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: CAPTURING PATIENT-REPORTED AREA OF KNEE PAIN: A CONCURRENT VALIDITY STUDY USING DIGITAL TECHNOLOGY IN PATIENTS WITH PATELLOFEMORAL PAIN
Review round: 1
Reviewer: 2

Output:
Basic reporting: The article is nicely written.  In the following areas, minor changes to the English language should be made, suggestions included:

Line 108-9; replace tablets with tablet
Line 120: replace density with densities
Line 152: please replace “their ambiguity of” with “the ambiguity of”
Line 186: please replace “X” with “X”s
Line 186: please replace it’s with its
Line 193: please replace “adjunct tool” with “as an adjunct tool”
Line 201: please replace i.e. with e.g.
Line 223, remove comma

Lines 182 to 194. You may want to describe the placement of “X”s as identifying the pain area by marking squares on a grid.
Experimental design: An interesting study, with a well considered design.

Lines 144-5: Please report the intraclass correlation coefficient (ICC) as opposed to Pearson / R-squared as this tests not just for a linear trend but that values are equal (i.e. gradient = 1).

Line 148, please provide the reference for SPSS.
Validity of the findings: Line 155: please provide mean and SD for BMI.

Lines162-4.  As this is an equivalence study, then the test of means is not on it's own sufficient - as identifying no significant difference does not enable us to conclude definitively that the means are the same.  The correct analysis should be conducted by comparing the 95% CI for the mean difference and seeing whether this lies within or outside the acceptance region – which I understand to be +/- 1% from your sample size calculation (line 111).  You quote the 95% CI as -0.159 to 0.157.  The units are unclear, but if this is -0.159% to 0.157% then your confidence interval for the mean difference falls entirely within the acceptance region of -1.0% to +1.0% and so it is possible to conclude equivalence based on your pre-defined acceptance criterion.  Please clarify this point, and adjust the text describing the results appropriately.  More details of this approach can be found in  Byrom and Tiplady.  ePRO: electronic solutions for patient-reported data.  Gower.  pp189-90.

Lines 165-8.  I agree that a linear correlation is present. Please be aware that Pearson’s correlation tests for a linear trend, but in an equivalence study we are looking to demonstrate that values are equal – i.e. to see a linear trend with a gradient of 1.0.  This is more appropriately assessed by using the intraclass correlation coefficient (ICC).  Please report the ICC instead of R-squared.

Lines 236 – 248.  In conclusion, it would seem a good future extension to automate the calculation of the pain area (pixel density) using an algorithm to interpret the drawing on the tablet if the approach is taken forward.
Additional comments: This is an interesting study with good potential for application in routine care and clinical trials.


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: CAPTURING PATIENT-REPORTED AREA OF KNEE PAIN: A CONCURRENT VALIDITY STUDY USING DIGITAL TECHNOLOGY IN PATIENTS WITH PATELLOFEMORAL PAIN
Review round: 2
Reviewer: 1

Output:
Basic reporting: The author's revisions have satisfied this reviewer's requests.
Experimental design: The author's revisions have satisfied this reviewer's requests.
Validity of the findings: The author's revisions have satisfied this reviewer's requests.
Additional comments: The author's revisions have satisfied this reviewer's requests.


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

Input:
Title: CAPTURING PATIENT-REPORTED AREA OF KNEE PAIN: A CONCURRENT VALIDITY STUDY USING DIGITAL TECHNOLOGY IN PATIENTS WITH PATELLOFEMORAL PAIN
Review round: 2
Reviewer: 2

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: Thank you for making the suggested revisions to your paper.  It is a well written and interesting piece.


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: CELLULAR PRION PROTEIN AND Γ-SYNUCLEIN OVEREXPRESSION IN LS 174T COLORECTAL CANCER CELL DRIVES ENDOTHELIAL PROLIFERATION-TO-DIFFERENTIATION SWITCH
Review round: 1
Reviewer: 1

Output:
Basic reporting: General comments

There are quite a few grammatical errors throughout the paper. These errors, especially those in the abstract make it difficult to follow the paper at times.

Minor errors

1.	Line 291 typo: astrocyte
2.	Line 234: Specify which software
3.	The "internal control" on figure 1D needs to be clarified
4.	Figure 2B is too wide
5.	Figure 2D's low resolution makes it difficult to see the scratch assay
6.	No scale bar for figure 2D
7.	The Y axis for the figure 2D should probably be Time (hr)
8.	Figure 2C has two graphs which share the same Y axis title "Conditioned media". It maybe better to title them " conditioned media from LS cells" or  something like that to differentiate the two.
9.	No Scale bar in Figure 3 images
10.	Adjust the size of Figure 3 graphs
11.	No Scale bar for Figure 4 images
Experimental design: The experimental design is appropriate.
Validity of the findings: Statistics mentioned are limited to identifying if the data have a statistical p value of less than 0.05. The P values should be mentioned and the data whose p value is below 0.01 or 0.001 can be highlighted

Comments regarding the discussion

While the cited Bussolino, Mantovani & Persico review article indicates that the angiogenesis involves changes in morphology, it does not mention EA cell line used in the manuscript. Is there any other reference that can show evidence that EA tube formation can be used as a model for the formation of new vasculature?

Is there any evidence that NO levels may affect the proteins detected in the secretome (Figure 5)? Or are they independent
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: CELLULAR PRION PROTEIN AND Γ-SYNUCLEIN OVEREXPRESSION IN LS 174T COLORECTAL CANCER CELL DRIVES ENDOTHELIAL PROLIFERATION-TO-DIFFERENTIATION SWITCH
Review round: 1
Reviewer: 2

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


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

Input:
Title: GENOME-WIDE IDENTIFICATION AND CHARACTERIZATION OF DOF TRANSCRIPTION FACTORS IN EGGPLANT (SOLANUM MELONGENA L.)
Review round: 1
Reviewer: 1

Output:
Basic reporting: This manuscript reports the Dof family identified in the genome sequence data of eggplant, Solanum melongena. This paper is too descriptive and new suggestions and insights into genetics and physiology of eggplant are rather limited. Most of all analysis is based on published data. Even though the authors performed RNA-Seq analysis across six varieties, the authors do not disclose raw sequence data, which is conflict to the guidline of submission instruction of PeerJ.
https://peerj.com/about/author-instructions/#data-and-materials
Experimental design: Details RNA-Seq analysis are required in Materials and methods section. What organs and stages, how many replicates, and what types of sequencers did the authors employ? How much data for each sample?
Validity of the findings: The concordant map rates in wild relatives, SIS_2007, AET_SG, and INT_2006 were low. Dose this result reflect gene expression profile correctly? DNA polymorphisms can disturb read mapping on the reference sequences of cultivated eggplant, resulting in underestimation of the expression.
Additional comments: The authors performed phylogenetic analysis, gene structural analysis, ortholog/paralog identification, and expression analysis separately. The results should be compared each other to evaluate that the subfamilies are evolved structurally and functinally parallel or not. How about regulatory sequences, like promoters?


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: GENOME-WIDE IDENTIFICATION AND CHARACTERIZATION OF DOF TRANSCRIPTION FACTORS IN EGGPLANT (SOLANUM MELONGENA L.)
Review round: 1
Reviewer: 2

Output:
Basic reporting: No comment.
Experimental design: 1. For in planta classification of eggplant SmeDof genes into different groups/subgroups such as A, B, C D etc, the authors have chosen 4 additional species, namely C. reinhardtii, P. patens, A. thaliana, O. sativa. However, the rationale behind this choice is not provided in sufficient detail. Thus, it is not clear if the classification results would change if additional or different species were included in the analysis. For example, the authors cite Moreno-Risueno et al., 2007, whose analysis indicates the presence of a subgroup G but the representative protein sequences from that sub-group are not included in the current manuscript. It is suggested that to maintain consistency with previously published results, the authors include representative sequences from all subgroups in their phylogenetic analysis. One way to achieve this would be to include all the species that have been used in previously published classifications. Ideally, this would include the species at least from the papers that the authors have cited, namely Cai XF et al. 2013, Guo and Qiu 2013, Huang et al 2016, Kushwaha et al. 2011, Lijavetzky et al 2003, Ma et al. 2015, Malviya et al 2015, Moreno-Risueno et al. 2007 and Shigyo et al. 2007 etc. Alternatively, the authors should better justify their choice of species and protein sequences.

2. What plant tissue was used as the source of RNA for the RNA-Seq experiments? This needs to be clearly mentioned, otherwise the statements such as in line 306 “SmeDof-23 had high expression levels in the other five eggplants but did not express in MEL_LYQ”  do not remain very meaningful.

3. In the methods section, the authors state in lines 140 to 142: “The phylogenetic relationships of the six eggplants were determined by the alignment rate between their RNA-seq reads and the eggplant genome sequence (Hirakawa et al. 2014) using BWA software (Li et al., 2009).” Although sequence alignment rate is indicative of evolutionary relatedness, it is not a metric for inferring phylogenetic relationships. The authors should accordingly replace the phrase “phylogenetic relationships” in the sentence with a more suitable descriptor.

