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: PATTERNS OF GENE EVOLUTION FOLLOWING DUPLICATIONS AND SPECIATIONS IN VERTEBRATES
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: Review of David, Oaks, and Halanych: “Patterns of gene evolution following duplications and speciations in vertebrates.”

Overview:
In this manuscript, the authors describe having inferred a large set of gene trees that include branchings due both to speciation and duplication events. They divide the branches in this fashion and estimate branch-specific values of the selective constraint (Ka/Ks) for all branches. They then explore the pattern of shifts in Ka/Ks after speciation and duplication events.  They argue that the pattern of increases in Ka/Ks after duplication events is consistent with the orthology conjecture: that orthologs maintain more similar functions over time than do gene duplicates.

Major comments:
The authors have generally responded to my prior concerns, but I still think the manuscript overstates its case in a couple of places:

Lines 45&46: I do not agree that selection for higher dosage, escape from adaptive conflict and selection to maintain dosage balance can or should be squeezed under a neofunctionalization/subfunctionalization dichotomy (1).

Lines 171 and following: As another reviewer also pointed out, an asymmetry in selective constraint between duplicates is not solely explicable through neofunctionalization but is also compatible with, at a minimum, subfunctionalization.  The authors response here is not compelling: A duplication of a broadly expressed gene where one copy retains expression in most tissues and the other is expressed only in a few or at lower levels could quite easily generate the pattern the authors describe in the response. The reason is that in many cases the dominate force in shaping omega is not protein structure per se but expression level: see(2) This section needs to be revised.

References:
1.	Hahn MW (2009) Distinguishing among evolutionary models for the maintenance of gene duplicates. Journal of Heredity 100(5):605-617.
2.	Drummond DA, Raval A, & Wilke CO (2006) A single determinant dominates the rate of yeast protein evolution. Molecular Biology and Evolution 23(2):327-337.
Additional comments: No 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: PATTERNS OF GENE EVOLUTION FOLLOWING DUPLICATIONS AND SPECIATIONS IN VERTEBRATES
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors have properly addressed our points.
Experimental design: Na
Validity of the findings: Na
Additional comments: Na


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: PATTERNS OF GENE EVOLUTION FOLLOWING DUPLICATIONS AND SPECIATIONS IN VERTEBRATES
Review round: 2
Reviewer: 3

Output:
Basic reporting: No specific comments
Experimental design: No specific comments
Validity of the findings: No specific comments
Additional comments: The authors have answered all reviewer comments to my satisfaction. This is a nice addition to the literature on the ortholog conjecture and on patterns of protein selection.


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: PLANTAR SUPPORT ADAPTATIONS IN HEALTHY SUBJECTS AFTER EIGHT WEEKS OF BAREFOOT RUNNING TRAINING
Review round: 1
Reviewer: 1

Output:
Basic reporting: References
References in the main document should be revised.
Experimental design: Method
The 8-week training should be better explained. For instance: Who watched the runners perform every minute of the training? It has been controlled? Have they been done individually or in groups?

line 205: It is said that the participants have no experience in barefoot running but they appear not to be experienced in running too with only 5 to 10 kms a week. The mentioned adaptations could be part of the changes produced by the adaptation to the race and training, and not to the barefoot running?
line 126: The running speed in the test was 3.1 m·s. It is well known that the speed and fatigue influences the foot strike. If participants are not experienced runners, How does it was taken in account? Not all runners are comfortable with the same running paces or volumes.
line 145: At the end of the 8 weeks protocol, How was the measurement? It is supposed that the participants ran again the 20 minutes in the treadmill as the accutte changes protocol.
line 155: It seems that when the data is taken in 15 meters of stroke with the baropodoscope, the initial running conditions on the treadmill change (f.e. the 5% slope) Could that affect the data?
line 156: “A music guide was used to indicate to the subjects at what time they had to start running and at what time they had to put their feet on the platform”. This part of the protocol should be further explained. What is the role of music exactly?
line 159: “...when the natural running mechanics was not altered (in step frequency and amplitude)”. How has the amplitude and stride frequency been controlled?
line 199: The mortality of the sample is quite high. Why was it?
Validity of the findings: line 205: If there are no significant differences between dominant and non-dominant feet, could it be better to present the data of the average of both feet?
Additional comments: Dear authors, first of all, I congratulate you on the manuscript. I have listed some general comments below for your consideration. Please consider them as constructive recommendations to enrich your 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: PLANTAR SUPPORT ADAPTATIONS IN HEALTHY SUBJECTS AFTER EIGHT WEEKS OF BAREFOOT RUNNING TRAINING
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: This manuscript entitled “Plantar support adaptations in healthy subjects after eight weeks of barefoot running training” primarily aimed to analyze the modifications undergone by the Total Foot Contact phase and its Flat Foot Phase in subjects beginning the practice of barefoot running, in its acute and chronic effects. The authors bring an interesting study, but there are still some problems that can not up this review to a publishing level. Some suggestions are listed in the specific comments below.

Specific comments:
1.	In the abstract section, line23-24, delete ‘20.0 ± 1.5 years, 70.9 ± 10.4 kg of body weight, 1.7 ± 0.1 m tall, and a BMI of 24.1 ± 2.5 kg•m²’. Line 46, remove ‘and not in an acute manner’.
2.	Too many abbreviations in this manuscript, abbr such as ‘duration of TFC (DTFC)’ is unnecessary.
3.	In the introduction section, line 59, 30%-75%. Line 81, make revise for ‘of activity electric muscular activity’.
4.	Line 82, ‘plantar pattern was modified from the hindfoot to the forefoot’. It should be noticed that the change from the hindfoot to the forefoot is the foot strike pattern, not the plantar pattern.
5.	In the methods section, ‘activity, aged 20.0 ± 1.5 years, 70.9 ± 10.4 kg body weight, 1.7 ± 0.1 m tall, and a body mass index (BMI) of 24.1 ± 2.5 kg•m-².’ Is the demographic info for men, women or all subjects, please make it revised.
6.	Line 118, 134, acute effect protocol, chronic effect protocol
7.	Line 120, 126, ‘for’ instead of ‘during’, with a slope of 5%. Line 131, these trials.
8.	Line 137, why all participants chose the self-chosen speed in their training sessions?
9.	Line 159, I recommend the authors rewrite ‘from the images obtained of the support’ to make it more understandable.
10.	Line 183, which t-test was used for the statistical analysis? Effect size. Line 195 the statistical analysis software SPSS.
11.	In the results section, line 199-203, remove it from ‘results’ or it can be briefly described in the methods. Was it only 2 people who suffered the sole abrasions and bruises? As on one have previous experience of barefoot running. Line 205-207, should be written in the methods part.
12.	I suggest the authors trim down the results section.
13.	Please check the language and grammar mistakes throughout the whole article to improve clarity.
14.	The reference format needs some adjustments.
15.	Full-long lines is needed for table 1,4.


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: PLANTAR SUPPORT ADAPTATIONS IN HEALTHY SUBJECTS AFTER EIGHT WEEKS OF BAREFOOT RUNNING TRAINING
Review round: 1
Reviewer: 3

Output:
Basic reporting: The article presents a clear experimental approach to understanding the adaptations due to barefoot running in a 8 week period. The specific outcomes and conclusions are clear and relevant to the larger knowledge pool.

While in certain sections of the paper, the English could be improved to keep the flow clear, overall the paper is written and presented well.
Experimental design: The design of the experiment is comprehensive and the statistical analysis is sound.
Validity of the findings: Findings are valid and the statistical analysis carried out is appropriate and detailed.
Additional comments: The language can be improved in certain sections and the meaning of certain phrases can be improved on

Suggested modifications
line 26, at the end 'of' a running

Please clarify the following;
 line 38-39, what is meant by "the last evaluation values"
line 77, what "that practice" refers to
line 80, sentence is too long and tends to lose meaning. Simplify sentence
line 88, 'hat' should be' that'
line 97, significance of 8 weeks in the context of this study
line 335-339 Sentence can be written shorter and clearer
Figure 2B Y axis (% label is missing)


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: PLANTAR SUPPORT ADAPTATIONS IN HEALTHY SUBJECTS AFTER EIGHT WEEKS OF BAREFOOT RUNNING TRAINING
Review round: 2
Reviewer: 1

Output:
Basic reporting: I’m glad receive a revised version of the manuscript. Overall, this manuscript almost shows a fair enough level for publication, but there are still some works that need to do for a better version. Further revisions are needed.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: General comments
1.	Line 35-36, ‘a significant increase (p = 0.029)’.
2.	Line 42-43, ‘and between Post-8-week Training and…’.
3.	Line 44-46, The adaptation took place after the 8-week training. The adaptations to running barefoot were characterized by causing an increase…
4.	Line 53-54, Giving the deadline year of the data (55 million practitioners in the United States). ’55 million’ rather than ’55 millions’.
5.	Line 56, mainly in the lower limbs.
6.	Line 59, remove ‘mainly’.
7.	Line 82-83, barefoot running ‘produced’ on variables that affect plantar support.
8.	Line 91, using abbr. TFC, FFP.
9.	Line 92, ‘in its acute and chronic effect (20 min VS 8 weeks training of BFR)’.
10.	Line 101, ‘The subjects were running regularly between 5 and 10 km per week’. Line 132, running at a self-chosen speed.
Why the authors replied that the subjects had no experience as runners?
In this case, why the 20 min acute running did not use the same speed (self-chosen speed).
11.	Line 133, delete ‘during eight weeks’, as it is duplication.
12.	Line 157-158, line 162-163, change the sentence order as: It was considered as a valid attempt only when the three judges gave their approval and five valid attempts were recorded for each foot of the subjects.
13.	Line 188, Showing the classification of ES.
14.	Giving the subtitles for results for the clarity purpose.
15.	Adding the calculated ES values to the results.


