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: PHYLOGEOGRAPHY OF DICTYOTA FASCIOLA AND DICTYOTA MEDITERRANEA (DICTYOTALES, PHAEOPHYCEAE): UNEXPECTED PATTERNS ON THE ATLANTIC-MEDITERRANEAN MARINE TRANSITION AND TAXONOMIC IMPLICATIONS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The article is generally well written. I just found a few mystakes which are annotated in the attached pdf. Find also annotated in the pdf suggestions for rephrasing.

References used are complete and the background provided is clear and sufficient to show the state of the art of the topics addressed. The article clearly contributes to the topic with its results, and in fact proves false a previous hypothesis, so they are no doubt relevant to the topic.

Introduction
Line 40: I suggest rephrasing, as genetic data cannot be “a step” per se: “the increase of molecular genetic data has been a key step to achieve a better understanding of biodiversity, including fields like taxonomy and systematics”. If not, replace has by have as data is plural.
Paragraph 1: consider including the concept of integrative taxonomy as it claims for taxonomy to reflect evolutionary patterns (Stuessy, 2009, Plant Taxonomy, second. ed.; Schlick-Steiner, 2010, Annu Rev Entomol, 55: 421–438)
Line 61: I suggest “recolonized” instead of “reinvaded”
Line 90: “based on a fairly limited inter-population sampling for these taxa”: the sampling does not seems too low compared to the present study: 18 sites for D. fascicola and 7 for D. mediterranea (for the psbA analysis, table S1 of the cited study). Please specify what is meant with this statement. How is your sampling significantly better?
Line 94: I suggest rephrasing: “as the only example of Dictyota species having evolved from a common ancestor and also sharing their distribution along the Mediterranean Sea and the Atlantic Ocean” -> “as the only example of two sister Dictyota species occurring along the Mediterranean Sea and the Atlantic Ocean”. And please consider moving it as the last sentence of paragraph
Line 107: I suggest rephrasing: “we discuss the contribution of our results to the knowledge about the Atlantic-Mediterranean transition…”

Material and Methods
Line 156: cite PopART: Leigh, JW, Bryant D (2015). PopART: Full-feature software for haplotype network construction. Methods Ecol Evol 6(9):1110–1116.

Results
Line 199: uniform the paragraph please. When describe the parameters you can choose whether to put the values in brackets or not, but do the same for all parameters. I would suggest to at least write the symbol used in the table, while the values are probably not necessary since they are easy to reach in Table 2.
Line 201: nucleotide diversity is average number of nucleotide differences per site, so in my opinion a difference of 0.0008 should not be considered. I would be better to say that the 2 regions showed very similar nd.

Population codes: using simpler population codes would help the reader (e.g. M1.1: D. mediterranea pop 1 individual 1).

Figure 2: Please consider to invert the tree in B, so that the terminal nodes are on the left and the root on the right. This would help to graphically compare the two trees. In this case the pairs of incongruent individuals could be linked with lines to make it easier to find them.

Figure 3: See annotated comments in the pdf. I suggest to make a loop between M13 and M10, or any other graphical resource to avoid the crossing lines. Indicate the number of unsampled intermediate when there are more than 4-5 would be helpful. Same fig S1.
Experimental design: Sampling is said to be one of the majors improves of the study. Although sampling is indeed relatively high, sampling sites are often extremely close to each other and the sampling of Tronholm et al 2010 seems to be not that lower (18 sites for D. fascicola and 7 for D. mediterranea). Sampling for phylogeographic purposes should be planned in a way that the distribution of the species analysed is representatively sampled. I am however aware that it is not always possible to plan a perfect sampling campign. However I would suggest adding the D. mediterranea sequences from the Adriatic (from Tronholm et al. 2010) to partly fill the sampling gap for this area.

