARING HAND: VESTIBULAR STIMULATION DOES NOT IMPROVE HAND LOCALISATION
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: The point on the effects of walking on CVS is now addressed and clearly the experimental choice is backed up. The addition in the manuscript will be helpful should a reader have the same doubt.
Validity of the findings: Thanks for taking into account the potential issue of false positives. I think that the use of corrections for multiple comparisons make your findings stronger. Indeed the result and the corresponding explanation make sense.
Additional comments: I would like to thank the Authors for carefully considering my comments and addressing the concerns, both in their letter and in their manuscript.


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

Input:
Title: THE EFFECTS OF SPATIAL AND TEMPORAL REPLICATE SAMPLING ON EDNA METABARCODING
Review round: 1
Reviewer: 1

Output:
Basic reporting: The ms meets all the requirements of basic reporting.
Experimental design: The ms meets all the requirements of experimental design.
Validity of the findings: My only major concern is that the relative magnitude of temporal heterogeneity is somewhat overstated. For example, in Line 32 (Abstract - Conclusions) they state “We conclude that temporal replicate samples were generally more dissimilar than spatial replicate samples, especially over longer time intervals...”. However, their analysis showed that temporal replicates at 1 week intervals were only significantly more dissimilar than spatial replicates if all MOTUs were considered (there was no significance if only considering metazoan MOTUs). Also, even the significant difference is not of great magnitude (Figure 3 “All MOTUs”). I think it would be better to conclude something more modest, such as “We conclude that the dissimilarity of temporal replicates at one-week intervals is roughly equivalent to that of spatial replicates, but that it becomes much larger as time interval increases.” I might also do an analysis to see at what interval the temporal replicates have significantly higher dissimilarity than spatial replicates in the metazoan dataset.
Additional comments: The manuscript “The effects of spatial and temporal replicate sampling on eDNA metabarcoding” estimates PCR, spatial, and temporal heterogeneity of community composition in two dune lakes. The authors concluce that there were relatively larger differences between temporal replicates than spatial replicates and these differences grew over time. This result suggests that it is very important to consider temporal scale when designing or interpreting eDNA metabarcoding studies. Heterogeniety between PCR replicates was present, but was relatively smaller than either temporal or spatial heterogeneity and did not influence dissimilarity patterns.

This is an important and interesting study that increases our understanding of the heterogeneity in freshwater phytoplankton and zooplankton eDNA communities and informs eDNA metabarcoding applications. The paper is well written, clear, and includes the appropriate citations and context. Besides the one major issue I have describe above (re over-stating the temporal dissimilarity somewhat), I only have a few minor comments/suggestions:

Minor Comments/Suggestions
Line 50 – delete “Especially”

Line 62 - “suggests”

Line 64 – delete “and creating many”

Line 92 – How were the subsites chosen? About how far apart were the subsites?

Methods question – Were any field blanks taken? What steps were taken to prevent cross-contamination between subsites and sites?

Line 294 – add temporal eDNA citation Lacoursière‐Roussel, Anaïs, et al. "eDNA metabarcoding as a new surveillance approach for coastal Arctic biodiversity." Ecology and Evolution 8.16 (2018): 7763-7777.

Figure 1 Legend.  Please add the y-axis metric and explain the pie chart width metric.

Figure 4 Legend. Spearman rho is given in plots A and B but r is given in C and D – is this a typo or does it reflect a different analysis? Also, explain the error around the prediction lines.


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 EFFECTS OF SPATIAL AND TEMPORAL REPLICATE SAMPLING ON EDNA METABARCODING
Review round: 1
Reviewer: 2

Output:
Basic reporting: 1.1 Article is well structured and written. Some sections lack a bit of clarity due to wording and missing information, but it's a minor issue.

1.2 Environmental surveying context, sampling issues and motivation are very well referenced. Methods are unclear at some points (sampling site, PCR and sequencing design and bioinformatic pipelines). Data is well explored and all major results and method biases are discussed with plenty of relevant references.

1.3 Structure follows guidelines. Figures, tables and supplementary materials are all according to text. Figures S2 and S3 are blurry. Table S2 could include the tagging so experimental design could be better evaluated.

1.4 Proposed research was tested and discussed. No deviations from main concept.
Experimental design: 2.1 Research subject is within biological sciences (molecular ecology)

2.2 Question is well defined and relevant. Original sample design includes questions (PCR replicates) already answered in literature, but time correlation is the main point of the study and is well answered.

2.3 Experimental design lacks in clarity and controls.

A few notes:

Lines 91-93
Sampling sites were chosen at random every time or sampling occured in the same spots each week? It's not clear in the text or figures.
What were the distances between the sampling sites? Were the samples collected always in the same order? If the sampling sites are too close, collecting water in spot 1 can influence spot 2 and 3 results due to water movement and the collector itself carrying residual eDNA. Did the authors control for this bias?

Lines 112-114
Assay should be specified as final concentration instead of volumes.
* Did the assay use 0.05 nM of dNTPs for a 40 cycle PCR? There should be almost no amplification with this little dNTP amount.
* Used Taq polymerase should be specified as units instead of volume.
* Neither primer stock/ aliquot concentration or final assay concentration are specified (same for line 124)
* Sample concentration range should also be noted. Same issue for line 122. Why was the volume fixed instead of concentration?

PCR plate designs are not shown, so the cross-contamination controls cannot be assessed. Weeks are divided in 4 groups, each containing 5 sampling weeks (1-5,6-10,11-15 and 16-20). Each group uses the same inline barcode primers (BF/BR). This can lead to amplicons jumping within groups undetected if plates were designed poorly. Possible effects are: (a) similarity between groups will be overall smaller than it should, (b) MOTUs will jump betweeen locations and weeks as false detections and (c) PCR replicates for each sub-sampling site can show the same diversity, even if the real eDNA distribution is different.

Barcode combination design were optimized to split locations, PCR replicates and weeks within groups? Since chimeras removing parameters in UNOISE3 were not specified, a bad filtering can lead to MOTUs jumping between the ingroups (and even between groups if the filtering fails to detect inline indexes).