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

Input:
Title: TOUR BOATS AFFECT THE ACTIVITY PATTERNS OF BOTTLENOSE DOLPHINS (TURSIOPS TRUNCATUS) IN BOCAS DEL TORO, PANAMA
Review round: 1
Reviewer: 1

Output:
Basic reporting: This manuscript is very well written - clear, concise, and professional. The topic is important and timely and will make a nice contribution.

There are a couple of areas that need support from references (included in the attached pdf). There are also a couple of issues with citation formatting.

I suggest replacing the use of "impact" with "effect", in several instances (see attached pdf), which are often misused in animal behavior literature. Effect may be more appropriate for the short-term behavioral patterns that are described in this manuscript. For clarification, please refer to: Beale. 2007. The Behavioral Ecology of Disturbance Responses. International Journal of Comparative Psychology 20.

Another terminology issue exists - code of conduct "recommendations" are different than laws/regulations. Recommendations are not enforceable because they are not required by law. Please clarify in the inrtoduction and discussion sections what is currently in place - if recommendations only, then your text about compliance and enforcement in the discussion section do not make sense in this context.
Experimental design: This original research has well-defined questions and methods that are mostly described with sufficient detail. However, there is a huge range of tour boats that were considered "present" with dolphin groups, ranging from 1-18. Please include more explanation in the methods and results - for example, what is the sample size breakdown of tour boats per group - how many sequences with 2, 3, 4, etc. tour boats? Binning all of the boat number categories together as "present" or "absent" may skew results. There are examples in the literature that show that the NUMBER of tour boats may affect dolphins differently (e.g., Bejder, L., A. Samuels, et al. (2006). "Decline in relative abundance of bottlenose dolphins exposed to long-term disturbance." Conservation Biology 20(6): 1791-1798.)

Word choice when describing Chi-square results should be addressed. Chi square results are presented as one thing occurring more or less than "expected by chance". It is not a direct measure of one thing occurring more or less than another thing. See McHugh, M. L. (2013). The chi-square test of independence. Biochemia medica: Biochemia medica, 23(2), 143-149.
Validity of the findings: See comments in section 2 (Experimental design) on number of tour boats and how they are handled in the analyses.
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: TOUR BOATS AFFECT THE ACTIVITY PATTERNS OF BOTTLENOSE DOLPHINS (TURSIOPS TRUNCATUS) IN BOCAS DEL TORO, PANAMA
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript is logically and clearly written and provides sufficient background information to be informative.
The study covers a clear and important question.

The English language is good and ensures that an international audience can clearly understand the text. There are some minor parts where the English language should be improved to increase the article's clarity. One examples where the language could be improved includes lines 159 (“a 10m two stroke engine”) and 378, where the current phrasing makes comprehension difficult.
A detailed background supported with strong references is provided.

The raw data is present, as well as the R script of the analysis. Both are well organised.
The hypothesis is clearly outlined, tested, and discussed using the data collected.
Experimental design: The experimental design is described in detail with a clear structure.
The research question is relevant and meaningful.
There are two areas where I would appreciate further detail. It was unclear to me what was meant by “A group was defined as > 1 dolphin” on line 165, as in line 169 it is written “the speed of the dolphin(s)”. Does it mean that you did not take into account if there was a single dolphin, which would make sense as a single dolphin would not show a behaviour change towards socializing? Also, what did you do in case you had a resighting of the same group of dolphins? Please add a more detailed description.

The statistical methods used are fully described in the article. The R packages used in the statistical analysis are not presented or cited in the article. R packages within R can be cited using the code "citation(package = "PACKAGENAME")" for example "citation(package = "poLCA")".
In line 243 you should use correct referencing, instead of “Lusseau (2003)” use (Lusseau, 2003) or “…following Lusseau (2003):”
Validity of the findings: The findings are solid, and are an important investigation of an increasing problem in the field of marine mammal conservation. I think the results are communicated with clear figures and tables, helping to understand paragraphs of text with reporting of parameters.
All underlying data have been provided, they are robust, statistically sound, and controlled.
Results are accurately presented and conclusions are linked to the original research questions.
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: EVALUATION OF A TYPE 2 MODIFIED LIVE PORCINE REPRODUCTIVE AND RESPIRATORY SYNDROME VACCINE AGAINST HETEROLOGOUS CHALLENGE OF A LINEAGE 3 HIGHLY VIRULENT ISOLATE IN PIGS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Ref. No.: PEERJ_2019_42841
Title: Evaluation of a type 2 modified live porcine reproductive and respiratory syndrome vaccine against heterologous challenge of a lineage 3 highly virulent isolate in pigs
By Fu-Hsiang Hou et al.
Post-Overview and general recommendation:
This article describes the evaluation of a type 2 modified live porcine reproductive and respiratory syndrome vaccine against heterologous challenge of a lineage 3 highly virulent isolate in pigs. It suggests that vaccination with Fostera PRRS MLV confers partial cross-protection against heterologous challenge of a virulent lineage 3 PRRSV isolate. This is an original article on this topic both in its content and timeliness. The authors draw on recent results using several studies on epidemiological studies of PPRSV. The article suffers somewhat from the absence of scientific discussion in part of the introduction and the obtained results. This article was written in clear English, unambiguous, technically correct text. The article conform to professional standards. The abstract is clear. This article is ok for the statistic approches.
Introduction
The introduction is clear, but it deserves to be improved. The authors did not take into account the recent work published by Bernelin-Cottet C et al on these vaccine approaches in pigs to protect them from PPRSV. It is good to refer to this work. This introduction also lacks more epidemiological data and the impact of the PPRSV on wildlife (wild boar).
Materials and Methods
The material and methods lack explanations and descriptions of the techniques used and more specifically the histology part. The histological figures are important for the comprehension of the article and I advise to put them directly in the text and add a photo on the 3rd Mock group.
For the discussion, the immunology part on the vaccine response needs to be further developed in order to better discuss the host bioaggressor relationship, but also the protective response against the virus. Have you tested other cytokines following vaccination and vaccination + challenge?
Last point :
It is absolutely necessary to better explain the legends of each figure for a better understanding and to complete the bibliographical references in connection with the additions of the introduction and discussion.
Supplement data
There is an error in the Excel table of supplement data. On the last Excel sheet IFN-g-SC, it is UnV and not UuV.
The legend of the histological figures and the scale are missing. It must be introduced in the results by explaining the difference between the two. The thickening of the pulmonary epithelium and the massive infiltration of immune cells (figure B) are very important for the interpretation of the results and to show the vaccine efficacy.
Experimental design: The article is needed to find vaccines against PPRSV, a real problem in pig farms. The design of the study is correct and answers the initial question in accordance with the expectations of the review. The authors did indeed have their project evaluated by an ethics committee to take into account animal welfare, which is paramount in research.This article is necessary for the scientific community working on the same subject. It provides answers on the vaccine aspect.
Validity of the findings: The conclusions of the work are fair and appropriate. Th data are robust and based on the good statistics. There is a lack of clear explanations in the material and methods on scorings for the clinical and histological part.
Additional comments: This article is very interesting and very important for the scientific community. It deserves to be published when the authors complete the various points I have quoted. The literature on this virus is still scarce and this type of work is needed to better.


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: EVALUATION OF A TYPE 2 MODIFIED LIVE PORCINE REPRODUCTIVE AND RESPIRATORY SYNDROME VACCINE AGAINST HETEROLOGOUS CHALLENGE OF A LINEAGE 3 HIGHLY VIRULENT ISOLATE IN PIGS
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: 1.Deng et al. 2015you use in Line 53 is not the most appropriate for this situation. Please review the latest reference to understand the epidemiology of PRRSV in recently years in Taiwan.
2.The TSYM-strain for use in pig challenges in line 81 to 82 needs more detail. I suggest that you should add plaque purification test.
3.In line 151, a severe anaphylactic reaction was only found in MLV group. Is there a relationship between vaccine immunity and virus infection,please give your opinions.
4. The UnV group presented persistent fever (≥ 40 ℃) from 1 to 13 DPC(line 162-163)  were not in accordance with the Fig.1A(The temperature was less 40℃ from 2 to 4 DPI),please check.
5. In line 191, please check Fig. S1. You should provide the pathologic pictures of different groups.
6.In line 211,MLV group had significantly higher (P<0.05) antibody titers than those of the UnV group at 7 DPC. Please explain the reasons in discussion part.