4. In the section “Phylogenetic relationships of the Dof proteins among Solanaceae species”: the authors have tried to correlate the genome size with the number of Dof proteins. However, a more suitable comparison would be with the total number of protein-coding genes in each genome, instead of the raw genome size. This is because even without whole-genome duplications, genome size can increase dramatically due to increase in non-coding repeats, and transposons etc.
Validity of the findings: It is not clear if the classification into different subgroups such as A, B, C D etc is robust to the inclusion of additional plant species. See the issues raised in the “Experimental Design” section above.
Additional comments: Some language/typo suggestions:
1. Line 37: Please change “anti-biotic stress” to “abiotic stress”.
2. Line 124: Shouldn’t “E-value larger than the 0.05” be “E-value smaller than the 0.05”?
3. Line 136: Please change “RNA-Seq transcriptions” to “RNA-Seq transcriptomes” or similar.
4. Lines 239-240: It is not clear what do the authors mean by “attributed to alternative actions”, please rephrase and explain.
5. Line 288 and elsewhere: Instead of “different eggplants”, it is suggested that the phrase “different eggplant subspecies” or similar should be used.


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: CHANGES IN NUTRIENTS AND DECAY RATE OF GINKGO BILOBA LEAF LITTER EXPOSED TO ELEVATED O3 CONCENTRATION IN URBAN AREA
Review round: 1
Reviewer: 1

Output:
Basic reporting: English must be improved
More information on results of other studies should be added and an hypothesis too
The structure is OK
An hypothesis is missing
Experimental design: It fits with the scope of the journal
Research question well defined but more information on its relevancy must be added
IMethods are well described
Validity of the findings: Data and statistics are correct.
Conclussions should be improved

See General comments to the authors for more comments....
Additional comments: The paper addresses the question of how ozone affects the chemical composition and decomposition of Ginko biloba litter. The topic is in principle interesting as there are not many studies on this issue. The experiment was carried out in Open Top Chambers in China. However, there several relevant questions that makes the paper not acceptable for publication in its current form. Here there are some recommendations:
Some English expressions are somewhat awkward, and I strongly recommend that the paper is revised by a native speaker. Some examples: line 31: has a toxicity to plants that is widely concerned; line 67: every treatment was repeated, maybe: every treatment was replicated; line 69: management measurements…were consistent, maybe: Management measures such as irrigation were the same for the two treatments; line 95: through use, maybe: through the use; line 157: changed insignificantly compared test in ambient air (sentence not clear); line 153: , as is in agreement (better: which is in agreement); lines 174-178: unclear sentences; line 189: thereof is conducive (unclear meaning).
Lines 6-7: Is the topic of this paper forest ecosystems or urban vegetation?. The meaning of “Profoundly influenced” is not clear, what is the type of effect?, or remove.
Line 8: Ginkgo biloba was -> maybe: Ginkgo biloba saplings were used
Line 11: improved->increased
 Line 11: reduced->and reduced
Line 16: of->maybe: caused by
Line 28-29: Although this is the case for Asia, it is not true for Europe, as the increase has stabilized recently (see Cooper et al., 2014. "Global distribution and trends of tropospheric ozone: An observation-based review." Elementa: Science of the Anthropocene 2, 000029).
Line 50: A clearer hypothesis should be proposed, rather than “change”, better put increase or decrease. The hypothesis should be proposed after introducing previous studies. Based on previous studies, it should be expected that there is an increase or decrease. Then, test if that hypothesis is correct or not.
Line 65-66: The fumigation regime appears unrealistic for urban trees as 120 ppb is quite a high value sustained for about 4 months (please, add the dates). Please, justify what such high values were used.
Line 76: what is the regular litter bag method?. Please, add a reference
Line 99: Can you add a reference for this model?
Line 118-121: add that the changes in N and P were not significant.
Line 123: obviously->significantly
Line 144: at 0.05 and 0.01 levels->better refer to significance here, not to levels
150: Calatayuda is Calatayud (check also other parts of the paper)
150: In this context the following paper by Shang et al. (2018) can be relevant: "Elevated ozone affects C, N and P ecological stoichiometry and nutrient resorption of two poplar clones." Environmental Pollution 234, 136-144.
Line 202: please, add supporting references
Discussion: Several issue have not been well explained
O3 usually reduces N content of older leaves as there is a translocation of this nutrient to upper leaves which increases senescence. Why N increases in the litter?. Is it the case that more litter is produced?. Please, explain this.

Other important improvements are:

The discussion is descriptive of the results and there are comparisons with other studies but one does not get a complete picture of the processes behind such results. Please, try to give a more comprehensive picture of the processes.
Conclusion: Besides summarizing the results, what is the relevancy of these results for urban forests?. A wider scope is needed in the discussion.


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

Input:
Title: CHANGES IN NUTRIENTS AND DECAY RATE OF GINKGO BILOBA LEAF LITTER EXPOSED TO ELEVATED O3 CONCENTRATION IN URBAN AREA
Review round: 1
Reviewer: 2

Output:
Basic reporting: Language: although generally clear, the manuscript language could be improved at several points towards professional standard for scientific writing. Specific comments to the sections requiring language revision are reported below.
Literature references: insufficient field background is provided, particularly at specific points in the Introduction and Discussion sections, where substantially relevant information about previous evidence is missing. Please see specific comments below.
Article structure is mostly satisfactory. Specific comments aimed at improving figure and table style and quality of presentation are reported below for each item. Raw data are also presented as supplemental excel files, which is appreciable, but not described by metadata.
Experimental design: Some major flaws in the study rationale and experimental design are highlighted in the comments below (see sections 3 and 4).
Research question are potentially relevant & meaningful, although not well defined. It is not explicitly stated how research could fill the knowledge gap about O3 effects in urban forests.
Methods are not described with sufficient detail & information to replicate.
More details in comments below (section 4)
Validity of the findings: Statistical analyses should be re-done.
The absence of a true control weakens the authors’ conclusions.
Raw data are provided as excel files, which include all graphs presented in the manuscript figures. However, these are based on aggregated data (means and standard deviations) that are not linked to raw data by formulas, so that it is not possible to assess the correspondence between raw data and data presented in the manuscript figures. Possibly easily addressable by providing appropriate metadata (which raw data are used for each data point in each figure?)
Conclusions are concise, but at the current state of the manuscript they are not completely supported and in the case of litter decay acceleration by high O3 concentration could even be mistaken.
Speculation is widely present in the Disucssion section, not always identified as such.
More details in comments below (section 4).
Additional comments: Title
The manuscript title is not informative of the study results. As the study is about leaf litter, this should be included in the title. Suggestion: “Exposure to elevated O3 concentration enhances nutrient content and decay rate of Ginkgo biloba leaf litter”

Abstract
The abstract section could be largely improved, by including a more effective reference to the background, clearly stating the tested hypothesis, and adding missing information. Please revise this section along the line of the following specific comments:
LL 6-8: “has profoundly influenced” does not clarify the effects of exposure to O3 on forest ecosystems. Please rephrase including a general description of these effects. Which forest ecosystem are more affected, which response metrics are affected, by which magnitude and direction?
L 8: “still less known” perhaps better “still poorly known”
LL 9-10: “to investigate the impact…” at this point the study objective is not clear. The authors should consider to clearly define the scientific hypothesis underlying their study. What are the expected impacts on litter chemical composition and decay rate?  See comment to Introduction LL 49-53.
Language issue: please consider adding “concentration” to “elevated O3” throughout the text.
The authors here refer to “leaf litters of the urban tree”, inconsistent to the preceding background sentences that refer to “forest litters”. In facts, the manuscript reports on a manipulative experiment in an arboretum. This should be clearly stated.
Information on experimental conditions and the methods used to assess litter chemical composition should be included. At this point, it is not clear what the authors mean by “OTCs simulations”.
LL 11-12: some quantitative data (e.g. mean and sd of treatments, p-values for statistical significance) on the litter content of N, K, phenols, tannins and sugars should be included. Please consider rewording according to  the following suggestion: “Exposure to high O3 concentration, compared to the ambient air control, enhanced the litter content of N (mean ± sd vs mean ± sd, P < xxx) and K (mean ± sd vs mean ± sd, P < xxx), while reducing that of total phenols (mean ± sd vs mean ± sd, P < xxx), condensed tannins (mean ± sd vs mean ± sd, P < xxx) and soluble sugars (mean ± sd vs mean ± sd, P < xxx).”.
Please also consider adding a comment about the result of lignin shown in Fig. 3.
LL12-13: “Furthermore, elevated O3 significantly decreased the residual rate of dry weight by 11.9% of G. biloba litters (P < 0.05)”. Awkward sentence. Please reword, along the line of the following suggestion, more consistent with results presented in Fig. 2: “Furthermore, percent mass remaining in litterbags after 150 days of exposure to high compared to low O3 concentration was 11.9% lower (P < 0.05)”.
Please note: a) the use of “percent mass remaining”, which is the established definition Y = At/A0, in place of “residual rate” or “residual ratio”, that the authors inconsistently use throughout the manuscript; b) the inclusion of experimental conditions under which the result was observed. However, about this specific  result, see comment to Fig. 2 below.
L 14: What are the “nutrient components”? Consider using simply “nutrients”. Possible rewording: “Differently, net nutrient release from litter during decomposition was unaffected by O3 treatment.”.
L 15-17: current version: "as is mainly related to"; prudent alternative version "may be related to" (or something like that). I recommend writing prudently, considering that the authors only showed that lignin content decrease with litter decay under high O3 concentration, but they did not directly test the occurrence of lignin oxidation, nor reported previous evidence about this possible explanation for the observed enhanced litter decay. Speculation could be admitted, but should be identified as such. Please also consider discussing results on litter chemical composition as affected by treatments.