PCR duplicate is a well known technical artifact that increases the abundance of a sequence over others randomly. In metabarcoding context, it can lead to random MOTUs dominating over others. Usually, to avoid duplicates, samples (and/ or indexes) are marked with Unique Molecular Identifiers. This allows the identification of over amplified UMIs by random and removal by bioinformatics. In case UMIs are not used by lab design, PCR duplicates should still be assessed (using tools like Clumpify/ Picard/ SAMTools) and both data sets (with and without filtering) compared.

Why did alpha was set to 0.5 for UNOISE3? Any previous analysis? Setting the alpha this low for a primer that inclusive is probably generating too many MOTUs that belong to the same species. In this case, ZOTUs should be used. If alpha comparisons for this data set were ran, they should be presented in supplementar material.

Lines 168-169 Why did the authors keep cross-contamined wells instead of removing it completely?

Lines 171-172 Why were the MOTUs with stop codons or frame shifts removed? Degenerated primers (such as used) are known to cause polymerase slips and increase sequencing bases by a few pairs (Elbretch et al. 2018).

Is it possible that algae dominated the readings due to filter clogging? Manajeva et al. (2018) shows that small pore size favors sequencing of algae and bacteria, and that in this case, the samples should be pre filtered. Since PES is a surface filter, did the authors control for clogging (line 355)?
Wilcox et al. (2015) also shows that eDNA particle size is a key point for survey design. He suggests that bigger pores (up to around 1 micrometre) increase the filtering of animal eDNA particles as it doesn't retains smaller unicellular organisms. How the authors predict temporal correlactional will respond to changing eDNA particle size?

This one is just a small comment

Lines 257-260 PCR replicates from the same DNA extract being heterogeneous is a amplification (by primer design) and sequencing bias. Different DNA extracts for the sample sample yielding different MOTUs is a extraction method bias. Currently, there are several studies comparing eDNA extraction methods and their results, but there’s no consensus on which is the best. Sampling effect should also be noted from DNA extraction effect, as the sampling methodology is also not optimized.
Validity of the findings: 3.1 Study shows 2 contributions to metabarcoding and environmental DNA area:
1) Temporal replicates are shown to be an important part of a metabarcoding surveilance design
2) Communities have basal MOTUs that are constant and can be used as control for other experiments, or as biological indicators

3.2 Data should be reanalized to reduce noise according to 2.3. DNA sequences are not deposited yet.

3.3 Study is limited to the proposed question. Ecological statistics are well discussed, but genetics need to be refined (which leads to new analysis). Text is well written and fundamented by results.

3.4
Suggestion and speculation:

The irregular pattern of occuring (lines 307-311) is very interesting. Due to described stochastic nature of PCR, it could be possible that the original DNA extracts has the original sequence that clusters in the MOTUs, but didn’t amplify. In this case, being able to detect it by backtracking (species specific primers or probes) could be an interesting addition to the study that strenghthens the temporal component.
Additional comments: Study is very relevant and interesting. Identification of basal communities based on genetics and how it can be applied to metabarcoding surveys is a major for experimental design. It's the highest point of the study.


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 EFFECTS OF SPATIAL AND TEMPORAL REPLICATE SAMPLING ON EDNA METABARCODING
Review round: 2
Reviewer: 1

Output:
Basic reporting: This manuscript meets all the requirements for basic reporting
Experimental design: This manuscript meets all requirements for experimental design.
Validity of the findings: The authors have improved their discussion of the findings. The manuscript now meets the requirements.
Additional comments: This paper is a very interesting and well-written contribution to the growing field of eDNA metabarcoding. All of my previous concerns have been satisfactorily addressed in your rebuttal and resubmission.


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 EFFECTS OF SPATIAL AND TEMPORAL REPLICATE SAMPLING ON EDNA METABARCODING
Review round: 2
Reviewer: 2

Output:
Basic reporting: 1.1
>English is clear and sentences are well constructed.

1.2
>Context and motivation are well referenced. Methods are clear and satisfactory after revision.

1.3
>Structure is cohesive. Figures and tables are in adequate format. Supplementar material is complete. GenBank sequences are deposited.

1.4
>Figures  provided are in good resolution and show what is proposed by the text and analysis
Experimental design: 2.1
>Research subject is within biological sciences (molecular ecology).

2.2
>Main question of the study (time correlation) is well defined and relevant. Study answers the question satisfactorily.

2.3
>Experimental design is much clearer after revision.

2.4
>Methods are complete after revision.

A few notes:

1)	I asked about the water flow because I didn’t find any info on the water dynamics of the lakes. I was also curious because it is a factor that can induce methodogical bias. Since there seems to be no DNA carryover and it wasn’t controlled, it should not be a problem for the manuscript.

2)	I originally asked for DNA concentration because some samples can swamp and inhibit PCR. I was a little bit worried because of the clogging increasing the DNA concentration by a major amount, but after revision, doesn’t seem a problem.

3)	PCR plate design doesn’t seem to be a problem with the information provided. I was worried with the cross contamination methodogical bias. Results are very interesting, and it could be undermined by uncertainty introduced by amplicons jumping around.
4)	Filtering and chimera control are also ok.

5)	By PCR duplicates, I meant the overamplification of a haplotype by random. In a quantification context, It’s important to track this kind of interaction. Since the primer design didn’t include UMIs, it’s very hard to analyze. PCR replicates should be enough to control for this effect then, since it isn’t a absolute quantification study.

6)	My mistake on the alpha for UNOISE3. I inverted a parameter when calculating this, so the threshold (B) should indeed decrease very quickly with a 0.5 alpha given d differences between sequences, leading to less MOTUs.

7)	Not entirely convinced on the cross-contamination... But since it seems to be an internal issue, instead of a methodological bias, it shouldn’t be a problem. It isn’t clear in the main text though.

8)	No problem with the slippages then. I’m not the editor, but I think it’s very interesting to explore the pseudogenes. Maybe not in this paper, but it has a lot of potential for another study if the authors are interested in such kind of analysis.

9)	Since pore size was the same for all the samples, temporal correlation should not be affected. It was more of a methodological question, as some studies suggest that composion and abundance can change with the pore size.

10)	Extraction replicates not being as impactful as amplification and sampling replicates is very interesting! Since it was tested, should not be a problem too.
Validity of the findings: 3.1
>Study shows 2 contributions to metabarcoding and environmental DNA area:
1) Temporal replicates are shown to be an important part of a metabarcoding surveilance design
2) Communities have basal MOTUs that are constant and can be used as control for other experiments, or as biological indicators

3.2
>Data noise and possible biases are explained after revision.