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: EVALUATION OF A TYPE 2 MODIFIED LIVE PORCINE REPRODUCTIVE AND RESPIRATORY SYNDROME VACCINE AGAINST HETEROLOGOUS CHALLENGE OF A LINEAGE 3 HIGHLY VIRULENT ISOLATE IN PIGS
Review round: 2
Reviewer: 1

Output:
Basic reporting: Ref. No.: PEERJ_2019_42841 revised version
Title: Evaluation of a type 2 modified live porcine reproductive and respiratory syndrome vaccine against heterologous challenge of a lineage 3 highly virulent isolate in pigs.
By Fu-Hsiang Hou et al.
This article describes the evaluation of a type 2 modified live porcine reproductive and respiratory syndrome vaccine against heterologous challenge of a lineage 3 highly virulent isolate in pigs. It suggests that vaccination with Fostera PRRS MLV confers partial cross-protection against heterologous challenge of a virulent lineage 3 PRRSV isolate.
 This is an original article on this topic both in its content and timeliness. The authors draw on recent results using several studies on epidemiological studies of PPRSV.
The enrichment of the introduction on the wildlife aspect of the pathology and on the immune response allows the objectives of the article to be well framed and reinforces the scientific content.  Modifying the set of legends for each figure by clearly explaining the statistical analyses and describing what they illustrate helps to better understand the article. The additions and explanations in the histology section are much clearer and understandable.
Experimental design: The design of the study is correct and answers the initial question in accordance with the expectations of the review.
Validity of the findings: The conclusions of the work are fair and appropriate.
Additional comments: I would like to thank the authors of the article for taking into account all the corrections I have proposed to improve the text and strengthen the missing immunity part. This version is acceptable for publication.


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

Input:
Title: EVALUATION OF A TYPE 2 MODIFIED LIVE PORCINE REPRODUCTIVE AND RESPIRATORY SYNDROME VACCINE AGAINST HETEROLOGOUS CHALLENGE OF A LINEAGE 3 HIGHLY VIRULENT ISOLATE IN PIGS
Review round: 2
Reviewer: 2

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


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

Input:
Title: A BIOGEOGRAPHIC FRAMEWORK OF OCTOPOD SPECIES DIVERSIFICATION: THE ROLE OF THE ISTHMUS OF PANAMA
Review round: 1
Reviewer: 1

Output:
Basic reporting: The authors use a phylogenetic approach to test the influence of the closure of the Isthmus of Panama on speciation processes in octopods. The paper is excellent, I really enjoyed reading it. It will represent a very nice addition to the existing litterature on the topic. Working hypothese are clearly stated, the experiment was well conducted with a large sample size across a broad geographical area and the analytical approach is sound and robust.
The authors placed their findings in the context of existing litterature and provided sufficient background. Figures/tables are clear, and the raw data is available. My only suggestion would be to have the text reviewed by a native engish speaker to correct small language mistakes throughout.
Experimental design: This work is very relevant, and performed to a high technical standard.  Questions and aims are well defined and methods are clearly explained,
Validity of the findings: Results are robust and statistically sound. Conclusions are well stated and results are discused in the context of existing litterature.
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: A BIOGEOGRAPHIC FRAMEWORK OF OCTOPOD SPECIES DIVERSIFICATION: THE ROLE OF THE ISTHMUS OF PANAMA
Review round: 1
Reviewer: 2

Output:
Basic reporting: The article is clear and the analyses are presented in a straight forward manner.

Genbank accession numbers are not provided in Table 1 as stated.  The accession number range should be included in the main text, and the breakdown of accession numbers inserted into Table S1.
Equally importantly, no voucher specimens are referred to.  Given the difficulties of correct identification of octopod species, it is essential that vouchers are submitted to museums for all the newly sequenced material.  I suggest adding an extra column to S1 to accommodate this.

Please explain why the last occurrence of ammonites controls the upper bound of argonauts given these two groups are very unrelated.  Generally, more detail in the fossil constraints would be helpful.  For example, why was Vampyronassa chosen? No reason is given.

Figure 1 contains several errors.
Authors are advised to follow the convention of using two letters for genus abbreviations where they include more than one genus starting with the same letter.   For example, use Op. for Opisthoteuthis, and Oc. for Octopus.  There is a general inconsistency in the figure over using genus abbreviations (or not).  "longibrachus" is spelt incorrectly in the eastern Atlantic.

Authors should take care to cite early works and credit those authors that had the earliest thoughts about Trans-Panamanian geminate species, not just recent papers.

Attention to English language is required throughout.  Examples of errors included "waters exchanges", "octopods species", "stripped" instead of "striped", "comprise of" rather than the correct "comprises", misplaced definite articles, and incorrect pronouns.  Sentences on lines 118-120, 393-397, and 406-409 don't make sense.  Some family names are italicised, and some genera are not.
Experimental design: Authors are commended for including three of the four species pairs recommended as potentially trans-Panamanian by Naef, 1978.
Validity of the findings: The 95% HPD limits are very wide, with the highest bound of each clade ranging between 11 and 30 Ma, thus authors should take care how they interpret the data.  Line 401 they state that divergence processes occurred until 5 Mya but that depends rather on where the true date lies in their 95% HPD.
Additional comments: The second author published a paper revising octopod taxonomy, yet the authors do not adhere to that new (and as far as I am aware accepted) systematic scheme.  Octopodidae now refers to a restricted group and does not include Muusoctopus species.  Authors are recommended to carefully revise the text so that their higher taxonomy reflects WoRMS and Strugnell et al. 2014.

"dispersion" has a very special meaning in biology related to the spatial distribution (uniform, random, clumped).  It should not be used as a synonym for dispersal.

Line 357, no species have planktonic eggs.  Octopuses lay their eggs attached to substrate and these may hatch into planktonic paralarvae in some species.

Please ensure that the way abbreviations are used in a sentence makes sense when they are expanded and that abbreviations are correctly pluralised.  Failure to do this makes the text difficult to follow.


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

Input:
Title: A BIOGEOGRAPHIC FRAMEWORK OF OCTOPOD SPECIES DIVERSIFICATION: THE ROLE OF THE ISTHMUS OF PANAMA
Review round: 2
Reviewer: 1

Output:
Basic reporting: While generally very well written and clear, there remain many places where language correction is required.  The paper needs to be proof read and corrected by the native English speaker among the authors.

There is a disparity between the text and table 2 (17.24 or 17.44)
Experimental design: The science is sound
Validity of the findings: No further comments to make
Additional comments: The science in this appears to be sound and it will be a useful addition to the literature.  There are just a couple of points raised in the previous review that have not been addressed:

My previous comment: "Genbank accession numbers are not provided in Table 1 as stated. The accession number range should be included in the main text, and the breakdown of accession numbers inserted into Table S1."

The authors have included the Genbank accessions they have in Table S1 and will add others in proof.  But Supplementary material becomes separated from the paper over time (as academics share pdfs) and it is essential that the range of Genbank numbers (e.g., COI FZ654009-FZ654092; 16S FZ654093-FZ654142 etc) be included in the main text.  This does not take up much space but will be enormously useful to future readers.

My previous comment: "The second author published a paper revising octopod taxonomy, yet the authors do not adhere to that new (and as far as I am aware accepted) systematic scheme. Octopodidae now refers to a restricted group and does not include Muusoctopus species."

The authors say “We described the Family of Muusoctopus species in the line 249”, but this doesn’t address my point.  In line 106, the authors say “The greatest diversity of benthic octopuses occurs in the Family Octopodidae with more than 300 species world-wide”.  The authors here, following the taxonomic reorganisation of Strugnell et al. 2014, are referring to the Superfamily Octopodoidea (this is clear because if they are referring to Octopodidae, i.e., excluding Enteroctopodidae, Eledonidae, Megaleledonidae etc, then there aren’t more than 300 species - more like 150…)


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: PROBABILISTIC MODEL BASED ON CIRCULAR STATISTICS FOR QUANTIFYING COVERAGE DEPTH DYNAMICS ORIGINATING FROM DNA REPLICATION
Review round: 1
Reviewer: 1

Output:
Basic reporting: Literature references up to date.
Figure adhere to a high quality.
Manuscript would benefit from restructuring, see General comments for suggestions.
Experimental design: Authors propose a tool based on circular statistics to infer information on growth of prokaryotic population from genome sequencing coverage.  Analysis well planned, rigorous investigation performed.
Validity of the findings: Few aspects need to be clarified, see for details General comments.
Additional comments: Major remarks:

Methods proposed  by (Koremet al. 2015) and (Brownet al. 2016) have been criticised in (Gibson et al. 2018): “In principle, these measures of cells performing DNA replication could be used to estimate the doubling time of bacteria in the wild. However, it is unclear how or whether the methods can be calibrated.”  An example of such calibration was done in (Forsyth et. al 2018), figure 3D, where a linear model was constructed as growth_rate = 1.678*PTR - 1.772.
Therefore I think that in the manuscript “pPTR” should not be substituted by  “growth rate”.  Say on line 597: “estimating a correlation between computational and experimental growth rates as per a previous study (Korem et al., 2015)” - This should be “ between pPTR and experimental growth rate”. If You look how (Korem et al., 2015) phrases it, it’s “PTRs were correlated with the measured growth rate, preceding it by 30 min”  and “1/log2(PTR) [] correlated with measured bacterial generation time, confirming PTR as its proxy, even when the replication time is unknown.”, as well as “The relationship between PTR and growth rate extends to other commensal strains, as we found that PTR and temporal measured growth rate were significantly correlated ...”
The same applies to lines 603, 605, 607, 614 etc.