Considering that the sampling is partly overlapping with that of Tronholm et al 2010, it seems that a more efficient approach would have been to use their published sequences and sample the extra populations sampled. Thus resources could have been directed to sequence some more regions, which as the authors state (line 239), is one of the weaknesses of the study. This would have increased the relevance of the present study. However, I am aware that this would be a different study and therefore I do not ask for any change in this direction.

To improve the replicability, it would be helpful to use an aligning algorithm at least for the first step (e.g. as implemented in Clustal), and thus minimize the adjustments made by hand.

I suggest an additional phylogenetic ML analysis to complement the Bayesian Inference. That will most probably not change the essential results but obtaining redundant results from two different approaches increase the robustness of the results.

I agree on not concatenating the two alignments, since they are loci that undergo independent evolution. I suggest however to mention it and cite some of the revisions discussing the topic (e.g. Degnan & Rosenberg, 2009, Trends in Ecology and Evolution, 24(6), 332–340). It would also be useful a short description of the properties of the two genomes analysed here (ie cpDNA and mtDNA), such us inheritance pattern (see e.g. Peters et al., 2007, Europena Journal of Phycology, 39, 235-242).

PopArt default settings: since not only the most parsimonious paths are considered, it would be useful to describe these settings with more detail.

Line 163: n=18 is a value you choose or it is fixed by the program developers? please argue why using 18 if you chose it.
Validity of the findings: The manuscript is a significant contribution to the systematics of the seaweed species studied and to the biogeography of the Atlantic- Mediterranean transition. Despite the existence of two comprehensive phylogenetic studies on the genus Dictyota, the current paper contributes with a wider sampling and a zoomed perspective that contradicts a previous hypothesis on the biogeography of the 2 species, although it could be better explained how the current study overcome the previous ones (see my previous comments on sampling).

The authors in general interpret well their results, propose hypotheses to continue the study of the topics raised and properly stress the limitations of their own study, avoiding too much speculation. However, a limited sampling and the use of only 2 molecular markers challenges the prevalence of the results obtained here.

Furthermore, I suggest to add an alternative interpretation of the phylogenetic results in the discussion. Reading the section: “Systematic and taxonomic implications”, it seems that there is no congruence at all between taxonomy and the phylogenetic trees obtained. Instead, the major pattern shown in the figure is a monophyletic D. fascicola nested in a paraphyletic D. mediterranea. This pattern could be reflecting an anacladogenetic speciation process, which often renders an ancestral paraphyletic species. Several studies discuss this type of speciation and the need of accepting paraphyletic taxa (e.g. Hörandl, 2006, Taxon, 55: 564–570; Crawford, 2010, Taxon, 59(5), 1413–1423; Anacker & Strauss, 2014, Proc. R. Soc. B, 281, 20132980; Kuchta et al., 2018, Zoologica Scripta, 47(3), 285–299; Smith et al., 2018, Plant Systematics and Evolution, 304(4), 511–519). This hypothesis should be also considered and discussed by the authors, additionally to the already discussed hypotheses of a complex of cryptic species and a single polymorphic species.

Although it is true that there are a few individuals of both species that are nested with individuals of the other species, this is more an exception than the rule. A bit more discussion on this specimens should be conducted. Some of the possible lines of discussion are:
•	Misidentification: as said in line 83 the two species are difficult to identify, have the identity of the “misplaced” individuals been rechecked? In this case it would be good to mention in to rule out the possibility of misidentification.
•	Putative hybridization is vaguely introduced in lines 235-238, but it should be more developed. What kind of conflicts are the authors talking about? Were there intermediate individuals? Were they included in the molecular analyses?
•	e.g.: ind M-Bany2 is placed with D. fascicola. In the same population the 2 species co-occur, and the rest of D. mediterranea individuals from Bany are far from M-Bany2. I would therefore suspect hybridization or misidentification in this case.

Although it is not necessary to speculate the reasons for every misplaced individual, some explanations are necessary. Furthermore, a short discussion on the possible source of incongruence between the two markers is also necessary. I appreciate the final remark of the section, indeed nrDNA and morphological analyses might be needed to clarify the conflict pointed out by the authors.