Introduction
This section should be re-arranged in order to improve the logical order of presentation of different arguments. Please follow specific comments below.
LL 30-31: This sentence is scarcely informative. What is the point in reporting that O3 toxic effects on plants are widely concerned, when such effects are not reported? The authors should either insert a synthetic sentence that describes O3 toxicity, or delete the sentence.
LL 31-32: Again, “direct or indirect influences” is meaningless without specifying which influences we are speaking of, and which are direct or indirect. The authors should either insert a synthetic sentence that describes the “influences”, or delete the sentence. In case the sentence is not removed, please consider replacing “material cycles” with “biogeochemical cycles”. “Catalayuda” is misspelled. Please use the correct author surname “Catalayud”.
LL 34-44: this paragraph is a mix of different topics whose order of presentation is hard to follow, and lacks of appropriate reference to the literature. The paragraph should be re-written leaving out topics out of context, such as climate change, increased CO2, nitrogen depositions. I suggest to include the following argumentations, with appropriate citations where needed:
Effects of O3 on forest ecosystems productivity and feedbacks have been widely investigated worldwide (reviews in Chappelka & Samuelson, 1998; Paoletti, 2006; de Bauer and Hernández-Tejeda, 2007) and recently synthesized (Wang et al. 2016). Differently, O3 effects on litter decomposition are much less known. Indirect evidence comes from litter photodegradation studies in semi-arid and arid ecosystems  (e.g. Austin & Vivanco, 2006 and following). Among the few studies carried out in natural forest ecosystems, Parsons et al. (2008) showed that, over a 23-months observation period on leaf litterbags of aspen and birch reciprocally transplanted to separate substrate quality from environment effects, increasing O3 concentration by fumigation slowed down both aspen and birch litter decay rate, exhacerbating the effects of elevated CO2 concentration, but accelerated birch litter decay under ambient CO2. A negative effect of O3 fumigation on litter decay rate was also observed for holm oak leaf litter in Mediterranean forest (Baldantoni et al. 2011). Such observations were explained by CO2- and O3-mediated changes in litter chemistry, particularly carbohydrates, nitrogen, and tannins. O3 effects on litter decomposition in urban forests have not yet been explored. Filling such gap is particularly important, as in urban ecosystems, where tropospheric O3 concentration can be very high due to photochemical air pollution, urban trees play a fundamental mitigating role (e.g. Manes et al. 2012). Their leaf litter, if decaying faster when exposed to high O3 concentration, would improve soil chemical properties and promote nutrient cycles, therefore affecting the sustainable development of urban areas (Nikula et al., 2010; Xu et al., 2012).
Cited references:
Austin, A.T., Vivanco, L., 2006. Plant litter decomposition in a semi-arid ecosystem controlled by photodegradation. Nature 442, 555-558.
Calfapietra, C., Fares, S., Manes, F., Morani, A., Sgrigna, G. and Loreto, F., 2013. Role of Biogenic Volatile Organic Compounds (BVOC) emitted by urban trees on ozone concentration in cities: A review. Environmental pollution, 183, pp.71-80.
Chappelka, A.H. and Samuelson, L.J., 1998. Ambient ozone effects on forest trees of the eastern United States: a review. The New Phytologist, 139(1), pp.91-108.
de Bauer, M.D.L. and Hernández-Tejeda, T., 2007. A review of ozone-induced effects on the forests of central Mexico. Environmental Pollution, 147(3), pp.446-453.
Manes, F., Incerti, G., Salvatori, E., Vitale, M., Ricotta, C. and Costanza, R., 2012. Urban ecosystem services: tree diversity and stability of tropospheric ozone removal. Ecological applications, 22(1), pp.349-360.
Paoletti, E., 2006. Impact of ozone on Mediterranean forests: a review. Environmental Pollution, 144(2), pp.463-474.
Parsons, W.F., Bockheim, J.G. and Lindroth, R.L., 2008. Independent, interactive, and species-specific responses of leaf litter decomposition to elevated CO2 and O3 in a northern hardwood forest. Ecosystems, 11(4), pp.505-519.
Wang, B., Shugart, H.H., Shuman, J.K. and Lerdau, M.T., 2016. Forests and ozone: productivity, carbon storage, and feedbacks. Scientific reports, 6, p.22133.

LL 46-48: please rephrase avoiding the use of passive voice. E.g.:  In recent years, we assessed the effects of elevated O3 concentration on the leaf eco-physiology of this species (He et al., 2009; Lu et al., 2009; Li et al., 2011). Here, we aim to complement such previous studies with a manipulative experiment testing O3 effects on G. biloba leaf decomposition and chemical features.

LL 49-50: expected outcomes are too broadly reported. The authors should specify at least the expected effect direction, or even effect magnitude, on the base of previous evidence, their own argumentations, or both. In this respect, it has to be considered that the only previous experiment directly testing O3 fumigation effects on leaf litter decomposition (Parsons et al. 2008, see above), though carried out on different species and under different conditions compared to the present study, produced different results for different conditions. This should be explicitly reported, and expectations clearly and unambiguously defined and justified.

LL 51-53: Main objective 1 is well defined, although language can be improved, e.g. replace “check the changes in” with “assess changes of ”. Main objective 2 should be more accurately defined, such as: 2) to assess leaf litter decomposition rate of this urban tree, as affected by O3 fumigation treatment.

Materials & Methods
L 57: “where there is” maybe nearby?
L 58: you mean a mean elevation? Is it the region that covers the urban forest, or vice-versa?
LL 59-63: please add appropriate reference for climate classification and data. Consider replacing “per annum” with “yearly”.
L 65: Replace “A total of two” with “Two”.
LL 66-67: A description of the fumigation system, or at least a reference to one of your previous studies where such system was applied, would be appreciated. Treatment naming is confusing. If you applied O3 fumigation at 40 ppb, I do not see the point of naming such treatment “ambient air”, unless you show that ambient air in natural conditions contains a constant ozone concentration of 40 ppb. Consider using LOC (Low Ozone Concentration) and HOC (High Ozone Concentration) to name the treatments. Your experimental setup suffers the lack of an untreated control, which prevents from assessing litter chemistry composition in truly ambient air, that is in not fumigated leaf samples. Indeed, based on your experimental setup, deviations from natural conditions attributable to ozone treatments cannot be assessed, which weakens your conclusions.
LL 67-75: Treatment repetition means that you replicated the treatment over three different time periods. This is not the case, isn’t it? If I correctly understand your experimental design, you just considered three independent replicates of both treatments. Please confirm and change the text accordingly. Please add information on seedling history before planting: five years are missing from the story, and could affect your results. “management measures were consistent”, poor language and incomplete information. Do you mean that cultivation practices were either consistent among treatments and replicates or consistent over time? Or both? Please add quantitative information on irrigation rate and timing and other cultivation practices, if any. I would not use “seedlings” for five years old plants and “trees” for five years and 60 days old plants. Please use consistently either of the two. Replace “fumigated by” with “fumigated with”. “All yellow leaves…” you mean senescent standing leaves or fallen leaves? Please clarify. “These yellow leaves…” you mean that you physically divided (cut?) each leaf into two parts? Please reword to clarify, e.g. “leaves from each OTC were subdivided into two pools”. Replace “initial chemical composition” with “chemical composition of undecomposed litter”. Remove “sufficiently” and replace with information on drying time. Replace “next year” with “the following year”. Where and in which conditions was the litter material stored before the decomposition experiment?
LL 76-88: Remove “regular” (is there any “irregular” litter bag method?) and add appropriate reference for this method and possible modifications. “each of the 15 bags was placed into each OTC”: presumably poorly written, as literally meaning that litterbags were moved from one OTC to another, which is unrealistic.  How many OTC were used for the decomposition experiment? And how many bags per OTC and in total? What do you mean by “close to the soil surfaces”? Please indicate if bags were placed above or below the soil surface and at which height/depth from the ground level.  “in a random order” is also poorly written. A correct random and reproducible procedure should be described as “N pools of K litterbags were randomly selected out of a total number of N * K, and each pool was randomly assigned to one of N different OTCs, where litterbags were randomly placed X cm above/below the soil surface.” Considering the soil depth (i.e. 0-10 cm), I would rather speak of “topsoil”. Replace “deep” (adjective) with “depth” (noun). Where do data in Table 1 comes from? Either you collect OTC topsoil samples and measured chemical-physical properties of collected samples, and in this case you should add information on topsoil sampling and chemical-physical measurements, or those data were previously published, which is not an issue, but in this case it must be appropriately referenced to a paper where methodological information can be found. The same applies to micro-environmental data reported in Table 2. “These litter samples” avoid the use of “these”. About pulverization and filtering, please add information on mesh size as related to sample particle size.
LL 89-97: I would replace “Measurements”, which is too generic, with “Chemical analyses” in the subsection title. Which elemental analyzer was used? Please indicate model name and producer, so that information on precision and accuracy can be retrieved. Please specify with more detail or appropriately reference the methods used to determine lignin, phenols, condensed tannins and soluble sugar content. In the case of lignin, please note that the accuracy of UV/Vis spectra can be highly affected by the type and structure of lignin (i.e. relative abundance and linking of H, S and G residues), the used solvent, and the pH of the solution. Please justify your methodological choices with respect to alternative methods (i.e. for lignin they range from spectroscopic techniques, such as ICP-Mass spectrometry or reflectance IR spectroscopy, to simple and fast analytical methods, such as proximate analysis or acetyl-bromide method.
LL 98-104: “Decomposition rate of litters was represented by residual rate (%) of litter and were calculated by using the Olson litter decomposition index model.” Consider rephrasing as “Litter mass remaining over time, expressed as percentage of the initial value, was calculated according to the well-known simple exponential model formulated by Olson (1963).” (see comment above to the Abstract section LL 12-13). Don’t need to define the Euler’s number. Please note that if you express Y as monthly, then units of measure of t and k are months and months-1, respectively, not days. Also note that the “fitting parameter” a is not included in the Olson’s model. By the way, according to your equation, at t=0, Y = ae-kt = 1, with e-kt =1 and a=1. Therefore, a cannot be a parameter to fit. Half-life time equation is also wrong, as well as the subscript notation: the correct conventional notation is t½ or t0.5 and it is calculated as t½ =ln(2)/k (note that k is a positive number, as –kt is negative and t is positive). Complete decomposition time is questionable, and in the case of this manuscript is useless, given the short experiment duration.
Cited reference:
Olson, J.S., 1963. Energy storage and the balance of producers and decomposers in ecological systems. Ecology, 44(2), pp.322-331.
LL 105-112: The authors cite a paper by Brandt et al. (2010) to justify the mass balance equation used to calculate “release of nutrient element assay” based on “residual rate of elements”. However, neither such equation, nor the terminology is reported in the cited paper, where “% of initial N” is calculated (Brandt et al. 2010, page 771, Fig. 5). Please justify.
LL 113-115: description of performed statistics is inaccurate, likely as being too short. Based on what is reported, the authors tested differences between two means (AA vs EO) with a one-way ANOVA plus LSD (post-hoc) test, which is basically wrong, as ANOVA is intended to test differences among three or more treatment levels. Also, given the small sample size (presumably N=3, which is the number of OTCs, for each of the two treatments), ANOVA assumptions of normality and homoscedasticity cannot be tested, therefore leaving room for using a non-parametric test for two independent samples (e.g. Mann-Whitney-Wilcoxon test). However, given that the dependent variables were measured for both treatments at 6 sampling dates (i.e. 30, 60, 90, 120 and 150 days of decomposition, plus initial values in undecomposed litter samples), using a one GLM model for each dependent variable (litter mass remaining, N, P, etc.) would be more appropriate, including fumigation treatment as a fixed effect with two levels, and litter age as a continuous covariate. See e.g. Bonanomi et al. 2013, Soil Biology & Biochemistry 56: 40-48, 2013 as an example of this type of statistical analysis applied to litterbag decomposition under different treatments.