Gibson B, Wilson DJ, Feil E,Eyre-Walker A. 2018 The distribution of bacterial doubling times in the wild.Proc.  R.  Soc.  B285: 20180789.http://dx.doi.org/10.1098/rspb.2018.0789
 Forsyth VS, Armbruster CE, Smith SN, Pirani A, Springman AC, Walters MS, Nielubowicz GR, Himpsl SD, Snitkin ES, Mobley HLT. 2018. Rapid growth of uropathogenic Escherichia coli during human urinary tract infection. mBio 9:e00186-18. https://doi.org/10 .1128/mBio.00186-18

Line 613: “Secondly, we tuned the parameters concerned. The window and stride size of the median filter were optimized…” & from supplementary material:  “The purpose of the filter is to reduce the variance in the coverage depth and outliers. As such, if filtering is performed, ideally, the average depth will be maintained and the variance will decrease.” I don’t see why You need median filtering when You are fitting coverage depth to a function?  Zero depth shouldn’t be an issue, as function is fitted to all data and keeping in mind a limited number of parameters to estimate, it is mostly influenced by high coverage areas. As You show in  Supplementary fig. 6, 10, your fitting results are sensitive to the choice of window length.

It is nice to see the theory extended to a multi-origin case. However, I would argue that the maximum of wPTR is more appropriate index as a proxy for growth rate.
“These scores are based on the hypothesis that replication stops if the replisome is faced with another replisome (Wendel, Courcelle & Courcelle, 2014).” - this is a reference on a single origin bacteria.
Synchronous initiation of replication origins has  been reported in Sulfolobus species (Lundgren et al., 2004), but not in H.volcaniil (Hawkins et al., 2013). Furthermore, the difference in height of fitted mixture components indicates difference in initiation of replication (figure 4 a  & Supplementary fig. 21 a-f).

Other remarks:

Introduction.
Although lines 166-170, 363-367,  Figure 2 etc talk about PTRC, DEMIC, bPTR, iRep, and GRiD, these are not properly introduced.  Add a paragraph to describe previously used indexes (not just PTR). Make it clear that You are benchmarking Your method against existing ones and that You have made a package for Your method.

 Materials & Methods.
I can’t find if coverage data have been binned into 10kbp regions as in previous studies? If yes, then methods need to be revised, as i is not nucleotide, but a bin on the chromosome. By the look of Figure 3&4, these don’t seem to be at 1 nt resolution.

Move subsections “Software” and “Availability of data and material” to the end of Materials and Methods. Move lines 166-168 to “Software”, i.e that SPHERE is Your package for computing directional stats from coverage depth data.

Start “Materials and methods” with subsection “Circular distributions and statistics”, as this is the main aspect of the manuscript.  Put all probability density functions into a table with the following columns: (i) the name of the distribution, as used in Figure 2; names are missing from lines 179-183, but then they are used on lines 204, 255 etc and results section; (ii) formula of probability density function; (iii) analytical expression for PTR (given on lines 199-203), or numerical method used for calculation; (vi) parameters defining probability density function;  and (v) and reference for this probability density function.

Line 222:  “Fitness of the model was improved..” -  was this done for fitting to the data to models? Then lines 222-225 should be moved to “Parameter estimation”.

I would suggest moving lines 516-578, i.e subsection “Statistical model to estimate replication rate” to “Materials and methods”, and adding it after “Parameter estimation”.

Line 520: “We fit a generalized  linear model (GLM)..” This is confusing - i thought You’re fitting probability density functions defined in subsection “Circular distributions and statistics”?

Line 563: “This score represents the latent bias of probability for the position at which a nucleotide is observed around the replication origin.” Can you please clarify? As p_max(theta) and p_min(theta) are point estimates of minimum and maximum of probability density function, why there should be “latent bias” around replication origin?

Line 566: .”Following the model, the coverage depth at a given position can be modeled by the binomial distribution...”  Which model?

Move lines 570-578 to supplementary materials.

Lines 579-593 - make this into a new subsection “Creating an artificial coverage depth” or similar and keep this in “Results” section. I really like Supplementary fig. 3, would be nice to move it into the main text. But provide parameter values for all distribution in the figure legend.

Line 674: “The relatively weak negative correlation between relative abundance and growth rate, became stronger when the dataset was transformed to log-scale (Supplementary fig. 14).” Can state values of correlation (i.e. -0.1172 & -0.215) rather than “ became stronger”.

Line 787: “Although it was more fragile than when..” What do you mean by “fragile”?

Line 794:  “replication balance among multiple origins” - i don’t think that this has been estimated or interpreted in biological context. As well  on line 946: “One interesting finding was the balance of replication activity among multiple origins.”

Figure 1: I don’t think that comments “Previous methods of in situ growth rate estimation used linear trend regression in the log-transformed Euclidean system.”  or “Similar to previous methods” are necessary in the legend.
Figure 2: “PTR error rates” - how these were calculated for iRep, GRiD & DEMIC indexes? Can You plot raw values with extra bars plotted for “Full coverage”, i.e Estimte_reference and Estimate_modified as in equation (3)?
Figure 4:  can You make ranges smaller, so that mixture distribution overlays coverage depth, in a similar as it is shown in Figure 3?

Minor corrections:

Line 87: “ piecewise polynomial linear regression” - it’s either polynomial or linear. In the case of Korem et al., 2015, it is a piecewise linear function (page 7, supplementary materials).

Line: 141: “Mixing directional distribution” - Using a mixture of directional distributions

Line 178: Define what is theta and T.

Line 242: “skewness or peakedness parameter” - is that the same parameter - lambda?

Line 751:  “by mixing circular distributions” -> a mixture of circular distributions

Line 771: “we determined the number of components in both datasets as 3 because it yielded the best score on average (Supplementary table 9)” - there’s some mixed-up with tables, as “peerj-42075-Supplementary_table_9.xlsx” contains “Supplementary table 8: Two comparison test result of growth rate in three metagenomic cohort. We used Welch's t-test for the p-value, and Benjamini-Hochberg procedure for FDR correction. “


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: PROBABILISTIC MODEL BASED ON CIRCULAR STATISTICS FOR QUANTIFYING COVERAGE DEPTH DYNAMICS ORIGINATING FROM DNA REPLICATION
Review round: 1
Reviewer: 2

Output:
Basic reporting: 1.	There are multiple instances in the paper where the grammar and structure of the sentences is not good and makes it hard to follow.

2.	The introduction is argumentative rather than informative and could be further shortened and clarified.

3.	The discussion need not repeat each and every result in the paper. It is way too long in my opinion.

4.	L99-104 – prediction of the origin of replication was actually done in Korem et al, using their statistical model (see their Fig. S9).

5.	L74-76 – Where is the limitation? What is the issue with applicability via single metagenomic samples?

6.	I suggest to put Fig. 2A with bPTR in the supplementary and focus on the others for readability. Also, in Fig. 2B, it’s impossible to understand what are the methods compared.

7.	Fig. S9B – there is no point in taking the X-axis up to 50%. It’s not even the same microbe at this point. The fact that pPTR even output something at these rates is alarming and indicates that there isn’t a good control for the actual presence of the species in the data.

8.	L273-277 - What is the procedure to determine if top 1% should be filtered?

9.	L284 – what correction procedure is being referred to?

10.	L362-363 – It’s not clear where that error rate was used and what it was defined for.

11.	L3375-383 – It is not clear what is “randomized direction” and “single direction”.

12.	L402-403 – what are “points”?

13.	L485-487 – It is not clear what “filtering out” procedure is being referred to here.

14.	L532-534 – This is the only place in the results where the MRL is mentioned. The authors treat it as if it is another method (e.g., in the discussion) but it is never used in the manuscript.

15.	L539-L543 – Not clear how this sentence fits in here. Are the authors trying to justify non-linearity of the distributions? It’s not clear.

16.	Legend of Fig. S4 is insufficient to understand panels c-e.

17.	L595-L612 – the authors should explain the experiments whose data they are using even if these were described in previous studies.

18.	What is “ptr” on the legend of Fig. S8?

19.	Fig. S10B – how many points are at the (1,4) and (4,1) coordinates? Could be multiple overlaying.

20.	L646-L656 – Don’t assume that the readers know that it’s the same dataset with different sample size analyzed in both this and Korem et al.

21.	L674-L676 – Where does this result fall within the narrative of the analysis?

22.	L78 – DNA increases considerably…via DNA deep sequencing? I would say the increase is biological.

23.	L362 – It’s not clear what mutations are being referred to.

24.	L579-580 – The sentence is not in proper English.

25.	L663-L666 – What does “confirm the trends” mean? What trends? “This trend was reproduced in the estimates” – I don’t understand what this means.
Experimental design: 1.	The authors assess the robustness of their method on a very limited and clean datasets of monocultures. This is valid independently, but there is no point in comparison to different methods that were optimized to work on metagenomic datasets, perhaps making decisions that improve robustness in “dirty” metagenomic data in favor of accuracy in monoculture data.
This subject came up in previous review and the authors claimed they need a ground truth measurement. This is true for assessing accuracy, but absolutely not true for robustness. The point in robustness is to see whether the method is sensitive to low coverage, mutations, increased coverage, etc., regardless of how accurate it is (accuracy is assessed separately). In fact, the authors are not using the ground truth in the robustness analysis at all. The legend of Fig. 2 clearly states that they are comparing PTRs on subsampled data to PTR on full data - ground truth doesn't play here. This was done in Korem et al., for example, where all the robustness analyses are done on a metagenomic cohort.
In my opinion, the authors cannot claim that their method is more robust than other methods unless they perform this comparison on metagenomic data.