3.3
>Study is limited to the proposed question and answers it satisfactorily. Text is well written and discussion is well fundamented.

3.4
>The following topics discussed in the first revision and this one seem very promissor for future studies:

- Effect of clustering method on the replicate strategies
- Effect of DNA extraction replicates
- Pseudogenes in metabarcoding
- Backtracking MOTUs “jumping” betweeen weeks

While it may not belong to the scope of this study, it certainly can lead to 2-3 more papers for the authors, as data is already available.
Additional comments: Since the methodological biases don't seem to be a problem, basal communities for surveying seems even more promising.


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 EFFECTS OF A 4-WEEK MESOCYCLE OF BARBELL BACK SQUAT OR BARBELL HIP THRUST STRENGTH TRAINING UPON ISOLATED LUMBAR EXTENSION STRENGTH
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: This is a well-written paper. I especially enjoyed the intro and discussion sections. I would like to see this paper get published as it shows that hip extension exercises, contrary to popular opinion in S&C, do not sufficiently strengthen the erectors. I have just a few minor points for the authors to address prior to accepting the article for publication:

Abstract page 6 line 47: put comma after strength coaches

You may want to point this out as I found it to be intriguing. The individual who gained the most squat strength (25 kg) was someone from the hip thrust group, and the individual who gained the most hip thrust strength (55 kg) was someone from the squat group. Gaining 25 kg on the squat or 55 kg on the hip thrust in 8 sessions over 4 weeks for someone with at least 6 months of training experience is very impressive.

Last thing. I wonder if any particular joint angle of lumbar extension increased significantly. I understand that the area under the curve (strength index) didn't, but maybe the measurement for neutral (0 degrees) did. Did you guys look into this at all? If so, please address it. If not, please list it as a limitation. I appreciate you mentioning the brevity and small sample sizes in the discussion.


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 EFFECTS OF A 4-WEEK MESOCYCLE OF BARBELL BACK SQUAT OR BARBELL HIP THRUST STRENGTH TRAINING UPON ISOLATED LUMBAR EXTENSION STRENGTH
Review round: 1
Reviewer: 2

Output:
Basic reporting: The authors completed a very small study powered to look at within group changes to two different forms of exercise. The article is very well written and the authors are very clear that they are underpowered to look at between group differences. The authors demonstrate the principle of specificity but also suggest that there may be some degree of cross-over to other strength tasks. Below, I offer suggestions that may be able to improve the manuscript

Introduction line 58: I wonder if it makes sense to say that 50% of the variance is shared rather than “it explains 50% of the variance.”  Perhaps this is semantics but sometimes this leads people to think in a “causal” way.

Methods: For your analysis, I think it would be quite useful if you were able to provide time matched control assessments for your 1RM and hip thrust strength tasks. For example, even using an estimate may be better than nothing. For example centering it around 0 and then add in what you might expect that SD of the difference to be…that would allow you to calculate if your within group comparisons are different than noise. I think this would be much stronger than just stating that it is different than 0 (pre to post difference). It would be much stronger if you could build in your “reliability” to an actual statistical comparison.

You could do the same thing with your SI index. I was a bit confused on the reliability that you propose in the text because it is in Nm/degrees whereas data is represented as Nm only.

Results line 211: I agree that nothing is going on here, or very little, but I do not think it is necessarily because the mean does not exceed the minimal difference. Typically that is used to determine the confidence of one person exceeding this threshold but I do not think the entire mean change has to surpass the “minimal difference”.  I think you could do like the earlier suggestion, and just compare the effects of the treatment to that of the “pseudo control”.  Same comment at line 232.

Figures: if you could add in a line to denote “zero”…that might be easier for readers to orient themselves to the figure.
Experimental design: see above
Validity of the findings: see above
Additional comments: see above


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 EFFECTS OF A 4-WEEK MESOCYCLE OF BARBELL BACK SQUAT OR BARBELL HIP THRUST STRENGTH TRAINING UPON ISOLATED LUMBAR EXTENSION STRENGTH
Review round: 2
Reviewer: 1

Output:
Basic reporting: All good
Experimental design: All good
Validity of the findings: All good
Additional comments: Great job with the edits and explanations.


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 EFFECTS OF A 4-WEEK MESOCYCLE OF BARBELL BACK SQUAT OR BARBELL HIP THRUST STRENGTH TRAINING UPON ISOLATED LUMBAR EXTENSION STRENGTH
Review round: 2
Reviewer: 2

Output:
Basic reporting: see below
Experimental design: see below
Validity of the findings: see below
Additional comments: I appreciate the authors addressing my questions. My only remaining comment is that SI index is still Nm in the figure rather than Nm-deg and same comment in Table 2.


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: METABAT 2: AN ADAPTIVE BINNING ALGORITHM FOR ROBUST AND EFFICIENT GENOME RECONSTRUCTION FROM METAGENOME ASSEMBLIES
Review round: 1
Reviewer: 1

Output:
Basic reporting: The article "MetaBAT 2: an adaptive binning algorithm for robust and efficient genome reconstruction from metagenome assemblies" describes an algorithm for metagenome binning. The main purpose is to describe the improvements made to the algorithm previously published as MetaBAT and to evaluate the performance of this algorithm on three artificial data sets and a large number of real metagenomes.

The article is well written with a language that was easy to understand. I also find the structure and presentation to be well organized. However, the following suggestions may improve the language even further.

Line specific language comments:
Line 38: "tools become available" -> "tools have become available"

Line 50 states: "MetaBAT is the … most robust software" but on line 52: "due to its lack of robustness", this seems slightly contradictive to me. Please, adjust it.

Line 50: "can scale up to large" -> "can scale up to handle large"

Line 54: Please expand the abbreviation 'TNF' as this is the first time it is mentioned

Line 60: I believe "docker" should be "Docker" as it is a company name

Line 70: Line numbering seems broken here

Line 70: "where nABD represents" -> "and where nABD represents"

Line 83: "The LPA was modified so that the partitioning is deterministic since the search order is decided by edge strength". Check grammar: "since" does not fit perfectly.