Some more specific comments are listed below:
Section “Phylogeography and diversification within D. fasciola – D. mediterranea complex”
Lines 256-257: or could not, take into account the uncertainty of dating. The HPD ranges approx. from 10Ma-4Ma in Tronholm et al., 2012.
Lines 256-265: the possibility of survival for seaweeds in the Mediterranean during the MSC has to be discussed, as it has been stated in the introduction that the most accepted hypothesis is no survival in the Mediterranean. The authors have to discuss how their results contradict this hypothesis and refer to other studies reaching similar conclusions.
Line 262-265 and 268-270: Although I admit that colonization of the Atlantic Ocean after the MSC seems feasible, remember 1) the uncertainty of dating in Tronholm et al. (2012) and 2) that your phylogeny is not dated and you cannot therefore infer when the colonization of the Atlantic occurred. The data shows clearly that it had to occur later in the diversification of the complex, but not exactly when.
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: PHYLOGEOGRAPHY OF DICTYOTA FASCIOLA AND DICTYOTA MEDITERRANEA (DICTYOTALES, PHAEOPHYCEAE): UNEXPECTED PATTERNS ON THE ATLANTIC-MEDITERRANEAN MARINE TRANSITION AND TAXONOMIC IMPLICATIONS
Review round: 1
Reviewer: 2

Output:
Basic reporting: In general, the manuscript is clear, well written and structured. However, I think there is an overuse of sentence connectors such as indeed, this way and in this context. The use of many connectors gives the perception of a slow pace and a bit distracting. I would recommend to use them only when it is necessary to emphasize an idea.

The references are sufficient to explain the problem to be addressed and provide a good background in the field.

The results obtained are relevant and addressed the objectives of the paper.
Experimental design: The aims of the paper are in general well defined, although the second one is a bit confusing (see PDF file attached).

The analyzes are sufficient for the purposes of this work. The images are clear and self-explanatory.

The methods are short but well described and referenced.
Validity of the findings: This paper helps on the understanding of the diversification processes in seaweeds, particularly Dictyota fasciola and D. mediterranea in the Mediterranean Sea. The results obtained are also intriguing due to what were considered two distinct species, the new evidence suggests a complex of species with rich genetic diversity. In addition, this work will contribute to understanding the diversification of species on the coast of the Mediterranean Sea and Eastern Atlantic.

Here I include some comments that must be clarified.

The species complex D. fasciola and D. mediterranea.
Tronholm et al. (2010) are cited as the main reference for the taxonomic status and phylogenetic relationships of the two species. At that time, there is no evidence of any species complex and including it in the introduction is bias.

The speciation of D. mediterranea.
Tronholm et al. (2010) are cited again to test the hypothesis of speciation, diversification, and invasion of these two species. However, the authors in that paper only speculate about possible scenarios given the available data. It is also speculated that the speciation of D. mediterranea from D. fasciola occurred within the Mediterranean Sea as D. mediterranea is not distributed in the Atlantic. This should be considered for the discussion.

I feel the discussion regarding the discovery of the species complex is short. The evidence provided by Tronholm et al. showing a clear-cut between both species of Dictyota seems convincing (many molecular markers and morphology). Is there any other reason other than sampling to explain such differences?
Additional comments: In general, the manuscript is clear and well written. However, I think there are some issues that must be addressed and clarified before publication (see report and PDF file attached). In addition, the captions for Fig. 3 is exactly the same as Fig S1. Please add further descriptions to differentiate them properly

I hope the comments on this work would help to improve the manuscript.

Best regards.


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: PHYLOGEOGRAPHY OF DICTYOTA FASCIOLA AND DICTYOTA MEDITERRANEA (DICTYOTALES, PHAEOPHYCEAE): UNEXPECTED PATTERNS ON THE ATLANTIC-MEDITERRANEAN MARINE TRANSITION AND TAXONOMIC IMPLICATIONS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The revised manuscript fullfil the requirements and the authors have worked well through the comments. The present version improves the otherwise already good version submitted in the first round, by including a new phylogenetic analysis and a very comprehensive discussion, which tackles in my opinion all the relevant hypothesis that may explain the obtained results.