2.	The authors make arbitrary decisions when selecting microbes for the analysis of metagenomic datasets (e.g., abundance larger than >0.5%, high coverage), that are unacceptable. There is also no point in selecting taxa with already significant results at the relative abundance level for further analysis, esp. as Korem et al showed that relative abundances are not correlated with growth rates and that they found multiple significant correlations between PTRs and host phenotypes in bacteria whose relative abundances were not correlated with the same phenotype. The justification the authors provide in L476-478 is not acceptable: if the method presented here is so sensitive that it cannot distinguish taxa that are really present in the sample from those that are not, it should either be amended, or the authors should not claim it is useful for metagenomic analyses. Finally, these arbitrary decisions result in very small reference datasets – the authors basically compare 10-20 taxa – while previous methods work with thousands.

3.	Fig. S4, c-d: (i) it is not clear what data was used here. (ii) From the methods, it seems that the authors used the abundances when calculating the experimentally derived growth rates. The log abundances should be used. See e.g. Hall et al. (2013) Mol. Biol. Evol. 31(1):232–238. (iii) The authors claim that their correlation was better (L607-L609), was this statistically significant?

4.	L609-612 – there are no experimental growth rates in this experiment. It is impossible to calculate growth rates from relative abundance due to compositionality of the relative abundances.

5.	L539-L543 - What is the point of Fig. S1? How does it relate to the sentence? Do the authors claim they can see non linear trends there? Can they prove it? Seems like a reproduction of Fig. 1C from Korem et al.

6.	Why is FDR correction necessary for Fig. S11? (L645) Seems like just one test?
Validity of the findings: 1.	The authors should either prove that their method works well on metagenomics datasets or limit their claims to monocultures. The authors fail to show that their method works on metagenomics on multiple accounts:
a.	The robustness analysis is done only on monocultures.
b.	pPTR seems incredibly sensitive to mutations at specific locations, that are typical in metagenomic datasets (Fig. S9C, S9E).
c.	The analysis the authors do perform on metagenomic cohorts is handicapped by arbitrary selection of species to work on, resulting in a very small reference datasets (2 orders of magnitude vs. previous methods). The authors even justify that by the deficiency of their method to work in this setting (L476-478).
d.	The analysis the authors perform on these metagenomic datasets is not compared to any other method. I read the authors essay about this in the rebuttal. While 80 vs 100 significant association is perhaps not a good argument to make towards the superiority of any method, if one method discovers multiple significant associations and another recovers just one or two then this is a different situation.

2.	The characterization of Korem et al in L85-93 is inaccurate. Korem et al limited the coverage due to reduction in accuracy in low coverage (see their Fig. S7). The use of “division by zero” by the authors makes it seem like some error in the code, which I assume is inaccurate and even if so not very relevant. This returns in L814-816 – there is no zero division error anywhere. The limit of 0.05x was due to accuracy. These cases are filtered out and do not result in an error. This is very clear in the manuscript of Korem et al.

3.	The authors also cannot claim that their method is robust to very low coverage of .005x, if they themselves limit the applicability of their method to .1x which is higher than previous methods. Their arguments to the contrary in the rebuttal are unacceptable - the bottom line is what matters. If there method cannot work with coverage <.1x  - it is not applicable for coverage up to 0.005X.

4.	How can the authors say that parameter estimation was independent of sample size (L635-636)? Fig. S9F clearly shows increased error with lower sample size!

5.	L647-652, L850-L854 – PTRs cannot be used to compare between different bacteria. This appeared in previous peer-review which the authors reject for not good reason. PTR = 2^C/G (Korem et al, Cooper and Helmstetter), where G is the generation time, and C is the replication period, that is, the time it takes a microbe to completely replicate its genome. The C-period is different between different microbes. Therefore one cannot say that one microbe is growing faster (smaller G) than the other just based on PTR, without knowing C. This argument is valid only within a species where one can assume that the C-period is similar.

6.	L651-652 – What exactly is consistent between the two analyses?

7.	L806-809 – The comparison was done on a monoculture, while these methods were developed for metagenomics, and thus is invalid.

8.	L856-L857 – this sentence is false. On a 10M 100bp reads sample and a 5Mbp genome, relative abundance of 0.1% corresponds to coverage of 0.2X, which is not low.

9.	L868-L870 – this is not supported by the analyses, as mentioned above.
Additional comments: I have reviewed this manuscript before. The general notion of using a statistical model to model growth rates is an appealing one. The authors try to make a claim regarding the superiority of pPTR to previous methods and its applicability to metagenomic datasets that is not supported by their findings and analyses. They cannot claim that these analyses are just "an evaluation", and then put claims of superiority. There are multiple other inaccuracies that I point out in my review.


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: PROBABILISTIC MODEL BASED ON CIRCULAR STATISTICS FOR QUANTIFYING COVERAGE DEPTH DYNAMICS ORIGINATING FROM DNA REPLICATION
Review round: 2
Reviewer: 1

Output:
Basic reporting: Literature references up to date.
Figure adhere to a high quality.
Article structure and language has impoved for the revised version.
Experimental design: Authors propose a tool based on circular statistics to infer
information on growth of prokaryotic population from genome
sequencing coverage. Analysis well planned, rigorous investigation
performed.
Validity of the findings: no comment
Additional comments: Minor comments:
Line 78: "bidirectional progression" -> "bidirectional progression of replication forks".
Line 80: add before "iRep used a.." a sentence "Few mathods have been proposed to quantify growth of bacteria from genomic data."  or similar.
Line 94: Change "Another growth rate estimation method, which fills the gaps, could be utilized for broader.." to " A novel growth rate estimation method, which is less sensitive to coverage depth, could be..."
Line 104: What does "DNA quantity" mean?
Lines 261-264:  I appreciate that the authors wrote a thorough response on my comment on multiple origin model. With regards to the following assumptions: "This model means that each replication origin is not responsible for the entire DNA sequence, but rather only for a certain region. This assumption results in the probability of regions that are not related to the origin approaching 0 or being extremely small. Hence, the probability distribution of each origin becomes very steep, increasing in the concentration parameter and unweighted PTR." But look at Supplementary figure 20 (a)-(f), all of them have one of unweighted PTRs dominating the fitted mixture.
Line 784: Define what is "the center of gravity of the replication origin position".
Line 801: "replication activity balance among multiple origins" - again, not according to supplementary figure 20.
Line 801: What do You mean "In a field"?
Line 849-850: "for DNA to be more concentrated around the replication origin" -> "for multiple rounds of replication around the replication origin".
Line 856: Plese clarify what You mean by "the increase in density around the replication origin should
be resolved".
Line 861: Please clarify: "Thus, the degree of density around the origin of replication increased when both replication and cell division were active."
Line 875: Please clarify: "If a certain single origin had strong activity".


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

Input:
Title: PROBABILISTIC MODEL BASED ON CIRCULAR STATISTICS FOR QUANTIFYING COVERAGE DEPTH DYNAMICS ORIGINATING FROM DNA REPLICATION
Review round: 2
Reviewer: 2

Output:
Basic reporting: The section on “Shape, peakedness, and skewness of coverage depth” is very descriptive, based on a very limited samples size,  and unfocused. I recommends that it is significantly shortened, with technical details and discussion moved to the supplementary material.

I recognize that the authors have improved the English throughout the manuscript. I appreciate their efforts but there are still places in the manuscript where phrasing, grammar, etc. do not conform to professional standards. Some specific examples:

L44-45: It is not clear what this assertion of target range and measurement properties means and even if it was clear, where it was assessed.

L71 – stable isn’t the right word. Perhaps static.

L76 – after reading the authors’ response, their phrasing doesn’t say what they mean. What they want to write is something like “Meanwhile, the peak-to-trough ratio (PTR) of coverage of WGS reads mapped to reference genome sequence provides an estimate of growth using WGS reads coming from just a single sample”. The current sentence indicates something completely different and is unclear.

L77-78 – the increase is not via bidirectional progression, the increase is via replication, bidirectional progression creates the coverage signal across multiple microbes.

L78-79 – The “as well as DNA deep sequencing” part has no connection to the sentence.

L121 - The phrasing of “small number of coverage depths” is not in correct English;  the “among other methods” has nothing to do with the text.

L125-126 - implies that you made sure that previous studies are reproducible: instead what you did was making sure that your method is robust to coverage and noise using data from previous studies.

L178-179.- “the low coverage depth issue” is referenced but not defined beforehand.

L278 – this sentence is unclear. What is “the package for all of the data”?

L315 – the moving median filter should be defined before it is referenced and justified.

L317-319 - "peaked noise" is not a clear term.

L320 – should be “based on a previous study”.
Experimental design: The “Statistical model to estimate replication rate” section should emphasize that the coverage is actually binned and not at the nucleotide level.