Results
LL 118-125: The authors should re-analyze their data based on the comments reported above, and re-write the Result section accordingly.
LL 126-138: Please avoid wordy expression such as “the Olson attenuation index model”. Simply use “Olson’s model”. Remove “complete decomposition time”. The use of such indicator is a bit stretched: no litter decomposes completely, especially in a time frame as that tested in this manuscript. What the authors mean by “small differences (Table 3)”? Are those differences significant or not (easy to test by an ANCOVA model)?
About the rest of the result section, please see specific comments on Tables and Figures as follows:
Table 1. Besides comments on methods for assessing topsoil features described above, I would suggest to include information on additional features possibly affecting litter decomposition (e.g. texture, salinity, organic matter content, etc.). Moreover, as a table footnote specifies that soil data are mean of 3 replicates, standard deviations should be added. Given the very similar feature values, differences in topsoil properties between the two treatments are likely not statistically significant. This should be explicitly stated, at least in a footnote, since apparently it was not directly tested.
Table 2. Not sure that all this information is properly presented in its current form. I would suggest to move these data in one Figure with two panels, one for each treatment, showing time profiles throughout the observation period for O3 (both concentration and  AOT40) and microclimatic variables (T, RH). Such figure would be more informative of time-dependent variability of the experimental conditions, currently not clear from the Table. Among microclimatic variables, I would add light irradiance time profile (possibly as a further or supplementary figure). Indeed, irradiance effects interacting with O3 treatments might not be excluded in relation to possible litter photodegradation. These effects may also vary in relation to canopy cover variability at the litterbag placement sites, therefore I would be pleased to see irradiance time profiles (or at least canopy cover data) for each litterbag of each OTC.
Figure 1. Consider rewording the caption as “Leaf chemistry changes in Ginkgo biloba as affected by O3 concentration. Data refer to mans and standard deviation of the content of carbon (C), nutrients (N, P, K), C-to-N ratio, total phenolics (TP), condensed tannins (CT) and soluble sugars in leaf samples (N=3) of the target specie after 3 months of daily exposure to high O3 concentration (120 ppb), expressed as percentage of corresponding data observed at lower O3 concentration (40 ppb). Significant changes are shown (*, P < 0.05; **, P < 0.01).”
Figure 2. I am puzzled by the fact that the authors base most of their conclusion about O3 accelerating effects on litter decay on this figure. Indeed, the authors correctly report higher mass remaining in the 40 ppb treatment compared to 120 ppb after 150 days of decomposition (but see above about the need to re-analyze the data to properly assess statistically significant effects). However, it should be equally stressed that litter mass loss dynamics for the two treatments are not different over 120 days, indicating a large independence of decomposition from O3 for most of the observation period. Moreover, it is not correct to describe the difference observed after 150 days as an “acceleration” of litter decay under high ozone concentration. Indeed, in the 30-days period from 90 to 120 days, litter mass remaining in EO passes from 61.4% to 51.5% of the initial value, for a mass loss of 10%, while in the 30-days period from 120 to 150 days, litter mass remaining passes from 51.5% to 49.5%, for a mass loss of 2% (see supplemental raw data file Data1.xls). These data indicate that litter decay in EO slows down in the last 60 days. But what is more surprising, is that at the corresponding dates AA treatment shows a mass increase! In other words, the result does not show an increase in litter decay rate under elevated O3, but that litter material does not decompose, and even increases in mass, under low O3 concentration. Such observation indicates either a possible inclusion of exogenous material in the litterbags (microfauna? microbial biomass? other?) or a misprocessing of the sample. How the authors explain such unexpected result? This is particularly relevant, as the manuscript conclusions mostly rely on this point.
Minor comment: not sure about the asterisk indicating significant difference of litter C content in treatments after 150 days. Since standard deviation bars overlap, and sample size is low (3? 6?), standard errors and confidence interval widths should not be so small to produce a significant difference. Please double check after re-analyzing data according to the comment at LL 113-115 above.
Figure caption should be re-worded as “Mass remaining and C, N and P content in leaf litter of Ginkgo biloba daily exposed to either higher (HO, 120 ppb) or lower (LO, 40 ppb) O3 concentration for 150 days. Data refer to mean and standard deviation of N replicated litterbags for each treatment. Significant treatment-dependent differences are shown (*, P < 0.05; **, P < 0.01).”
Figure 3. As this figure is completely consistent with Figure 2, in terms of layout and content, I would merge the two figures into one, being a 2 x 4 panel. Caption of the new figure should be arranged as commented for Figure 2 above.

Discussion
LL 149-162: Subsection title, “composition” is singular. The sentences at LL 157-160 should be synthesized and moved at the beginning of the paragraph, in order to briefly resume your results prior to a comparison with previously reported evidence.
“Catalayud”, see same comment about the Introduction section. Delete “of chemical composition” at L 151, as well as “as in” and “in this study” at L 153. Use past tense when reporting previous observations (i.e. “demonstrated” at L 155). “…compared test in ambient air.” Change with “…compared to tests in ambient air conditions”.  The use of “obviously” when commenting results about N and K should be either justified, or avoided.
The comment at LL 160-162, in its current formulation, is too broad and obvious: of course experiments carried out under different conditions can produce different results. The interesting point here is to what extent (and direction) such conditions and results are different and how the different conditions could explain the observed differences in the results. Any mechanistic hypothesis?
LL 163-165: this is a repetition of LL 157-158. Could the authors extend and clarify their hypothesis about “litter stress resistance” at L 165-166?
LL 166-169: potentially interesting, but requires a more accurate formulation (e.g. what is intended for “leaf transformation mechanisms”?) and more logical argumentation. Btw, in the cited paper by Maurer et al., intraspecific variability in leaf N content, as affected by different fertilization regimens, affect resistance to O3 damage. Are there possibly cause-effect misinterpretations? Maurer’s observation is about a leaf trait (high N content) that (possibly) lessen resistance to O3 damage. Here the authors show that exposure to O3 causes higher leaf N content. Not exactly the same cause-effect relationships. Could the authors clarify?
LL 179-180: “some”, “certain”… either specify which, or delete the sentence.
LL 203-220. This paragraph suffers a substantial lack of reference to previous, well established knowledge on litter decomposition as related to C, N and lignin dynamics. It should include at least the argumentation suggested above when commenting the introduction (see comment at LL 34-44) and discuss the results in comparison to previous evidence (Parsons et al 2008, Baldantoni et al. 2011), which is apparently contrasting with the presented results. I suggest to start with a statement resuming your results (e.g. a reduced version of LL 205-209, but please avoid to explain the interpretation of correlation results). About the relationship between litter N content and decay rate, many precious references are available after Melillo (1982). Please have a look to  Berg, B., and McClaugherty, C., 2008. Plant Litter: Decomposition, Humus Formation and Carbon Sequestration, second ed. Springer-Verlag, Berlin, Heidelberg. As this source, being a book, may be possibly unavailable, a good discussion of C and N topics can be also found in the works by Bonanomi et al.: 2010, Plant and Soil, 331(1-2), pp.481-496; 2013 Soil Biology and Biochemistry, 56, pp.40-48, 2014 Plant Soil (2014) 381:307-321.