Line 158: "from a very diverse environmental sample" -> "from very diverse environmental samples" or "from a very diverse set of environmental samples"?

The authors also show a great understanding of the field and that they are up to date with the latest advances. However, I find the article lacking in available raw data. Especially in the form of code used to perform the comparisons and analysis throughout the article, but also the results behind figures 2 and 4 where only a subset of the samples are shown should be made available as supplementary tables. Regarding the code, while the code for the MetaBAT2 tool itself is supplied as a bitbucket link (unfortunately without a doi, commit hash or detailed version number), I cannot find any code corresponding to how the different tools were ran.

Specifically I'd like to see:
Scripts or commands showing how the data sets were preprocessed for the different tools
Scripts or commands showing how the different tools were run
This should be supplied for both the CAMI datasets and for the IMG100 dataset.

Furthermore, these scripts should make it clear whether the IMG100 samples have been binned separately, without coverage information from additional samples. If this is the case, this should also be clarified and discussed in the main text. This is especially important since binning of individual samples is quite a different task than binning using the information from multiple samples.

Additional comments:
Line 100: The versions are stated to be the "latest". Please add a date for when this check was done. At least for some of the programs (e.g. CONCOCT), more recent versions are already available.

Line 102: "All tools were run with their default parameters" - This comment is not a sufficient description on how the comparisons were made. Preferably, complete scripts to reproduce the comparison should be supplied. Especially, the commands for input preprocessing is important as this should differ between the tools.
Experimental design: MetaBAT2 is arguably the most used metagenome binning algorithm of today. To have a detailed description of the internals of this algorithm is of great value for the research community. This article sets out to fill that knowledge gap and goes a long way in doing so. However, some details need further clarification regarding the algorithm, specifically:

First part of "Line 70": "We use ABD to rank-normalize TNF (where the smallest TNF becomes the smallest ABD and the greatest TNF becomes the greatest ABD, and so on)" - I cannot understand what this means in its current form. I assume TNF and ABD means “TNF score” and “ABD score”. Although, with this transformation, does that mean TNF scores are replaced by ABD rank? Then it stops being a TNF score, right? Please expand on the concept of rank-normalization and what purpose it serves in the algorithm.

Line 70, the definition of w: My interpretation of this is that it will give more weight to the ABD score the more samples with sufficient coverage there is. Is this correct? In that case it might be worth pointing out to the reader.

Line 71: "S should be more accurate" - More than what? Why? Please clarify.
Line 71 - 72: "especially when the communities are extremely complex". Why? Please clarify.

Line 76: Please clarify if TNF in "constructed by only using TNF" is TNF score or the actual TNF.

Line 81: Are edges discarded if a node has reached it’s limit? How large is the subset per iteration?

Line 85: I was not able to find an explicit mentioning of Fisher’s method in the MetaBAT 1 paper. Please explain how this is used in MetaBAT 2.

Line 91: "correlation to member contigs from that bin is larger" Please clarify if this is intends the average correlation for all contigs in the bin or if just the maximum correlation value is intended?

Line 104: A more explicit description of "The genetic algorithm" would be beneficial. Currently only the parameters are listed.

Furthermore, as I've mentioned in above, scripts or commands on how the investigation was performed needs to be submitted. This is essential for the reproducibility of the results and should definitely be included as supplementary data. In the manuscripts current form, it is not clear how parts of the comparison was performed.

Furthermore, it seems to me (not entirely clear due to above mentioned reason) that the comparison on real metagenomes are not using coverage over multiple samples. The possibility to use coverage over multiple samples is what distinguishes the metagenomic binning tools released at around 2015 from the previous generation of binning softwares. To compare these tools without using that possibility (especially without explicitly commenting on it) seems to me slightly odd. Please, clarify why the real dataset was chosen so that multiple samples were not possible to use. Or if my understanding is in fact wrong, please make this clearer in the text.
Validity of the findings: MetaBAT2 is most likely the fastest binning algorithm available today, and the authors make that statement clear with the data included in the results section. I also greatly appreciate the good description on how this computational efficiency is achieved.

The quality of the results from MetaBAT2 is also most likely very good although I am not completely convinced that the evaluations included in the manuscript shows this in a fair way. I might have misunderstood the following, and in that case, please clarify in the manuscript so that this misconception is avoided. For example, it appears as Figure 2 includes a subset of samples where specifically MetaBAT2 was most successful. The result from the other samples (~90%) are not shown and not included in any supplementary data. Also, comparing the results of the competitors in Figure 1 to Figure 2, panel b in Szcyrba et al. 2017, all three competitors which are included in both comparisons (MaxBin2, COCACOLA and CONCOCT) show poorer performance in this manuscript than in the previously published article. Part of this might be due to a parameter optimization performed in the Szcyrba publication which is not reflected in the default settings of these programs. The settings used for Szcyrba et al. should be readily available from the corresponding biocontainers and I would argue that that would be a more fair comparison. The performance displayed by MetaBAT2 in this manuscript would still place it in the first place using the results from Szcyrba et al. 2017, but the competition would be more close.

Line specific comments:
Line 162: "only producing fewer than 5 genome bins" - for MetaBAT2 or for all softwares combined?

Line 163: "the top 13 metagenomes ordered by the number of genome bins identified" - for MetaBAT2 or for all softwares combined? From looking at of Figure 2, the different samples seem to be sorted based on the number of bins from MetaBAT2 only. If this is the case for how the samples shown were chosen as well, this is unacceptable. Choosing the samples where MetaBAT2 performed the best obviously bias the comparison in MetaBAT2:s favour. Please clarify how the 13 samples were chosen based on number of bins from all binning softwares or only from MetaBAT2.

Figure 1: "All the tools … were run using the default parameters": Again, I would argue that this is not enough. Especially since comparing these results to figure 2, panel b of (Sczyrba et al. 2017), all overlapping software (except metabat) performs worse in your comparison than in the already published comparison. In Sczyrba et al., all tools were submitted in reusable, reproducible containers. For this reason, it is very important that the authors of thus manuscript can show how the tools were run in their comparison to ensure that no mistakes have been made.