L561-565 – this procedure and its aim are completely unclear. Also: what theoretical score?

I couldn’t find the methodological details behind Fig. S3c/d

L665-667 – The text here does not correspond to the legend of S8d
Validity of the findings: L396 – this is far far away from evaluation of applicability. This is robustness pure and simple.

L669-670 – while the estimates were indeed as stable as previous methods, they were significantly less accurate (e.g. compared to ptrc, irep, grid). This should be stated and discussed.

L670-672 – Please also discuss the reduced accuracy compared to previous method. pPTR is problematic with respect to the type of variability observed in s8d/e/f and this should be clearly acknowledged.

L672-676 – It is unclear how this is addressed. The authors seems to insinuate that the simulations that they do have much more noise than real data, but
(a) one of their analyses is on monoculture sequencing of 3 well characterized laboratory strains (Text S8); this is not the same as sequencing of a metagenome. (b) their analysis deals only with absolute deletions, which is not the only source of noise in these scenarios.

Limitations with respect to metagenomic applications that the authors discuss in the rebuttal letter should be clearly presented in the discussion. This is currently not the case.
Additional comments: L81 – this is not true. iRep uses all the contigs.

L805-814 should be moved to supplementary

The authors need to make sure they have the rights to use fig s1 which is a reproduction of Korem et al

In general, this revision presents a much tighter, rigorous, and valid study with analyses that are better justified. The authors have removed statements of superiority and analyses that were not properly validated, and have addressed most of my comments.


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

Input:
Title: MIXED EFFECTS OF ECOLOGICAL INTENSIFICATION ON NATURAL PEST CONTROL PROVIDERS: A SHORT-TERM STUDY FOR BIOTIC HOMOGENIZATION IN WINTER WHEAT FIELDS
Review round: 1
Reviewer: 1

Output:
Basic reporting: -English language is, in general, good but needs some revision. Although I am not a native English speaker, I think that there is an unnecessary excess of “the” throughout the text (for example, lines 32, 33, 59, 166, 201, 236, among others). Furthermore, some complex sentences are difficult to follow or understand, such as in lines 69-70, 84-89, 103-106, 166-169, 299-303. Finally, more connection between some sentences in the introduction is needed. Therefore, I believe that a detailed revision of English grammar and an improvement of these pointed sentences can increase the quality of the paper.

 -Introduction and background mention relevant references and all necessary topics are introduced. Nevertheless, I think that the first paragraph, where the authors describe the general overview of the background could be benefitted by a clearer organization from general (cropland expansion, agricultural intensification) to specific (landscape effects on animals, biotic homogenization) aspects. Besides, in the of this paragraph is not clear what the authors meant with “the current opinion on the effects of biotic homogenization…”.  Are they trying to highlight the knowledge gap they studied? Probably no, because they mention community organization and this is not addressed here. Thus, I think that this last part could be changed to either emphasize the gap they are covering or at least improve the redaction.

On the other hand, EI is introduced in the third paragraph, but at the beginning of the second paragraph, they already mention that ecosystem services can replace external inputs and thus the concepts of EI are somehow mixed. I suggest that they focus the second paragraph on describing ecosystem services and their providers, and how landscape can influence them, and the third paragraph on EI.

-Structure of the article is good and follows the journal´s standards. Also, it can be considered as self-contained as well.

-Figures and tables are relevant and correctly labeled and in general, well described. Table 1 legend could be expanded to explain which information of the model selection process and each model is provided. Figures are, in addition, of good quality and clear. In figure 1 there is a small mistake in the legend, as it says that fertilizer is added in both F+ and F-. Also, it is not clear if the b panel is showing a zoom of one of the treatments, so perhaps it could be clarified in the text or showed in the figure with some arrows or other similar indication. In Figure 3, the drawings of the carabid and the spider could be added to panels a and c, respectively, to improve understanding. Also, in my opinion, the titles of Y-axis of figures 2 and 3 could be changed to “Aphid abundance (sqrt)”.

-Raw data and codes are provided with the submission as supplementary files and are clear. Nevertheless, I have problems to understand the codes of the farm names and how many data points per farm you had and in many cases, one code is mentioned at the beginning of the table and repeated later. I suggest changing this complex names for a simple number such as F1. In the case of data points, for many of the tables, the number of points per farm is not the same and it is not stated if some data were lost or it was not possible to obtain them. Finally, for aphid and pathogen data, a column stating the sampling date would be helpful too.
Experimental design: -The article reports the results from original research and fits well within the scope of PeerJ. Also, the investigation is performed following good technical and ethical standards.

-The main research question is considerably clear and the knowledge gap is established throughout the introduction. However, when the research question is described with more details, as in lines 103-109, I believe that the writing is not so clear. On one hand, the authors talk about “related pest control”, but pest control is not analyzed in the article. Therefore, they should explicitly say that they studied natural enemies or providers of pest control, but not the service per se. Specifically, in lines 103-104 they mention “enhancement of invertebrate assemblages” while they are meaning beneficial invertebrates. Concerning this, the title of the article mentions effects on natural pest control and I believe this should be changed too.

Secondly, the authors refer to the presence or absence of set-aside land as a measure of landscape configuration. In my opinion, this is not strictly true, because they addressed this presence as a local variable (set-aside neighboring the wheat field or not), whereas landscape configuration implies some measure at the landscape scale. Finally, while I think it is acceptable to talk about pests in general throughout the introduction, I think that in the objectives and hypotheses they should refer to aphids only, as this was the only group of pests studied and as it is written now it reads as they worked with many groups.

In connection with the research question, the hypotheses need some fine-tuning. In hypothesis 1, they mention an enhanced dispersal potential of natural enemies in relation to landscape composition. However, it may be better to refer to the connectivity in these landscapes that allows the dispersal, as it seems that they imply that dispersal abilities increase in complex landscapes. Or maybe they could talk about higher dispersal success. They also refer to higher pest control in this hypothesis, which was not directly evaluated. If they meant that this pest control could be reflected in pest abundance, they should say that clearly. Hypothesis 3 describes an interaction between fertilizer use and soil quality, but they did not test any interactions in their models, so they are not testing this hypothesis, which I believe it is very interesting. Please see below my concerns about statistical analyses for more details. Finally, no hypotheses for pests and pathogens are given, and no predictions for how yield can change at the landscape level (although they could expect an increase if pest control is higher, but this is not explicitly stated).

-Methods are described in detail and with sufficient information, though I have some minor comments. It would be good if they can include a figure showing a map of the study sites, at least as a supplementary figure. It is not clear if the with/without set-aside land treatment was assigned to seven pair of neighboring fields or if they are interspersed in the region. In connection to the field used for the study, they could also provide a supplementary table with the characteristics of each site in terms of proportion of semi-natural habitats. I consider that the separation of simple vs. complex landscapes is valid, but I have some doubts regarding the limit (less of more than 20%) because it can be the case that some of the simple landscapes are close to 20% and some of the complex too. Thus, these values are necessary for clarifying this issue and if there is not a clear separation between landscapes categories it may be a better idea to analyze landscape composition as a continuous variable. For the soil characterization, more details on the size of the soil cores and the distance separating these samples within each field.
Validity of the findings: -Data are of good quality and presented in their raw form, but I have some questions and suggestions about statistical analyses.
The authors stated that they used GLMMs but then transformed the data using square root. I believe that all these data could be used in the raw and analyzed with GLMMs with Poisson or negative binomial distributions using the same R package, except for harvest data which could be analyzed using a normal distribution without transformation if the data fit well. Although the square root may not be so bad as other transformations such as log, it still performs worst than using other distributions according to O´Hara and Kotze (2010).

My second concern with analyses is about data dependence. The authors used farm ID as a random factor, but this only considers dependence within each farm. For aphid and pathogens, they had several temporal repetitions that should be incorporated using sampling period as a random factor too. Furthermore, if there were multiple measurements within each fertilizer treatment, transect and/or distance to edge should also be considered, nested within farm.

Finally, I do not fully understand why the authors did not test models with more than one independent variable. I read that they wanted to avoid collinearity between variables, but many of the factors were under a crossed design and this should not be a problem, whereas soil characteristics variables could be included and Variance Inflation Factors calculated to test for collinearity. Moreover, one of the hypotheses was that soil fertility would interact with fertilizer use, and this should be tested using an interaction between these variables. I think that the model averaging approach they used is especially good for testing models with more than one variable and they could find some interesting results that are not covered by these single variable models. Also, a null model without any variables would be necessary to compare it against the other models.

-In general, the discussion is limited to the results and other related studies. The conclusions section could be shortened to focus specifically on the implications of their findings. I think that most of the conclusions section is based on their results except for the statement on lines 359-361 when they mentioned “detected asymmetric species flows” but they only worked with abundance, so I do not think this is the best way to describe it.

 -Negative or non-significant results are well discussed and considered as important, so the authors did a good job avoiding bias towards significant differences.
Additional comments: In this interesting article, the authors investigated how two strategies of ecological intensification (EI) of agriculture (set-aside land and fertilizer use), landscape composition, and soil conditions affect a group of pests (aphids), pathogens, natural enemies, and yield of wheat crops. The topic is highly relevant and has implications for management applications of agricultural landscapes. However, I found several aspects of the manuscript that in my opinion should be improved before it is ready to be published. Below, I describe these issues in the five sections suggested by the journal. I apologize in advance for the extension of my comments, but I hope that this will help the authors for improving this interesting work.