Please find just some minor corrections and suggestions for rephrasing in the attached pdf. Most of the rephrasing suggestions are indeed just suggestions subject to the author’s criteria. See also two comments about the methods (sect 2 experimental design). The manuscript is in my opinion almost ready for publication.
Experimental design: Lines 149-151: It is not stated how the sequence evolution models are selected. I guess it was directly done with PartitionFinder at the same time the partitions were selected.

Lines 159-162: Need to state that the partition is the same as in BI, or that has been chosen with PartitionFinder.  But why is the sequence evolution model different? Please provide some information here.
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: BIOCHEMICAL CHANGES THROUGHOUT EARLY- AND MIDDLE-STAGES OF EMBRYOGENESIS IN LOBSTERS (HOMARUS AMERICANUS) UNDER DIFFERENT THERMAL REGIMES
Review round: 1
Reviewer: 1

Output:
Basic reporting: Please see attached pdf file.
English needs improvement.
Article structure ok but Figures 1 and 2 not needed.
Discussion should be more focused and stick to actual results.
Experimental design: Please see attached pdf file.
In general, the methods should be better explained.
Not sure egg volumes and biochemical content were measured at the same times, although methods suggest this was the case.
Measurements of egg biochemical content did not extend to close to time of hatching in the "inshore" and "offshore" treatments, compromising strong conclusions about the adaptive value for embryo development of thermoregulation through active movement of ovigerous females.
Validity of the findings: Please see attached pdf file.
The data appear to be sound, although some information is unspecified.
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: BIOCHEMICAL CHANGES THROUGHOUT EARLY- AND MIDDLE-STAGES OF EMBRYOGENESIS IN LOBSTERS (HOMARUS AMERICANUS) UNDER DIFFERENT THERMAL REGIMES
Review round: 1
Reviewer: 2

Output:
Basic reporting: The basic reporting is well done. The writing style was a pleasure to read and the authors have done a good job with referencing the related background literature.

I will only make two points concerning the article structure:

1) I do not think the first two figures are really needed as they just show some photos of lab equipment.

2) I do not see why figure 4 shouldn't be incorporated as a line graph into figure 3, as it has the same predictor variables.
Experimental design: I think there are some issues with the experimental design:

1) The main issue is pseudoreplication. According to Hurlbert's review on pseudoreplication, the setup used in this experiment is classified as "isolative segregation" which is stated as one of the "various ways in which the principle of interspersion can be violated" (see Figure 1, Hurlbert, S.H., 1984. Pseudoreplication and the design of ecological field experiments. Ecological monographs, 54: 187-211). Ideally each lobster would be housed in is own separate tank and thus the eggs would be independent replicates that are randomly interspersed among other replicates. I think the authors will need to argue how their experimental setup can be valid despite the lack of interspersion of their treatments. Potentially the authors may be able to demonstrate that conditions (besides temperature) were similar among the treatments despite the fact they were isolated.

2) I had trouble following some aspects of the methods descriptions. Line 155-156 states there were 2 tanks used per treatment, and there were 3 treatments, so I was expecting 6 tanks, but line 165 states there were 4 tanks. Also, having 4 tanks does not seem to fit with having a total of 15 lobsters. Thus I was not able to work out how the experimental setup was organised, although it might become clear if the methods writing is changed/expanded and/or a diagram provided.

3) For this experimental design to work, all the treatments should have had eggs with the same level of development at the start of the experiment. From figure 3 it does seem the lipids and proteins were very similar among treatments, but in Figure 5 it seems there may have been differences in size at the 1st month. I would ask that the authors check (e.g. ANOVA) that all egg variables were statistically similar among treatments when the experiment started.