Line 204-207: Please clarify which "some tools failed on some large assemblies". Perhaps in a supplementary table showing the number of genome bins for all software and all samples?
Additional comments: While not necessary for publication in PeerJ, I feel this paper is of great value to the metagenomic binning research community. However, I find several points that need to be revised before it is fit for publication. While these changes are of great importance, I do not think they will be very difficult to make. To me, the most important point to improve is the transparency on how the comparisons were made and how the results of those are displayed. The second most important point is to expand and clarify the methods section to make it easier for readers to understand the algorithm.


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: METABAT 2: AN ADAPTIVE BINNING ALGORITHM FOR ROBUST AND EFFICIENT GENOME RECONSTRUCTION FROM METAGENOME ASSEMBLIES
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript is very minimalist in its descriptions of metaBAT2's algorithm. However, this is mostly sufficient considering the main algorithm is covered in detail in the original metaBAT1 publication.

It is important for the authors to provide all the shell commands and post-processing scripts used in this analysis. I recognize the simplicity for some of the benchmarks, however it is still crucial for re-reproducibility purposes.

A minor suggestion for the readability of Figure 2: visually separate the bars coming from different metagenomes to make the segments more distinct.
Experimental design: As expected, the performance benchmarks display the superior performance of metaBAT2 over other software. However, considering that this manuscript is meant to outline the improvements of metaBAT2 over metaBAT1, it is strange that performance of metaBAT1 itself is not included in most benchmarks (Figures 1 and 2, Table 1). Even if the differences are minor, it is important to include this information.

The authors also talk extensively about the scalability of metaBAT2 compared to metaBAT1. In my experience this is accurate, however it it would be nice to see this quantified. Beyond adding metaBAT1 to Table 1, it would be interesting to see a curve displaying the size of a data set vs either total compute time or maximum memory usage.
Validity of the findings: It is interesting to see the very sub-par performance of CONCOCT in the benchmarking of CAMI High, considering its relative success in the original CAMI challenge publication. I wonder if the bench-marking was done without breaking up the longer contigs into smaller sub-sequences, as was intended by the software. See discussion at https://github.com/bxlab/metaWRAP/issues/76.

The metaBAT2 software has already being applied in a variety of microbial metagenomic studies, and it is well known in the research community that currently it delivers unparalleled binning performance in most data. Compared to metaBAT1, which was also popular, metaBAT2 offers significant improvements in performance and computation efficiency in larger data. With this in mind, it is good to see that the manuscript has been written, especially considering the software has been available to the community for some time. However, the article and its conclusions could be greatly improved by a several added benchmarks that would put it into context with the already published metaBAT1 software.
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: METABAT 2: AN ADAPTIVE BINNING ALGORITHM FOR ROBUST AND EFFICIENT GENOME RECONSTRUCTION FROM METAGENOME ASSEMBLIES
Review round: 2
Reviewer: 1

Output:
Basic reporting: The manuscript has greatly improved from the previous version, and sufficient retails are provided.
Experimental design: Thanks to suggestions from Reviewer 1, the methods are more detailed and the authors provided scripts for reproducibility online.
Validity of the findings: Most comments have been addressed, and I thank the authors for providing  comparison between the performances of metabat1 and metabat2 to clearly outline what was gained by producing this new version.

One request that was not addressed was the inclusion of metabat1 run-time statistics in Table 1. Considering speed is one of the major improvements of metabat2 over metabat1, I feel it is important to include this comparison. Running metabat1 on default settings is probably sufficient for this.
Additional comments: No additional comments


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

Input:
Title: ASEXUAL REPRODUCTION OF A FEW GENOTYPES FAVORED THE INVASION OF THE CEREAL APHID RHOPALOSIPHUM PADI IN CHILE
Review round: 1
Reviewer: 1

Output:
Basic reporting: The article presents a nice comprehensive study of the genetic variation within Chilean R. padi populations, however there are some areas of concern which should be addressed

Notes to the author relating to their raw data:

Proportion of alate adult data: proportion data is bound at 0.0 and 1.00. Why does proportion data exceed 1.00, and go beyond 20? Is this percentage data?
Proportion of alate data: In host plant column should be . not , following the shortened genus names
Genotyping result data: data seems fine, but is hard to navigate/assess in current format I suggest the authors separate this into separate columns instead of a string of text in the first column.

Notes to the author relating to the introduction of the manuscript:
Line 77: "As described  for several pest aphids, the predominance of a certain reproductive mode in R. paid relies on geographic factors". The authors should provide a citation for this statement and briefly describe the examples they mention, this will also improve the introduction by providing a wider assessment of the literature
Line 82 - 95: I recommend the authors put more emphasis on the importance of their study, and the reasoning behind why a) assessing the genetic diversity in R. padi populations in Chile is important; b) how this can help improve crop protection (or inform pest management strategies)
Line 92 - 95: I think the manuscript can be improved if the authors more clearly describe what they did in the study and provide a comprehensive description of the key findings. For example: "In this current study we examine the genetic diversity in populations of the cereal aphid, R. padi, in the main cereal growing region in Chile. We show that...."

Notes relating to the materials and methods:
Line 164 – 167: This information would fit better in the introduction
Line 236 – 237: For the PGR equation, please ensure that log (ln) is written as ln not Ln

Notes to the author relating to the discussion of the manuscript:
Section 355 - 402:
I suggest the authors also include the Leybourne et al 2018 (doi: https://doi.org/10.1111/1744-7917.12606) study in their assessment of European R. padi genetic diversity (this study assessed genotype distribution in small R. padi population in Eastern Scotland, UK)
Also, as a potential recommendation, the authors could improve the manuscript greatly by extracting the genotyping information from all the studies they mention and to examine whether their Rp1 genotype (most distributed in Chile) has a similar distribution in other countries.
Section 414 - 448:
Line 439 – 446: Guo et al 2019 (doi: https://doi.org/10.1002/mbo3.817) and Leybourne et al 2018 describe endosymbionts in R. padi, these are more suitable references than the Sepulveda et al 2017 reference as they show that R. padi is able to form these relationships. Additionally, the Zepeda-Paulo et al 2018 study shows that in one of their aphid clones assessed (named Rp1) a small proportion of the aphids contained the endosymbiont R. insecticola, so the statement that secondary symbionts have not been found in Chilean R. padi is incorrect.