-In addition to all my previous comments, I have one more general comment regarding the title of the study. Besides the natural pest control part that I already commented before, the title is focused on biotic homogenization and I do not think this is strictly investigated here. Usually, this topic is addressed by community composition or beta diversity between samples/sites. But I believe that in your study the main focus is not on biotic homogenization but pests/diseases, natural enemies and yield. I believe that studying the effects of EI on all these variables is valuable and you should highlight this in the title.

Other minor comments are provided below.
-Abundance of adult carabids was used as a proxy for natural enemies, but non-predator species of carabids can represent a high proportion of individuals in arable fields in Europe, such as species from the genus Amara that feed almost exclusively on seeds. Do you think that this could have biased your results?
-The specific objectives of your study should be clearly defined in the abstract. Instead of using terms as “such as” (line 33), you could mention directly the EI, landscape and soil factors tested and the analyzed groups.
-Line 43: dispersal ability might be a more appropriate word than power.
-Line 68: space is missing in et al.
-Line 79: the correct spelling for the reference in Bartomeus.
-Lines 98-101: you state that most of the evidence is from Western Europe but Sutcliffe et al. title is about central and Eastern Europe.
-Line 127: you could either provide the real average value for all fields or the range of sizes.
-Line 129: the distance was from … to … might be a better option.
-Lines 163: check the reference, publication year was 2011.
-Line 181: where were pitfall traps installed, specifically? Pitfall sampling was performed only once? At which moment?
-197: these sub-samples are not clear to me. Did you take sub-samples of known weights? What were the final measures used for analyses?
-Lines 232-233: Please refer to all the results about aphids first and then to pathogens.
-Line 232: The differences in Fig. 2b does not look so significant, CIs are quite large and overlapping. Is this result or graphic correct?
-Line 248: Moreover could be replaced by specifically or something similar because you are talking of the same results as in the previous sentence but with more details. Otherwise, you could remove the first sentence and directly mention the effects for both measures of yield.
-Line 249: It may be better to show yield results in a figure after the natural enemies Fig. 3, to follow the order of the results and tables. Alternatively, you could merge all pests and natural enemies into one figure and show yield in a second one.
-Lines 253-254: here you also mention the relationships between pests and enemies, which were not tested directly.
-Line2 259-261: if fertilizer use did not affect on wheat yield, I do not see how this within field intensification can compensate for the differences between fields. Please explain further.
-Lines 275-278: better than saying the EI can be described by, the authors could say that this effect of this particular EI measure could be linked to other enemies or dispersal barriers.
- Line 305: why you mention this potential scavenger role of predators and beta diversity in the title? I do not see the link with beta diversity in your study, whereas it is not clear if the mention of scavengers is linked to the seed detection you mention and how is this connected with your results.
-Line 364: “Better” is not an ideal adjective to describe natural enemies. Do you mean higher diversity, more individuals, or improved effectiveness?

References cited:
-O’hara, R. B., & Kotze, D. J. (2010). Do not log‐transform count data. Methods in ecology and Evolution, 1(2), 118-122.


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: MIXED EFFECTS OF ECOLOGICAL INTENSIFICATION ON NATURAL PEST CONTROL PROVIDERS: A SHORT-TERM STUDY FOR BIOTIC HOMOGENIZATION IN WINTER WHEAT FIELDS
Review round: 1
Reviewer: 2

Output:
Basic reporting: There a few grammatical mistakes throughout the manuscript, although the text remains perfectly understandable. In the introduction, the connections between paragraphs and sentences are weak. Too often the authors resorted to conjunctive adverbs to add new details (I counted six “furthermore”, three “additionally/in addition” and one “moreover”). I think that too much information are added one after the other, and it is not clear what is most relevant for this work.

In the introduction ecosystem services are given by the acronym “ESs”, but the authors keep using the full name throughout the text. Also, US and UK spellings are mixed up (fertiliser vs. fertilizer, fertilisation vs. fertilization). Please, decide for one and be consistent.
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Important references are missing from the introduction and from the discussion:

Dainese, Matteo, et al. "A global synthesis reveals biodiversity-mediated benefits for crop production." bioRxiv (2019): 554170.

Karp, Daniel S., et al. "Crop pests and predators exhibit inconsistent responses to surrounding landscape composition." Proceedings of the National Academy of Sciences 115.33 (2018): E7863-E7870.

Those are recent papers that are very relevant for this study and should have been at least discussed.
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Tables are OK (maybe switching their order would be a good idea).
Figures could be improved. The captions are supposed to be self-explanatory but they are not always as such. For example, in Fig3 “Response of ground beetles (a, b), and spiders (c, d)” don’t clarify if it refers to abundance, density, species richness…
Please add the unit (“g”) in the vertical axis of Fig.2. The figures itself would be clearer if the mean is represented by a point rather than with a box.
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The discussions would have been clearer if referring to the hypotheses (accepted, rejected, etc.) whenever relevant.
Experimental design: The manuscript would fit the aims and scope of the journal.
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The research question is well defined, relevant and meaningful. However, the authors refer to natural pest control as if they measured it, and they are providing new insight about it (lines 69-70, 81-84, 90-95, 104, 111 in H1, but also lines 361-363 in the conclusions). Even the title mentions “the mixed effects ecological intensification on natural pest control...” and this is misleading. In this study, the authors measured the abundances of natural enemies (ground beetles and spiders) and of pests (aphids), which does not translate directly to measuring natural pest control. Tue authors may claim that the abundance of natural enemies could be considered as a proxy of natural pest control, but there is ample evidence that this is not always true. For example,

In barley fields: Rusch, Adrien, et al. "Predator body sizes and habitat preferences predict predation rates in an agroecosystem." Basic and Applied Ecology 16.3 (2015): 250-259.

In wheat fields: Mansion‐Vaquié, Agathe, et al. "Manipulating field margins to increase predation intensity in fields of winter wheat (Triticum aestivum)." Journal of Applied Entomology 141.8 (2017): 600-611.

Dainese, Matteo, et al. "A global synthesis reveals biodiversity-mediated benefits for crop production." bioRxiv (2019): 554170

Maybe the introduction should mention ecosystem services (ESs) without devoting excessive attention, as the only ES that was measured was crop yield. In fact, this experiment was not really designed to test H1 (on pest control), which is not followed up in the discussion.
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The investigation was carried out accordingly to the prevailing ethical standards in the field.
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Overall, the M&M section is OK. The authors should specify that leaf spot prevalence was calculated as the percentage of the plant with spots (was it?).

The statistical method used to analyse the data is good. The authors used multi-model inference and cited all the basic literature making clear what they are doing it. They also built models with only single explanatory variables to avoid potential collinearity between explanatory variables, which is a safe approach (although the reality may be more complicated than that). Nevertheless, it’s not clear if model averaging (in the two cases when it was applied) put together explanatory variables that were collinear. I am not aware of a solution to account for this, but the author should at least recognise it in the m&m or in the results. Did it happen that two collinear factors ended up together because of model averaging?
Validity of the findings: The parts were natural pest control is discussed should be removed, as natural pest control was not directly measured in this study.

Line 305. In the title of the subsection “The natural enemies are scavengers…”. It’s not clear what the authors mean, as they don’t discuss food preference and in any case spiders (that were also studied here) are not scavengers. The authors emphasise the spatial and temporal dynamics (spillover and seasonal effects) but neither of them was the focused of this study, which was only a “snapshot” of the season and that was not based on directional traps (which are particularly useful to determine spillover).

As I said before, the discussions would have been clearer if referring to the hypotheses whenever relevant. The authors spent more time discussing what they found to be the best models and the significant explanatory variables than discussing their hypotheses (for example, lines 257-259, the set-aside fields were expected to have an effect on natural enemies (H2), but the fact that no effect was found is not mentioned). I think the authors should speculate more about the reasons why their hypotheses were accepted/rejected. At least as much, if not more, than they speculated about what they found to be significant.
Additional comments: The introduction is confused. Starting by talking about biotic homogenisation immediately (lines 51-57 are unnecessary) would be an improvement. After discussing biotic homogenisation and how its effects on the community at different spatial scales have not been clarified (lines 64-65), the reader naturally expects to read what we know so far about the effects at within-field and landscape scales, but virtually all the attention is focused on the landscape scale.

Table 1 is already part of the results (the best models) and should be considered as such. I would suggest to switch Tables 1 and 2 and to start mentioning them from the results.
Another minor thing is that generalised linear mixed models should be changed to linear mixed models, as the authors normalised all responses and used always the Gaussian distribution.

Results could have been more informative, including mean and SD (or SE) when presenting the data.

There are several occurring mistakes in the reference list. The ones I detected are marked in the pdf.

Acknowledgments should not include the funders according to the guidelines of the journal.

Please find other, specific comments in the pdf of the manuscript.