Conclusion
As a conclusive remark, the authors could comment on the relevance of their findings within the context of decomposition studies, including possible effects of increasing tropospheric ozone concentration on the C budget at ecosystem level, at least in the urban forest where the study site is located. In other words, would it be possible to tentatively estimate or at least discuss the increase in CO2 emissions due to litter decomposition as affected by higher O3 levels? Although decisions are not made based on subjective determination of novelty and interest of the study, adding such information would certainly provide a remarkable added value to the study, making it likely interesting for a broader audience.


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: EFFECTS OF XYLO-OLIGOSACCHARIDE AND FLAVOMYCIN ON THE IMMUNE FUNCTION OF BROILER CHICKENS
Review round: 1
Reviewer: 1

Output:
Basic reporting: 1. In most journals, animal mRNA names should be italicized e.g. Infγ (Italic, isn't supported on this webpage). I am not sure the formatting of PeerJ.
2.  It is controversial to use gene name Tnfα in chickens and some researchers don’t think chickens have one. If you search on NCBI, you will see Litaf with alias of Tnfα. You’d better to write it as “Litaf (Tnfα)” at the first time if you really want to use Tnfα.
3. At line 135, you wrote “mean± standard deviation”, but at Table 3 legend, you wrote “mean± SE”. Please keep consistent across your manuscript.
4. At line 73, please add “antibiotic” before “flavomycin”.
Experimental design: Generally, the experiment design is reasonable. The mRNA data at jejunum are weak because most microbial activities and host immune response are in the low gut (ileum, ceca and large intestine).
Validity of the findings: 1. The authors’ findings are relevant to poultry industry. It is consistent that using antibiotic flavomycin (FLA) improved body weight gain and reduced IgG and IL-2 (a sign of lower inflammatory status), but it is interesting that FCR wasn’t improved by FLA.

2. The discussion section is better be more concise and focus on what is closely related to your data in the result section. For example: from lines 176 to 180, the authors don’t have any data related to XOS degradation/digestion and it distracts readers from your main points.  From lines 194 to 211, the authors discussed various bacteria, SCFAs, XOS, and FLA. The authors don’t have any data on the bacteria. It would be better to use 1-2 sentences for this paragraph.

3. The authors suggested that increased body weight gain in XOS chickens was resulted from increased SCFAs and reduced immune response, but it wasn’t much convincing. Butyrate doesn’t improve body weight gain in healthy chickens as showed by Zhang et al., Br Poult Sci. 2011 Jun 1;52(3):292-301. The authors showed jejunal immune genes but the main site should be ceca in chickens. The IgG and IL-2 are comparable between CTL and XOS birds at 42 days of age, suggesting similar immune status. The author may read works about XOS and inflammation from Hansen et al. J Nutr. 2013 Apr;143(4):533-40, or other related papers.
Additional comments: Please improve your writing, below are some examples:
At line 37, “not adding AGPs to poultry feed” changes to “poultry feed without AGPs”
At line 62-63, “during which proteins are secreted that regulate immune responses by ..” changes to “where secreted proteins regulate immune response on.. ”
At line 82, “Broilers were reared for 42 days, were free to feed and drink water” changes to “Broilers were received feed and water ad libitum for 42 days”
At line 144, “supplementary” changes to “supplementing”


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: EFFECTS OF XYLO-OLIGOSACCHARIDE AND FLAVOMYCIN ON THE IMMUNE FUNCTION OF BROILER CHICKENS
Review round: 1
Reviewer: 2

Output:
Basic reporting: Abstract
Lines 29-30: “reduced their susceptibility to disease” This is not a proper interpretation for this study in which no disease challenge was used or specific disease diagnosed.

Introduction
The introduction could be improved to more clearly introduce the hypothesis and justify the experimental design.  There is a lot of general information included that does not necessarily strengthen the justification of the experimental design.  There needs to be more direct emphasis on the test compounds and their potential effects.  Why was flavomycin chosen as the compound compare with XOS?
Additional changes to the Introduction below:
Line 44:  Change “Therefore, it is possible that adjust…” to “Therefore, it is possible to adjust…:
Line 46: It seems that “prebiotic” should be “probiotic”
Line 48: “proved” should be “proven”
Lines 48-52:  The last 2 sentences of this paragraph could probably be combined and/or streamlined to improve flow and clarity.
Line 65:  The sentence beginning “When cytokines..” is poorly phrased and can be improved.
Line 76:  “Explain possible beneficial effects of antibiotics…” XOS is not an antibiotic, so this should be revised accordingly.

Materials and Methods
What breed cross of broilers was used?
Details on housing/cages of the broilers are needed.  This has implications on the outcomes of the experiment as stated below.
How were the compounds added to the basal diet? “On-top” of the basal diet?
Was there any mortality? If so, the amount should be reported.  Was the feed conversion ratio corrected for mortality?

Results
Please state the actual P-values throughout the results section.
Line 143: “the highest ADG occurred with supplementary XOS…”  The ADG of FLA and XOS were not significantly different, so this statement should be revised.
Lines 151-152: “Supplementary FLA or XOS had no effect on cecal butyrate compared to the control.”  This is incorrect – according to Figure 1, cecal butyrate was higher for the XOS group than for the CTL or FLA groups at both 21 and 42 d.
Line 164:  “jejunum TNFa mRNA level was significantly higher in the FLA group than in the XOS group”  This is incorrect – according to Figure 2, the TNFa was higher for the XOS group than for or FLA group at 42 d.
Tables and Figures
The actual P-values should be listed in the table and figure descriptions, rather than P < or > 0.05. The should be accomplished by having a P-value column to correspond with each measurement.
Table 1
-	“ME” should be defined and reported as a whole number.
Table 3
-	The table description states that values are means of 6 replications, whereas the number of replicates in the Methods section was identified as 5. Please address.

Discussion
The discussion is somewhat disjointed and does not accurately provide solid interpretations of the results, backed by relevant citations.  It should be revised extensively.  Some specific changes include:
The inherent fiber content and “fermentability” of the diet used should be addressed?  Would same results be expected in a diet with higher fiber ingredients that are commonly used in broiler production?
Line 167-176: Address implication of feed intake response in opening summary paragraph.
Line 196: “reproduction of probiotics” It is likely the authors meant beneficial intestinal bacteria rather than “probiotics” per se
Line 201-202: The effect of XOS on butyrate and acetate production has already been mentioned in Line 182-184.  All discussion should regarding XOS and be combined into one subsection of the discussion, and this discussion referenced when necessary in describing other effects.
Line 217: NFkB is a transcription factor, not a cytokine.
Lines 233-238:  The discussion of deoxynivalenol is irrelevant to the current experiment and should be eliminated.
Experimental design: Overall, the experimental design is adequate to test the effects of XOS or FLA on the selected outcomes, but several issues should be addressed:

It appears that the chickens were reared in battery cages with raised wire flooring – this should be clearly defined in the Methods section.  It is only listed in the abstract as “cascade” cages.

If chickens were in fact reared on raised wire flooring, the significant responses to flavomycin, and to some extent XOS, are surprising.  Much of the interpretation is based on potential changes in the intestinal microbiota, but the environmental exposure to bacteria of birds in wire cages is much lower and different than that for birds reared on litter floors under commercial conditions.  The authors should provide further info and potential implications of selected housing system in the interpretation of results.

It would have been interesting to see the plasma responses of the same cytokines that were measured by gene expression in the intestine.
Validity of the findings: The difference between the 1-42d ADFI values for FLA (98.36) and XOS (101.06) is only 1.5x the SEM; thus, it seems unlikely that this difference would be statistically different.  The same could be said for the 1-42 ADG differences between CTL and FLA (~1.3x the SEM).

There were no differences in FCR, only AFDI and ADG.  Indeed, the XOS group ate the most feed and gained the most weight.  However, the authors do not address this in their interpretations in the discussion sections.  The improved ADG with XOS supplementation is attributed to “biological function” (Line 173), when the data indicate it is merely a function of higher feed intake.  The potential implications of XOS on SCFA production and feed intake should be thoroughly addressed in the discussion.
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: EFFECTS OF XYLO-OLIGOSACCHARIDE AND FLAVOMYCIN ON THE IMMUNE FUNCTION OF BROILER CHICKENS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors made relevant changes.
Experimental design: The authors made relevant changes.
Validity of the findings: The authors made relevant changes.
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: GENETIC DIVERSITY AND CONNECTIVITY OF THE MEGAMOUTH SHARK (MEGACHASMA PELAGIOS)
Review round: 1
Reviewer: 1

Output:
Basic reporting: The paper presents the first data on the genetic structuring patterns of the megamouth shark (Megachasma pelagios) using sequence data generated from the mtDNA COI gene and a nuclear microsatellite locus (Loc 6). Indeed, the megamouth shark is rare and obtaining adequate samples for a population genetics study is challenging; however, inadequate sampling compromises the population genetics analyses and interpretation of results.

Overall, the paper reads well although it could do with re-structuring of several sections such as the Methods and Discussion. Kudos to the authors for a thorough literature review on shark population genetics, although it is disappointing to note that the author did not use the available information when selecting their molecular markers.

The figures provided by the authors are of low quality and need minor edits. The authors are encouraged to check their tables again.