Notes to the author relating to the Tables and Figures of the manuscript:
Table 1: Host plant – for wheat please indicate which were collected from durum wheat and which from aestivum wheat
Table 3: For microsatellite loci – suggest splitting the heterozygous numbers to show more clearly the size of each allele (so change from displaying 322322 to 322 | 322) this would facilitate ease of readability. The authors should also define Psex
Some figures are rather large – too large to print. Figure 1 and 2 exceed A3 size, so would not be able to be easily printed and observed
Fig. 1: Legend: typo – should read “unique MLG” not “uniques MLG”.
Fig. 2: It is unclear what the blue, green, red, grey, and gold segments of the graph are showing

Some suggested English language improvements throughout the manuscript
Line 43: Suggest deleting "in following years"
Line 46 - 49: Suggest the authors rephrase this wording of this section to make their point more clear
Line 53 - 55: Suggest rephrasing to "their reproduction mode, which is based on parthenogenesis and can result in rapid population increases"
Line 62: delete "aphids"
Line 64: delete "the aphid"
Line 68: delete "the aphid"
Line 82 - 84: This sentence needs to be rewritten to improve the readability, for example "Understanding the genetic features of pest populations can help to inform which pest management strategies would be most appropriate"
Line 99: Rephrase to “R. padi individuals were collected…”
Line 100 – 102: Rephrase to “Sampling was carried out during spring…”
Line 106 - 107: Rephrase to “Sampled plants include the most commonly cultivated cereal crops:…”
Line 116: Please use the correct entomological terminology of “apterous” when mentioning wingless aphids, and “alate” when referring to winged aphids
Line 117 – 120: Rephrase to: “In addition, live individuals were collected from each plant to establish clonal laboratory lines for aphid performance assessment”
Line 121: change “in barley” to “on barley”
Line 123: Define what a “transparent plastic mica” is, this is not a usual term before
From Line 123: expand this section to include information on how the collected aphid lines were reared in the laboratory (moving this information up from the aphid performance section)
Line 136: replace “in 2 min” with “of 2 min”
Line 201: Delete the “of” in the two instances of “within of”
Line 228: Please rephrase to “The reproductive performance of Rp1 was assessed on …”
Line 235: please use the terms apterous and alate
Line 307 – I think it is best to state “comparing its PGR when feeding on four different hosts” use of the term “reared on” implies that the aphids were kept on the four different plants in culture, not during the experiment.
Line 330: delete “exhibiting”
Line 331: Rephrase to “rather than act as casual agents”
Line 331: typo, should be “latter” not “later”
Line 330 – 338: I think in general this section needs to be rethought and rephrased, it isn’t explicitly clear how this point relates to the findings reported in the manuscript
Line 340: typo, should this not be “founder effect”?
Line 340 – 342: This appears to be speculation, the authors should indicate that they are speculating
Line 343: add “the” so that sentence reads “that in the native” delete “widely”
Line 343 – 344: rephrase to “were reproducing most frequently by asexual reproduction”
Line 345 – 346: The authors should include a reference to the four/three most widely distributed grain and pea aphid genotypes. It is not clear if this is supported by the Figueroa 2005 and Peccoud 2008 references, or if these references just support the French lineage statement.
Line 359: typo, should be “formed of”
Line 368: rephrase to “the most common genotypes”
Line 376: rephrase to “is not very cold”
Line 378: Rephrase to “However, in China”
Line 380: typo, should be “predominantly”
Line 386: Reword to “Additionally” not “Differently”
Line: 388: “Are similar to Australian populations”
Line 389 onwards: I suggest the authors make use of an English language editing service
Experimental design: Certain aspects of the experimental design need to be addressed (or clarified) to improve the article. Some detail which would be required for full reproduction of the work are currently missing from the manuscript. Key information regarding experimental design and experimental conditions is missing (specifically related to the genotype fitness experiment)

Line 92 - 95: The authors need to more clearly define the purpose of the research/the research question more clearly at the end of the introduction
From Line 218: include information on the host plant from which each Rp1 clonal line was originally collected from. This is mentioned in the results – line 309, I suggest stating this in the methodology section not the results section.
Line 221 – 228: I suggest moving the aphid rearing conditions to the previous section (indicated by my point above)

Specific notes relating to the materials and methods section of the manuscript
Refer to Table 1 (the 17 sampling sites) in the methodology section where appropriate to facilitate reading
Line 104 – 105: What was the selection criteria which determined which of the initial fields were sampled in the subsequent sampling campaigns? Why weren’t all the fields revisited in the other years?
Line 129 – 130: To facilitate full reproducibility of the experiment please provide the Forward and Reverse primer sequences used in this study (even if they are reported in the Simon et al 2001 study). This can be in the form of an additional supplementary table
Line 135: What concentration (active Units of enzyme) of Platinum Taq were used?
Line 136: How was DNA concentration determined? And were 50 ng of DNA used in total, or 50 ng / ul
Line 140 – 143: please provide the full methodology for the capillary electrophoresis procedure, for example: were the PCR products diluted? How much of the sample was loaded into the reaction solution? How much dye was used (and what type of dye was used). What size standard was used? Were the samples suspended in Hi-Di Formamide?
Line 218 – 220: Please provide detailed information on the conditions used for these experiments – light intensity, temperature etc. Were the assays conducted in glasshouse or controlled environments? Assays carried out on the lab bench will not be appropriate as these conditions aren’t controlled
Line 229 – 231: Please indicate at which developmental stage (using the Zadoks et al staging key: https://dx.doi.org/10.1111/j.1365-3180.1974.tb01084.x) all plants were when they were used in the fitness experiment. Plant developmental stage can effect aphid fitness (for e.g. Leather and Dixon 1981 https://doi.org/10.1111/j.1744-7348.1981.tb03006.x and Stadler et al 2002: https://doi.org/10.1046/j.1461-0248.2002.00300.x). The results are only comparable across plant species if the experimental plants were at the same developmental stages. The authors should also discuss these results in relation to the findings of the Leather and Dixon paper mentioned above.
Line 232: Please provide the growth and environmental conditions of these controlled environment chambers
Line 233 – 242: Please provide information on whether this was glasshouse, field, or controlled environment experiment. Please provide explanation how aphids were contained onto each plant to prevent their escape
Line 234 – 235: Was the age of the adult aphids synchronised before they were used in the fitness experiments?
Validity of the findings: Genotyping results and interpretation - in general, I believe that these aspects of the study have been carried out robustly, analysed appropriately, and interpreted correctly. Besides the points mentioned above regarding providing more information on the experimental design, I have no major criticisms relating to this aspect of the work.