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

Input:
Title: MIXED EFFECTS OF ECOLOGICAL INTENSIFICATION ON NATURAL PEST CONTROL PROVIDERS: A SHORT-TERM STUDY FOR BIOTIC HOMOGENIZATION IN WINTER WHEAT FIELDS
Review round: 1
Reviewer: 3

Output:
Basic reporting: Structurally I think this needs some work, although it has a lot of promoise.  i would consider gettign a native english speaker to read though it before a resubmission. It needs a bit of polishing on this front.  The abstract needs a bit of work though, see comments below.
Experimental design: See gerneal commetns to authro, however this si a farily sensibel split pot design. Smapling effor is not crazy (10 days of pitfall trapping - only 1 year of sampling) but there is reasonable field replication.
Validity of the findings: See gerneal commetns to author, in particualr the comments about the stats section., I feel you need to pick one statistical approach (probaly thre multimodel averaging). the including of this with the univariate correlations seems unecisairy.  Note that if variabels are highly colinear jsut drop them enterly.  Multimodel averaging is as suceptabel as any other approach to this issue - although I woudl point out thats its a rare ecologcial study where all covariates are totally indipendent - some correlation is common.
Additional comments: Abstract : In general this needs a lot of work for clarity.  I would try and condense it more specifically into sections that define 1) what the problem is, 2) a simple summary of what you did methodologically; 3) what your key results were; and 4) the implications of this for sustainable intensification.  Its kind of there but comes across as a bit muddled and as such is hard to read.
L26: Its not immediately clear what you mean by ‘alternative ecological intensification in croplands’ and how this relates to your opening line about varying levels of homogenisation – i.e. is your assumption that heterogeneity and sust. intensification are by definition going to occur simultaneously.  I guess they could but not necessarily.  Further I am not sure what the ‘alternative’ bit relates to – you explain ecological intensification fine.
L27-30: I think I would split this sentence in two.
L31:  I think you need to be a bit more specific about what sust.int. management practices are being implemented.
L32’ ‘and local field management’
L33 ‘such as experimentally’
L33:  what fertilisers, NPK, liquid manure, solid manure?  Specify what you mean here.
L39-40:  This needs to be clarified a bit -  I am not entirely clear what you mean by this.  From 36-41 the narrative suggests for fields with no fertiliser being close to set aside decreased aphid abundance  -but for field s a distance from set aside the higher aphid densities required fertiliser to offset the reduction in yield.  Is this correct?  It neds work for clarity.
L43:  Do you explicitly test this?

General question is reduced aphid numbers correlated with increased yields. In the UK at least the main issue with aphids is in terms of disease transfer, rather than direct effects on yield.

Introduction
L54:  Why are crop lands hyper dynamic?
L54:  given your paper focus also see papers linking landscape to predator  - pest control attributes - Karp, D. S., et al  (2018). Crop pests and predators exhibit inconsistent responses to surrounding landscape composition. Proceedings of the National Academy of Sciences, 115, E7863-E7870.
L59 ‘decreasing system biodiversity and……………against disturbance.’   Would also make clear what you mean by disturbance, is this say pesticide use or cultivation.
L65-69: See also Woodcock, et al. (2014). National patterns of functional diversity and redundancy in predatory ground beetles and bees associated with key UK arable crops. Journal of Applied Ecology, 51, 142-151.
L79:  For actual demonstration of sust. Intensification see Pywell, et al (2015). Wildlife-Friendly Farming Increases Crop Yield: Evidence for Ecological Intensification. Proceedings of the Royal Society, Series B, 282, 20151740
L84-88:  A very good point, as you point out in many situations in wheat this is the main factor reducing yield.
L92-95:  I would re-write this sentence.  Lose the bit about ‘been introduced by scientists’.
L92:  Would not hurt to have some statistics in here about the value of natural pest control.
L102:  Delete this sentence, and start the next one (L103) with ‘IN this study we tested whether….’
L105-106:  This is quite muddled.  Its hard to follow. I would split into two sentences  (First sentence ends at L105 ‘intensity.’ – then go on to  talk about semi-nat habitat)– but in general you need to work a bit on clarity.  You sometimes have jumps of logic that are not necessarily wrong but for the reader they come out of nowhere.
L106:  Given the fact this saves one word I would just use Ecological intensification rather than EI, its clearer.
L 108 ‘management) as it interacts with landscape…’
L108:  I think the assessment of impacts on pathogens is a really important part of this work 9even if its no-sig).  I would mention it more in the abstract.
L110: At some point earlier than this I would maybe define probably what you mean by ‘intensive’. I know what you mean, but I think its worth doing.
L111:  IOk so this is one of those jumps in logic.  What enhanced dispersal capacity of predators?  Is this increased by semi-nat habitat or is this an innate property of them. If the latter this would seem to be only true in some cases as ‘nat enemies’ covers a massive range of taxa from highly mobile ballooning spiders to wingless ground beetles.  You need to tighten up the text in relation to these.  At the least justify what you mean.
L113:  So your logic doesn’t really follow here from L111, effectively you are saying they are poor dispersers, and so you would only expect increased effects close to semi-nat habitat (a reasonable assertion).
L114:  What type of fertiliser – inorganic, or manure, or liquid fertiliser etc.   they potentially have very different effects.
Methods
L126:  Might be useful to talk about common rotations, or length of rotations.
L131:  Maybe give full mixture and sowing rate in a appendix
L139:  But presumably in the control fields it was also at an edge (just there is no set aside for it to be close to).
L140:  The fertiliser statement seem contradictory to that in L143 what are you say you split this area and add NPK.
L147:  ‘We collected 15 cm soil cover to assess…’
L149: what’s the approx. date of this?
L151:  what did you assess, e.g. organic carbon, phosphorous etc?
L159: ‘We considered those landscapes surrounding experimental fields with more than 20% semi-natural habitat as structurally complex’
L161-163: I am not clear what you are trying to say here.  Also note that in the UK cover crops refer specifically to a situation when post harvesting a temporary mixture (eg. Oats or mustard) is sown to protect soils and increase fertility over the winter period before a spring sown crop is sown. Is this what you mean.
-	A general thought, are all your wheats the first wheat in the rotation (normally the most productive) or a second one?
L175:  delete ‘Moreover’
L185 –I thing you need to include sampling dates.
Stats:

L205: So why not use a poisson or negative binomial distribution rather than use a sqrt transformation
L205:  We created sets of….
L209-212:  You inver sampling suggests two sampling dates.  From what you describe you summed the two sampling dates (e.g. for the aphids) so that you have a single value per sub-plot position along the transect.
L212:  Ok so what random effects will depend on whether you included the points along the transect as individual data points, or whether you just summed these per sub-plot.  If you have got individual transect points I think you need a sub-plot classified nested within farmID.  If you have just summed across the transects (and across dates) then your current random effects look ok.
L216220: So I am happy for you to use the multimodel averaging approach. I think 2 AIc is a good cut off.  However, from what I can tell (L206) you just have models with a single explanatory variable (e.g. landscape complexity).  I think you should start with a maximal model with all your explanatory factors in it and use MuMin to assess all combination of these models (including the intercept) to derive you AIC delta 2 subset.   If you have genuine concerns that two factors are collinear drop one and simply state that they were highly intercorrelated and so were not included in the same model. Ecological data always has some intercorrelations, but I appreciate there is a cut off on this.  Looking at your explanatory effects there is nothing that jumps out to me as being crazy to include all in one model from which to get your subset for multi-model averaging.   To be honest I think you may have done this but the L206 suggests this is not the case.
Results
L237:  So if you are using multimodel averaging and a delta AIC subset this is not really compatible with the term significant as you are not undertaking a specific test of significance that produces an associated test statistic (e. F value) and p value.   Reading this now I think you are undertaking a significance test on the single factor models described in L205.  Firstly if you are doing this, what significance test are you undertaking.  My more relevant point is why are you doing this.  I would suggest that you pick one approach (probably the multi-model averaging approach) and  just present those results.  As I said if collinearity is a problem, this is a problem for your multimodel averaging approach as well so if there really are big issues here you need to drop explanatory variables.
-	Gerneal point:  So there are various test statistics worth presenting here to describe the importance of different explanatory variables in your  detla2AIC subset.  These focus on the summed wi score (although I think MuMin has a scaled version of this so that it adds to 1) and the importance score (which as I recall maybe the proportion of models in the delta 2 aic subset which include a given fixed effect.  Worth having a look at paper  Symonds, M. E., & Moussalli, A. (2011). A brief guide to model selection, multimodel inference and model averaging in behavioural ecology using Akaike’s information criterion. Behavioral Ecology and Sociobiology, 65(1), 13-21.  Note as this approach is relative (e.g. you will always have best fit model, even if it’s a poor fit) it may be worth considering a R2 measure of fit for your global model.  This paper also describes this.
Note earlier comments about not being clear if you are considering the transect data points separately, or different sampling dates (and how this affects you model).  From what I can see from your results you have either just summed these are averaged them.  In which case your random effects seem ok to me – but you still need to be clear what you have done.
General comments for Discussion.
So as some of the results are likely to be dependent on how you change your stat in response to my comments ill give some overarching comments here (also I have already given you lots of comments). I would work on adding more references in L253-267.  I would possibly consider structuring the discussion in relation to your hypothesis (maybe, this is just an idea).  I would potentially also consider if sampling effort or sample size may explain your absence of effects.  Also see the points I make above about refocussing to include just your multimodel averaging results (I don’t see the point of both doing univariate p tests and this approach).