I suggest that the paper is re-focused on the patterns of genetic diversity distribution, without making firm statements on the effects of biogeographic barriers on the genetic structure of the megamouth shark. This latter issue requires larger sample sizes for each studied sampling population.
Experimental design: I have many doubts on the adequacy of the sampling effort to achieve the set objectives stated in the last paragraph of the Introduction; “…to test (1) the effect of the East Pacific Barrier and closure of the Isthmus of Panama on the genetic structure of the megamouth shark and (2) whether it can travel from ocean to ocean.” With an overall sample size of 27 individuals the genetic diversity of the putative populations cannot be adequately represented.

Apart from issues surrounding sampling sizes per location, my criticism is on the choice and the polymorphism levels of the markers of choice, which are concerning. When sampling sizes are low, often, one screens several DNA markers to identify a highly polymorphic one. It is confounding that the authors would use a DNA Barcoding region as a marker of choice. Why? Moreover, why sequence a microsatellite locus?

Interestingly, the author constructed a table (Table 3) detailing examples of shark population genetics studies at a global scale, where the principal marker of choice was the mitochondrial control region. Within Table 3 the authors note that the control region and microsatellites were used in the present study; however, this is not true. Because of the low sample size and the lack of polymorphism of the both the COI gene (as to be expected intra-specific) and the nuclear microsatellite loci, the resolution is very low, which resulted in the failure to detect genetic differentiation.
Validity of the findings: Given the low resolution of the markers used in the present study several analyses that were done are flawed. First, the pairwise FST index cannot be conducted with such a low sampling population sizes. Second, the use of Bayesian Skyline Plot (BSP) analysis is only appropriate for informative enough data sets, thus the results obtained for such an analysis are misleading and SHOULD be omitted. Last, although no described in the Methods section, the use Tajima’s D test is also not appropriate, and the results are misleading. Frankly, I believe that all the interpretation based on all these analyses are in many ways ad hoc and speculative considering the intrinsic properties of the set of markers from which the results are derived. Also, it is not clear as to why the authors constructed phylograms using both Bayesian and Likelihood approaches. The logic escapes here.
Additional comments: Line 96: This does not make sense, unless a same individual was sampled more than once, and the duplicates removed. Please correct this.

Lines 155-157: Likewise, the mathematics does not check out here based on Line 96. Please amend accordingly.

Line 201-236: I fear that this entire section is detrimental to objectives of the study. All the discussion trying to link the lack of genetic structure with the ineffectiveness of the closure of the Isthmus of Panama in restricting gene flow is, in my opinion a bit confusing and speculative.

Lines 237-251: This section is not necessary, and it is also detrimental to objectives of the study, and I encourage the authors to remove it.


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

Input:
Title: GENETIC DIVERSITY AND CONNECTIVITY OF THE MEGAMOUTH SHARK (MEGACHASMA PELAGIOS)
Review round: 1
Reviewer: 2

Output:
Basic reporting: 1. Make sure to double check English language use, although most of it is fine some of the following corrections are slight improvements on wording to strengthen points and English language use, while others might help clarify

Line 22: You have mentioned no other proxies for indirectly measuring gene flow, consider removing another “This approach provides a proxy to indirectly measure gene flow between populations.”

Line 39: Consider rewording “… only about 120 specimens have been caught or documented.”

Line 40-41: “The only official published record is for Japan (Nakaya, 2010), which documents 40 records …”

Line 43: “The latest record is No. 117, from Komodo, Indonesia, in July 2017.”

Line 44: sources may be a more appropriate word than documents

Line 49: replace during with from

Line 49-52: consider breaking this up into multiple sentences to make the point more clear, it becomes a little confusing.

Line 56-58 Consider rewording “In addition, due to the scarcity and vulnerability of these sharks, satellite tagging has not yet been feasible. Therefore, information about their horizontal…”

Line 64: has frequently been found or is frequently found

Line 68-70—Sounds a little awkward, consider rewording. Also, maybe specify that Taiwan instated a National Plan of Action concerning sharks in 2006

Line 93: GenBank

Line 111: remove second gene

Line 123-124: “Two cox1 gene sequences of individuals from Indonesia (EU3938905) and Puerto Rico (KY392958.1) were downloaded from GenBank. In addition, a Loc6 sequence derived from a Japanese specimen was downloaded.” Or something similar, currently it

Line 130 and 131: in text citation format changes

Line 164: replace notes with nodes

Lines 181-184: Awkward phrasing... consider revising

Lines 202-203: Consider restructuring this sentence

Line 222: include Rhincodon typus

Line 223:  remove in “…showed no genetic structure within or between oceans, except the whale shark which showed…”

Line 226-231: Maybe divide into two sentences. “…gene haplotype found in the Pacific. This indicates that the…”

2. Figures and Tables are nice, but could use some improvement to adhere to standards. At this time the quality of some is lacking, and descriptions can be improved.

Figure 1: This figure could use some improvement, consistency with labeling would help, either do specific locations or country where they were caught, the combination could become confusing (for example have all of the labels as Japan, Indonesia, Mexico, Taiwan, Puerto Rico). Additionally, due to the focus on the Kuroshio region, it may be helpful to highlight this area on the map. The map in general could use some improvements, for the projection being used, the scale would not be consistent throughout the map.

Figure 2: Figure caption needs to be more descriptive, it provides no information about the figure, although there is description within the text this should be stand alone. Please specify that which tree and network A and B are, etc.  In addition, both the PDF version and eps file are blurry and difficult to read. Specify which order your values on tree A are in ML/BI or BI/ML

3. Make sure to double check the references.  Line 364 is not correct, and I think Line 376 might be part of that reference.
Experimental design: 1. Research Question
I think the first objective is very reasonable and that the paper covers results from this objective/question well.

However, Line 91: objective 2--- I’m not sure this is the best way to phrase the question, because you aren’t really examining if they can move from one ocean to another, you are looking more at genetic connections and structure between the ocean basins. Although this may suggest that they can move from ocean to ocean, without the tracking data this isn’t really the point you working towards. Consider rephrasing this.

2. Methods: It might be useful to define what differentiates the juveniles, sub-adults, and adults. What qualifiers were used in order to make this determination? This is especially relevant since this is brought up in the results.
Validity of the findings: For the majority, the findings are good and provide insight into popgen of a poorly understood species.

1. For the assessment of catch information: It is stated that megamouth sharks were generally caught between April and July, however there is no mention of when the fishing effort occurs. Is there fishing year round or is there only effort during that season? It may be out of the immediate scope of this paper, but knowing even rough catch per unit effort or if there is fishing in the areas where they are being caught year round may be helpful and would help to support your seasonality hypothesis. I completely understand that you may not have this data, but it may be worth considering to help strengthen your point about seasonality.

2. The conclusions are well stated. I think that there is an air of caution and the limited sample size's potential to bias the results and limitations of the markers are expressed.  However, this is once again where I feel that the second objective listed in the introduction is left out, and would warrant it either being removed entirely or stated in a different manner and discussed more.
Additional comments: I commend the authors for making the most of a limited data set and limited samples, I think it is a useful look into megamouth sharks that has the potential to be built on further throughout time.

However, I am concerned about the reliance on “sharkmans-world.eu” website for some of the information concerning occurrences of these sharks. I realize it may be the only source that comprehensively documents the occurrence of the sharks, but I would be more comfortable with more references to peer reviewed documentation (for example the listing of the first 14 occurrences in Amorim, Arfelli, & Castro, 2000) . Although it is considerably more work, it may be better to use previous peer reviewed reports and the direct sources of the sightings and create a supplementary table that creates a comprehensive source within this publication. In my opinion it would be a more compelling and trusted source than the webpage and would most likely be highly used in the future as well.


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: GENETIC DIVERSITY AND CONNECTIVITY OF THE MEGAMOUTH SHARK (MEGACHASMA PELAGIOS)
Review round: 2
Reviewer: 1

Output:
Basic reporting: The paper is much improved and focuses on reachable objectives given the small sample sizes.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: I congratulate the authors for their erudite contributions to the field of shark genetics and, providing the first and valuable genetic knowledge base on the megamouth shark.


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: GENETIC DIVERSITY AND CONNECTIVITY OF THE MEGAMOUTH SHARK (MEGACHASMA PELAGIOS)
Review round: 2
Reviewer: 2

Output:
Basic reporting: The paper reads quite well and improvements have been made. However, I still think some of the English could stand to be improved, especially within some of the areas where additions and changes have been made.

Line 39: Consider getting rid of “tracking”
Line 45-47: Consider rephrasing to improve the flow.
Line 82-83: Sounds awkward, consider rewording
Line 85-86: “…collected from Baja California, Mexico; 27 tissues samples from Hualien, eastern Taiwan; and published…”
Line 198: consider rephrasing to either “… genetic diversity to that of other sharks…” or “…genetic diversity with other sharks…”
Line 212: “which undergo” instead of undergoing
Lines 227-229 are awkward, consider rewording.

Line 188-189: citation for Red List assessment is missing (also not listed in the reference list). Should be Simpfendorfer, C. & Compagno, L.J.V. 2015

Supplementary Table: A great improvement, and useful resource. However, it feels out of place when mentioned in Lines 145-147 (as part of the results). Perhaps add reference to the supplementary table early on in the introduction (around line 47) when the resources sighted there are heavily referred to.

Figure 1: This has also improved; however the scale bar should be removed, it is not accurate for the map projection being used, and in this case is not necessary.
Experimental design: This area has had the most improvement. I think the new direction was a great suggestion and was beneficial to the manuscript.