Aphid reproductive performance - there are several aspects of this part of the manuscript which need to be addressed and clarified before it can be determined whether this aspect of the study was robust and statistically sound.

Notes on the statistical analysis of the aphid performance data
Replication level of five for aphid fitness data is rather low, however as this has to be weighed against the number of individual treatments (20) as such I believe this is adequate.

Line 233 – 242 – how were the reproductive experiments set out? Please include information on the experimental design of this (randomised, split-plot etc.). I suggest the authors re-run the analysis and take into account the experimental setup if possible, for example if the data is a fully randomised blocked design, I suggest the authors statistically account for the intra-block variation (for example, through the use of general linear/linear mixed effects models).
Line 239 – 242: How were the proportion data analysed? This isn’t mentioned in the manuscript. However, If the authors used two-way ANOVA (as they did with the PGR data) this isn’t the most appopiate statistical methodology to use for the analysis of proportion data. As proportion data is bound at 0.00 and 1.00 general linear models with binomial distribution (aka log-linear/logistic general linear models) are more appropriate for the analysis of data with this structure
Line 240: Was this Tukey HSD or Tukey LSD?
Line 241 – 242: What statistical software was used for this analysis?

Notes relating to the reporting of these results:
Line 310: the authors indicate that there are differences amongst the different host plants and on line 313 – 314 indicate that this is between Limavida lineage and all others (Fig. 4). It isn’t clear which lineages are significantly different from the others – The authors carried out Tukey HSD tests to test this, but do not report it. I suggest the authors either report the differences in this section in the text, for example by rephrasing the paragraph to state “The PGR varied among hosts (F3, 90 = 13.48; P < 0.001), with significant differences between Limavida and xxx (statistical results of HSD test)”. A simplified way of displaying this would either be in the form of an additional table reporting these which can be referenced to in the text, or by plotting the results of the post-hoc tests on Fig. 4.
Line 311: The same point as above should be taken on board by the authors with regards to the observed difference between the different aphid lineages. I suggest a table reporting the post-hoc results may be the most simple and appropriate way to report these results.
Line 311: “No interaction between factors was found” the authors should report the statistical results here, even if they are non-significant
Lines 315 – 317: Again, differences are indicated between the proportion of alate aphids in relation to host and lineage, but no post-hoc results are displayed to provide information on which differences these are. I suggest the inclusion of a "post-hoc" table detailing which factors are different for the PGR and the Alate proportion results.
Line 316: “but no interaction between both factors was found” the authors should report the statistical results here, even if they are non-significant
Fig. 4: This figure is not very clear, is hard to interpret and should be improved to aid the reader. Ideally, as they are not reported in the text, the post-hoc analysis needs to be included on the fig (or reported in a table if it is not going to be empirically stated in the manuscript). Fig. 4B is this percentage or proportion data? Proportion data would be bound at 0.00 and 1.00. Generally, the figure is hard to interpret. Other potential ways the authors could consider for displaying this data are:
o	A simplified table stating the mean and error, this could also simply be edited to include the results of the post-hoc analysis.
o	A dot plot – as sample number is low (5) dot plots will increase the transparency of the data.  It would, however, be difficult to clearly display the post-hoc results on this graph
o	Box plots – would make graph a lot larger, but would be greatly increase the readability of the figure and make it easier to interpret than current display, post-hoc results could also easily be added to this
Additional comments: In this study by Rubio-Meléndez et al. the genetic diversity of R. padi is assessed throughout Chile. Using a substantial sample size the authors show that one genotype, Rp1, is the the most prevalent in the regions assessed. The authors then carry out aphid fitness assays to examine the fitness of this aphid genotype in individuals collected from several regions.

The first half of the manuscript (analysis of aphid genetic diversity in Chile) has been adequately carried out and presents a nice, novel insight into the genetic diversity within Chilean R. padi populations. The prevalence of one genotype is specifically interesting. However, certain aspects of the aphid fitness experiment need rethinking and supplemented with addiitonal information as it is not currently possible to fully assess the validity of the findings as the experimental design of these experiments has not been adequately explained.

Overall, the manuscript needs a substantial rewrite to facilitate reading, use of English is confusing at times and sections of the discussion are often hard to understand. The overall quality of the manuscript would be improved by setting a research question/hypothesis in the introduction and using this as the basis of the discussion


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: ASEXUAL REPRODUCTION OF A FEW GENOTYPES FAVORED THE INVASION OF THE CEREAL APHID RHOPALOSIPHUM PADI IN CHILE
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript is well written and the paper is clear, but the text requires an additional round of polishing. Some typos I noticed include: sampling months in the Table 1 are written in Spanish, MICRO-CHEKER (Line 144), funder effect (Line 340), bluest (Line 790), interchangeable use of “its” and “their” is confusing (Line 92 – 95).

Using term “amplification” in the title may be misleading for the readers, as it might be related to DNA amplification. Also, term “clonal” is not the most suitable, as has been discussed by Loxdale (2008). Instead, “asexual” or “parthenogenic” could be used. The use of abbreviations (CP, OP, PGR) should be avoided, because they reduce readability of the text.

Please format the Main text and Supplementary Figures (consistent font style, font size, alignment, panel labels) to enhance representation.
Experimental design: Authors performed sufficient sampling for R. padi, the sampling scheme is adequate.
Number of replications for the analysis of reproductive performance is sufficient.

Readers would benefit from an explanation how 6 microsatellite loci were chosen and why their resolution is adequate (Line 130).

Authors assessed reproductive performance of the most common genotypes of R. padi on different host plants, but it is not explained (in the Introduction) which research question is addressed using this analysis.

While the Authors describe that the sampling happened during spring (Line 101), Table 1 indicates three sampling seasons: August 2013 – January 2014, September – November 2014, and August 2015. January 2014 does not belong to spring. The Authors write: “During the following years, the same set of fields were sampled” (Line 106) – but Table 1 suggests repeated sampling only for 3 localities (LI, VP, PE). The text in the Materials and Methods should be corrected to match the Table 1.

Methods described with sufficient detail, for the most part. Please provide details how the level of photosynthetically active radiation has been measured and controlled (Line 125).
Software used for the analysis of reproductive performance should be mentioned (Lines 240-242). Please also provide the raw data and code for this analysis to ensure reproducibility.
Validity of the findings: Repeated genotypes found in the study may result from cross-contamination between the samples. In the Materials & Methods, it is not mentioned how the authors controlled for the cross-contamination. Negative control (PCR master mix without DNA template) should have been run on a gel together with other samples to rule out potential cross-contamination. Please provide information how cross-contamination was controlled.

Are the same genotypes over-represented in different sampling seasons? This analysis is feasible for at least first two seasons: August 2013 – January 2014 and September – November 2014. This will help to assess the role of obligate parthenogenesis in explaining the finding of repeated genotypes.

For the linkage disequilibrium analysis (Line 180), correction for multiple testing should be applied. As highlighted by Halkett et al (2005) the LD analysis should be performed on datasets without repeated genotypes. It is unclear (Line 180) if the results shown in Table S1 were performs on dataset with or without repeated genotypes. Please provide details.

For the pairwise analysis of Fst (Line 198-202), correction for multiple testing should be applied. Please explain which formula has been used for FST estimation – and why it produces unusually low values (Figure S1).

Previously it has been reported (Delmotte et al 2002) that asexual lineages of R. padi form a distinct phylogenetic cluster. However, Figure 2 and Figure 3 suggest that candidate asexual lineages are polyphyletic. This contradiction should be addressed.
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: ASEXUAL REPRODUCTION OF A FEW GENOTYPES FAVORED THE INVASION OF THE CEREAL APHID RHOPALOSIPHUM PADI IN CHILE
Review round: 2
Reviewer: 1

Output:
Basic reporting: Following revision the manuscript is more clear, the addition of the paragraph at the end of the introduction (from line 103) greatly increases the readability.

I have made some additional (minor) suggestions below:

Line 118 - 119, consider rephrasing for clarity
Line 201: indices not indexes
Line 260: Consider rephrasing from "plants were disposed" disposed implies they were thrown out
Line 500: H. defensa not H. insecticola

Tables: For Tables reporting statistical results, I recommend the authors use <0.001 instead of 0.0000

Supplementary Data 1: Column C should read "nymphs"
Experimental design: No comment
Validity of the findings: No comment
Additional comments: In general, I believe the authors have made ample changes to the manuscript and have taken into account the comments and concerns raised by myself and the other reviewer.


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

Input:
Title: ASEXUAL REPRODUCTION OF A FEW GENOTYPES FAVORED THE INVASION OF THE CEREAL APHID RHOPALOSIPHUM PADI IN CHILE
Review round: 2
Reviewer: 2

Output:
Basic reporting: The text of the manuscript has been improved, couple more typos could be corrected:

Obligated parthenogenesis  -> obligate parthenogenesis? (Line 106 of the PDF)
Partenogenesis -> partenogenetic? (Line 95 of the PDF)
SupplementaryData1-Performance.csv: Nymfas -> Nymphs?
Figure 1, Legend: Uniques MLG -> Unique MLG?

The figures have been improved as well. I have further suggestions for improvement of the Figure 2A.

According to the Table 2, and the Figure 1, in the localities LIM, DO, VA, CA only the most frequent genotype Rp1 has been found. This agrees with STRUCTURE clustering: all the individuals from these localities belong to the cluster 1 (marked by the brown colour, Figure 2B).  But in the Figure 2A, in the circles representing these localities there are tiny yellow sectors. I assume they denote the frequency of 0 of the genotypes of the cluster 2? Then these tiny yellow sectors should be removed, as they are misleading (i. e. they suggest both clusters are represented in the localities).

Figure 2B would greatly benefit from increased resolution. The rectangles which denote the localities should have thicker black boarders, to improve readability of the figure (currently it is hard to distinguish between the localities). Please also use arrows to label localities with small number of individuals (LIM, DO, VA, CA).
Experimental design: Please mention and cite the specific R package used for the analysis of reproductive performance (Line 273-274 of the PDF).
Validity of the findings: Results:
Please report the frequencies of the most common genotypes in different seasons (454-457 of the PDF).

Table S2:
The reported significance level of 0.00033 has an extra 0, it seems to be a typo (for the Bonferroni correction 0.05/15 = 0.0033). Also, please explain why the p-values slightly changed in the revised version of the manuscript?

Eliminated Figure 3:
I suggest it is worthwhile to keep this figure at least as a supplementary material as it gives a good overview of how repeated and unique genotypes are related. I assume that the choice of colors used to mark “clades” is arbitrary (i.e. it does not relate to the Figure 1), so they could be eliminated. If my assumprion is wrong, please provide explanaton what do these colours denote.
Additional comments: The Authors provided a detailed response to all the comments and improved the manuscript.
Their new result of finding that the same most common genotypes were present in different seasons strengthens their conclusions about asexual reproduction of R. padi in Chile.


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: TIO2 NANOPARTICLES AFFECT THE BACTERIAL COMMUNITY STRUCTURE AND EISENIA FETIDA (SAVIGNY, 1826) IN AN ARABLE SOIL
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: This study focused on the effects of nanoparticles on bacterial community structure. Also how the presence of earthworms, oat seeds or their combinations affect soil properties and bacterial community were examined. This topic is interesting, which is helpful to understand nano-toxicity in the environment. Overall, this study is comprehensive, the experimental design and data can support the conclusion. The writing is also easy to follow. Some major issues need to be addressed before accept.
1)	Please rewrite the abstract. Now it is hard to find the novelty of this work through the abstract. Some main and important results are very vague. For example, the last sentence, I think if we did not do this study, we still have this kind of conclusion. So what are your special results in your study?
2)	Results and Discussion. The title of this part such as soil characteristics, earthworms, unamended soil et al. needs to be revised. The whole sentence is better to summarize the following results.
3)	Conclusion part needs to be rewritten. This part looks like an abstract since much specific results are shown in this part.
4)	The writing needs to be carefully checked. There are many gramma mistakes found in the manuscript. Probably a professional language editing company is helpful.