The new more reserved analysis of diversity and connectivity is more appropriate.
Validity of the findings: This has also improved, although I think it is important to continue to use great caution and not try to oversell the results too much, considering markers used and small sample size. In the conclusions these limitations are clearly pointed out. I am concerned about Line 217-218, I think it would be more beneficial to cautiously suggest the possibility of connections between the oceans. The current sentiment may be a misleading or slightly overstated.

Line 201-207: I find this section a little worrisome. Although the table of comparisons to other COI studies is interesting, the direct comparison to the A. pelagicus study seems to be a stretch. I am especially considering the drastic difference in sample size and number of sampling locations between the two studies. This caveat should be clearly explained or comparisons should be cautiously made. Also, the final sentence is not entirely clear and could use a bit of work.
Additional comments: Kudos to the authors for the great improvements that have been made.

Please make sure to double check formatting within the tables and figures, there still seem to be some minor details that need to be attended to. In Table 3 one of the scientific names is not italicized (whale shark). In Table 1, Date of Collection would sound better than Date of Collecting.

The use of the Florida Natural History Museum is a vast improvement over the previous website.


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: EXPRESSION PATTERN OF WIF1 AND Β-CATENIN DURING DEVELOPMENT OF ANORECTUM IN FETAL RATS WITH ANORECTAL MALFORMATIONS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Tang and co-workers have submitted a manuscript dealing with the expression and potential function of Wif1 in the anorectal region of rats. Wif1 is a Wnt-signaling regulator (suppressor) and known to play a role in mammalian development. Its role in anorectal development is of general interest, particularly if it plays a role in disease development, such as ARM (anorectal malformations), a relatively common disorder in human newborns.

The manuscript is presented in a relatively clear and logical manner and deals with a topic of relevance. Regarding the content, it is relatively minimalistic, showing the expression pattern of one factor (Wif1) in normal versus ARM rats at critical developmental stages. The authors show that Wif1 is dysregulated in ARM rats and from there, conclude that Wif1 could be involved in ARM pathogenesis. However, no effort is made towards deciphering how Wif1 might possibly be functionally involved, leaving the conclusion speculative without any empirical data to support a causal rather than effectual relationship between Wif1 downregulation and ARM phenotype.

A previous study by another group has already reported on a possible involvement of Wif1 in ARM pathogenesis, using mouse models to not only show dysregulated Wif1 expression, but downstream consequences where the Wnt/b-Catenin signaling pathways is disturbed. Although the present study is using the rat as a model, there is little to suggest that Wif1 function in anorectal development is in any way different between the two closely related rodent species. Thus, this manuscript only corroborates the finding that Wif1 is dysregulated in ARM, but does not corroborate additional functional findings, nor provide any additional knowledge about Wif1 function.
Experimental design: see 'general comments'
Validity of the findings: see 'general comments'
Additional comments: Specific Comments:
1)	The language and structure would benefit from further editing.

2)	Additional experiments should be performed to support the  view that WIF1 is functionally involved in urorectal development or ARM pathogenesis, not least since expression pattern and function of Wif1 has already been shown in comparative tissues in mice. Other factors should be analysed, such as Wnt/b-Cat signaling (as a minimum).

3)	Introduction should introduce properly what is known about Wif1 in anorectal development, and ARM, referring particularly to the paper by Ng et al (2014). Presently, the text reads that   “…dysregulated Wif1 expression caused septation defects (Ng et al 2014). These results suggest that Wif1 is required for urorectal development. However, the expression pattern of Wif1 has not been described previously in the embryogenesis of rat ARMs.” This is incomplete and misleading information.

4)	The discussion generally lacks references, makes unspecific statements and contains a lot of repetition

5)	Please use standard nomenclature for gene and protein names

6)	Materials & Methods: the group of rats that was ETU-free saline is referred to as “control” whereas it is referred to as “normal group” in the remainder of the paper. Use same denomination throughout.

7)	For antibodies, the catalogue numbers should be listed, as there are more than one available for WIF1. Figues also appears to be counterstained. This should be described in the protocol (as well as explained in Fig legends). Also list CAS number for 2-imidazolidinethione.

8)	Refer to Table 1 in Mat & Meth section where number of embryos for different experiments are reported.

9)	In line 75 it is stated that “One third of the embryos were fixed” while line 79 states “cloaca/hindgut of other specimens was dissected” does this then refer to the remaining two thirds? Please clarify.

10)	Gapdh was used as reference gene in qPCR experiments. Data confirming Gapdh is stably expressed under the experimental conditions should be provided, or a reference to other work having shown so. Further on qPCR results. Fig 6, it is unclear what samples the significant asterix denotations refer to. As it is depicted, one would assume controls at GD14 and GD15 are significantly upregulated in relation to control GD13, whereas I believe the authots mean to show difference between control and ARM samples at single time points. If so, the atserisks should be over ARM bars, or better still, with a line connecting the two samples the asterisk refers to.

11)	Western blot data in Fig 5 is not overly convincing, but ok when taking observations from IHC into account, But as commented upon under qPCR fig (above), the way the asterisks are shown, it is unclear (or wrong) what samples the statistical difference refer to. This must be corrected.  In Figs 5-6, also include statistical method used and what significance/confidence level the asterisk refers to. Also, the supplementary blots only show the cropped images. The full blots should be provided.

12)	All Figures: legends should contain more information, such topic/main finding, all marker stains, and n-numbers, in addition to what is already included.

13)	Lines 156, 160-161 & Figure 6: What is meant by “Optimal/optimum levels” or “Minimally expressed”?

14)	Line 155 & 161: GD13 should be corrected to GD14

15)	Tables 2 and 3 are redundant.


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: EXPRESSION PATTERN OF WIF1 AND Β-CATENIN DURING DEVELOPMENT OF ANORECTUM IN FETAL RATS WITH ANORECTAL MALFORMATIONS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The authors used ARM animal model to study the potential relationship between Wif1 expression and ARM. They found both the protein and RNA level of Wif1 were reduced in the ARM rats and concluded that Wif1 may play a role in the development of ARM. Overall, the text is fluent and this work is interesting in regard to the expression pattern of Wif1 in ARM rats. However, I still have some concerns.
1. The authors used t test to analyze the data. However, two-way ANOVA with Post-hoc test is more appropriate. The authors should re-analyze the data.
2. The authors tested the protein and RNA level of Wif1 in the cloaca/hindgut. They should also test the Wif1 level in other tissues. Since the authors used ETU to treat the pregnant rats, it is important to know whether the reaction of Wif1 is universal in the embryos or specific in certain tissues.
3. Since the expression level of Wif1 was changed in the ARM rats, the authors should test at least one downstream gene of this signaling pathway to further demonstrate the disruption of the signaling.
4. I have a hard time to evaluate Figure 1,2,3,4. It seems to me that there is no reduction of Wif1 in ARM rats. Could the authors use arrows to indicate the differences between the normal and ARM images?
5. As the authors mentioned in the text, previous study has already showed that Wif1 may play a role in the development of ARM (Ng et al. 2014). The authors should compare their studies and discuss more carefully.
6. Line 209-213: I think it is inappropriate to make such a conclusion in the text since the authors only find the expression change without any further investigation.
7. The images in Figure 1, 2, 3a, 3b are different from the raw data that the authors uploaded. They need to clarify this.
8. It is hard to evaluate the raw data of westernblot. The authors should upload the raw data of full-gel images with the protein ladders and proper labeling.
Experimental design: no comments
Validity of the findings: no comments
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: GENETIC VARIATION AND DNA FINGERPRINTING OF DURIAN TYPES IN MALAYSIA USING SIMPLE SEQUENCE REPEAT (SSR) MARKERS
Review round: 1
Reviewer: 1

Output:
Basic reporting: 1.	There is no problem with professional English language writing in this manuscript.
2.	The abstract clearly summarized the results and addressed the importance of this study. Please add one or two sentences for the content in discussion, which is also a crucial part of your manuscript.
3.	In the introduction, the author described basic information about durian, the need and the current progress for genetic characterization for durian. The author also talked very briefly about the objective and significance of this study. The literatures are well referenced and relevant.
Detailed comments on introduction for improvement:
There are few information the author mentioned in latter part of this manuscript that could be moved into introduction. Also, I would suggest the author to add a few information for clarification.
1)	Move line 257-260 to introduction paragraph one.
2)	Introduce durian’s reproduction biology characteristics, such inbreeding or outbreeding, clones or population.
3)	Introduce durian’s polyploidy-level and no. of chromosomes or even genome size (if available).
4)	Introduce if there is any next generation sequencing project done for durian (There is a draft genome just published one month ago). If no, the author could claim that the current study is the most advanced study in durian genetic variation study to date. If yes, the author could say that the next step is to utilize the available next generation sequencing data to develop more genetic markers. Either way, the introduction would be more informative.
5)	It would be better if the author can summarize what aspects will be discussed in the discussion at the end of introduction.
4.	Tables are relevant, well made and footnoted.
5.	Raw data are supplied.
Experimental design: The current research is original and within scope of the journal. The research questions are well defined, relevant and meaningful.
Detailed comments for improvement:
1.	The sample size (27 durian type) and number of makers (7 SSR markers) are small. It is acceptable in this case as the genetic resources for durian is limited. I would suggest the author to point out this situation either in introduction or discussion. This will help your research to stand out.
2.	The author should describe if the varieties you characterized are the same or different from Sales (2015). This will define the uniqueness of the study.
3.	The author should address the representativeness/importance of the varieties you used in this study. How much of the area in Malaysia are using these varieties.
Validity of the findings: The fact that only 7 SSR markers can distinguish 21 durian type is significant. The author listed detailed information of the DNA fingerprinting which is very helpful for understanding the context. Also, this validated the conclusion.

The discussion covered a various topics that related to this study, which makes this study more meaningful. The discussion also referred examples in other crops, situations in other countries and compared with recent studies, which makes the context well supported.

Detailed comments for improvement:
The author didn’t describe the morphological difference of these 27 durian types. Do they look similar or different? Does the SSR markers help to distinguish (or corresponding with) different phenotype?
Additional comments: Generally, this study is valid and significant. The manuscript is well written. A few improvements are needed as suggested above.


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

Input:
Title: GENETIC VARIATION AND DNA FINGERPRINTING OF DURIAN TYPES IN MALAYSIA USING SIMPLE SEQUENCE REPEAT (SSR) MARKERS
Review round: 1
Reviewer: 2

Output:
Basic reporting: Literature references:

Previous studies in this genus using different genotype markers are properly cited and compared. However, another paper (Santoso et al. Development of Simple-Sequence Repeats Markers from Durian (Durio zibethinus Murr. cultv. Matahari) Genomic Library) that also used SSR as the genetic marker in Durian just came out. Would be nice to add it to the background section (Line 77-80) .

Grammar:
This is minor - a subject-verb disagreement that I’ve spotted:

Line 58: While the ease of application of these markers make them.. (make -> makes)
Experimental design: No comment
Validity of the findings: No comment
Additional comments: In this study, Siew et al. have investigated seven SSR regions in different types of Durian. With the genetic data in hand, they were able to distinguish 21 different SSR combinations from 27 samples, and genetic diversity was observed within the same Durian species. This manuscript is well-prepared, and the results are interesting regarding to plant identification as well as other implications.

The authors do a good job presenting the rationales of using SSR as a DNA barcoding tool in Durian. The question is well defined, and the methods are standard and acceptably presented. Molecular cloning procedures, details and primers are provided for others to replicate the same experiments.

Minor point:

SSR variations were observed in the same type of Durian that were obtained from different orchards (Line 167-182), which is potentially interesting. Genetic distance plot, such as dendrogram or PCA analysis, will help to clarify the genetic relationship between types and geographic locations.


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: FIRST INSIGHT INTO THE VIRAL COMMUNITY OF THE CNIDARIAN MODEL METAORGANISM AIPTASIA USING RNA-SEQ DATA
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: This study aims to assess the virome of the model organism Aiptasia using previously published RNA-Seq data using an established pipeline to mine for viral sequences.  The authors describe a diverse but stable viral community across different stages of symbiosis.

One important area to address here is that the original RNA-Seq data was not obtained with the goal of targeting the viral fraction of the holobiont. Only a small fraction of the <3% of data, which was not host or endosymbiont, was viral. The inclusion of a discussion of a more targeted approach in the pre-sequencing preparation to capture the entire virome fraction would be appropriate. The absence of bacteriophages and members of the Caudovirales (which should be italicised also) is very surprising and I think this reflects the approach taken to screen for viruses in this dataset. This is really my biggest concern about this study and its findings. Bacteriophages of the Caudovirales, as the authors note here, are commonly the most abundant members of, for example, the coral holobiont, so I think this result (i.e. their complete absence here) is somewhat alarming. If bacteriophages are truly absent from Aiptasia (which I cannot really accept) the final sentence of the discussion is not fully the case either, as phages have been implicated in coral health in terms of controlling pathogenic bacteria. I feel this aspect of this study requires more detailed discussion and further justification of the methodological approach used is needed. Is there any additional data to explain this finding? Are phage signatures present before filtering/binning of eukaryotic sequences?

This approach also potentially misses retrotranscribing viruses that may be integrated into the discarded eukaryotic host genomes. Can the authors include some discussion on this point?

It would be relevant and of interest to include discussion/speculation on who the hosts for Partitiviridae may be within the coral holobiont.

Discussion of the species and clade of Symbiodinium used here in terms of any previous studies of the viruses infecting this alga should be included as this is relevant to the changes observed across stages of symbiosis.

More  discussion of the prevalence of giant virus-like sequences detected should also be included based on their detection in previous studies.

Picornaviridae have been found to be abundant and diverse in seawater (see Culley et al 2003) and inclusion of the results of such a study would be relevant here.

Fungi are members of the coral holobiont so in line 297 I don’t believe it is unexpected to observe viruses that target fungal hosts in the data. Additional information on what is known of fungi in the holobiont of Aiptasia or other related invertebrates would help in the discussion.
Additional comments: Minor comments

Ln 318 – Picornaviridae is misspelt
Ln 353: ‘receives’
Ln 363: Herpesviridae is misspelt


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: FIRST INSIGHT INTO THE VIRAL COMMUNITY OF THE CNIDARIAN MODEL METAORGANISM AIPTASIA USING RNA-SEQ DATA
Review round: 1
Reviewer: 2

Output:
Basic reporting: The authors mined previously published RNA-Seq data from Aiptasia with and without symbionts to investigate the viral community associated with this holobiont system, including whether the assemblage changes depending on symbiotic state and/or if the holobiont has a core virome regardless of symbiotic state.

The article is well written, the methods section was easy to follow, and I fully support bioinformatic data mining projects. However, my main concern for this manuscript is that these data sets limit how well the authors can address question they are trying to answer. I highly recommend the authors reconsider the scope of what they are actually able to report, given the limitations of the data sets.
Experimental design: The generation of the RNA-Seq data sets started with a Trizol/chloroform step for total RNA extraction, which was followed by an mRNA selection step to purify it from the total RNA in the samples. Unfortunately, both of these steps interfere with sample processing with respect to viruses. Chloroform does not interact with viruses uniformly, which likely changes how the natural viral assemblage is represented for all downstream sample and data processing. In addition, the top Trizol/chloroform layer is recommended for RNA collection, which greatly reduces the viral community those only active inside a cell. Finally, the mRNA selection step would miss any viral transcripts that do not have a poly-A tail, which include most (if not all?) bacteriophages. The analysis doesn’t return a single phage sequence, even though 90.97% of retrieved sequences from CLARK classification were annotated bacteria.

Except for the poly-A selection, none of this is mentioned in the manuscript...

Given that these data are now limited to select eukaryotic viruses, it would be very interesting to see how the list of viral taxa from this data set compares to a list of euk viruses known to have mRNA-poly-A transcripts.

I am also curious how these data annotate against a CLARK database that includes vertebrate sequences, as a test for possible contamination. Since vertebrate genomes are a bit messy, I realize that this might recruit false positives from the current virus annotations. However, as the authors report several vertebrate viruses, this could be informative and relevant.

Also, given that the relative abundance of viral groups were stable across symbiotic state, I would like to see intra/inter variance values reported. Several of the “within replicate” values in Table 1 were somewhat variable, and it would be nice to know if that was significant or not.

I am not up to speed on RNA-Seq data available for the associated Symbiodinium strain, but the data sets seem incomplete without accounting for the viral assemblage that will be present in those cultures, especially since they were added directly. E.g., there didn’t seem to be any attempt to “wash” the symbionts in virus-free seawater. The freshly hatched nauplii are of less concern, as they are not necessarily long-term member of the holobiont, but they are still a potential inoculation source.
Validity of the findings: Given the caveats in the experimental design, I would recommend revising the results section. Aside from that, I have a several additional comments.
First and foremost, I have reservations about the “stability of the viral community”. This can only be stated for the viral representatives present in these limited data sets. For example… Based on previous Symbiodinium/dinoflagellate virus work, I was very surprised to not see Phycodnaviridae represented. Unfortunately, we cannot know if their absence is a true negative or if those sequences were selected against during sample processing. All of this should be mentioned in the text, including a discussion on phycodnaviruses citing (for example) Correa 2013 and 2016, Weynberg 2017, Wood-Charlson 2015 (most already in ref list)

The authors find several viruses that annotate as Picornavirales, which they list as having vertebrate or plant hosts. I would caution against this. The order Picornavirales is vast and has been difficult to characterize in marine systems. Therefore, assignments to a particular family within the order could be a database issue. See Culley et al 2014 mBIO.

Re: Figure 2. I am not totally sold on this figure. The “increase” category includes representatives that increase between apo/partial but decrease again in the fully symbiotic state, indicating a temporary association. There is not enough evidence to relate this to the establishment of the association, nor can we know if they were only coming from the addition of the symbiont cultures (which are never axenic). Either way, they definitely don’t seem to be present (within the limits of these data) in the established symbiosis, so “increase” is misleading. In addition, B shows “decrease” but none of these are significant. This figure does not show the “other” category, where 2 were reported to have significant trend (Supp Tbl 2 – bold). I would like to see these data visualized – what pattern do they fit?

With respect to reporting, as mentioned above, I recommend the authors reconsider the scope of what they are actually able to report, given the limitations of the data sets.  For example, line 273-274 should be restated to reflect that this manuscript “provides a highly selective and incomplete view of the viral community”. I realize that greatly diminishes the potential important of this work, but it is a caveat that should be very transparent.
Additional comments: Line 223 – should this be Supp Table 2?
Line 254-256 – says “includes the four most” but only 3 are listed