4) ANOVA assumes homogeneity of data variance, which does not appear to have been tested (e.g. Cochran's test or Levene's test). It could also be argued that as only 15 lobster were used, the sample size is quite low in which case normality of distributions will also be an important assumption to test. I would ask that the authors please address these aspects of their statistics.

5) On lines 153-155 there is a brief comment that "A subset of the eggs in each clutch were viewed under a dissecting scope and staged... These samples also served as covariates for all subsequent statistical analyses". The use of covariates is never mentioned again in the Data Analysis section nor any results concerning covariates in the results section. If covariates are used then the analysis will be an ANCOVA (not ANOVA as stated on line 223). If the analyses really did have a covariate then I would ask that there is more description of why and how this was done.

6) In the lipid and protein analyses, the constant temperature treatment was missing the last data point because the eggs hatched, and thus the design is unbalanced (i.e. May data for only 2 out of 3 treatments); was the statistical procedure used able to handle an unbalanced design? It would be my preference to have finalized the whole experiment as soon as it becomes impossible to continue getting data from one of the treatments. Overall, the fact that the design was unbalanced needs to be discussed.

7)  I would say that the regression analyses (Figure 4) are not needed, as the significant effect of time and the pairwise tests in the ANOVA analyses provide all this information.
Validity of the findings: I would say that the validity of the findings is not certain at this stage due to potential issues with pseudoreplication and analysis assumptions, and the authors will need to do more convincing for the reader that their conclusions are valid.
Additional comments: Overall, if efforts were made to present this study in a concise way then it could have only 1 figure of results and perhaps 1 table. The discussion is well written and full of background information, but much of this is loosely related to the topic at hand and if this were reduced then the manuscript could probably only be considered as a short communication. Although it may be possible to work through most of the issues about experimental design, given that there are quite a few of them, and that the volume of results is quite low, I would be hesitant to support this manuscript without substantial and possibly fundamental changes.

here are a few further minor comments:

1) It would be very useful to see statistics tables and these will help the reader work out how the experiment was set up and the analyses done.

2) Line 221: Here you mention sampling of egg volumes was done at 5 times but Fig. 5 shows only 4 sampling times. Also on Fig. 5, why are the months different to the sampling months on the other graphs?

3) Please put SE bars on all data graphs.

4) I think all ecological laboratory studies should have at least some discussion/mention about the limitations of this approach. With enough effort it would be possible to measure lobster egg development in the field which would provide much higher quality results.


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

Input:
Title: BIOCHEMICAL CHANGES THROUGHOUT EARLY- AND MIDDLE-STAGES OF EMBRYOGENESIS IN LOBSTERS (HOMARUS AMERICANUS) UNDER DIFFERENT THERMAL REGIMES
Review round: 2
Reviewer: 1

Output:
Basic reporting: Please see attached review.
Experimental design: Please see attached review.
Validity of the findings: Please see attached review.
Additional comments: Please see attached review.


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

Input:
Title: BIOCHEMICAL CHANGES THROUGHOUT EARLY- AND MIDDLE-STAGES OF EMBRYOGENESIS IN LOBSTERS (HOMARUS AMERICANUS) UNDER DIFFERENT THERMAL REGIMES
Review round: 2
Reviewer: 2

Output:
Basic reporting: I have some suggestions to improve the basic reporting:

Line 58, 59: words “both” and “alike” maybe not needed
Line 62: change “as” to “are”
Line 66: remove word “also”
Line 88: I would say the word “lab” may be too colloquial
Line 102: change “we sought to test” to “we tested”
Line 150: maybe to increase succinctness remove “and placed in labeled plastic sample trays”.
Line 211: maybe remove “very”
Figure 2: please add standard error bars to the sampling points on the line graphs
Line 217: change “and these results are summarized in Tables 1 & 2” to “(Tables 1 & 2)”
Table 1: the means in this table appear to be exactly duplicating the information on Fig 2. I think the standard errors and post-hoc tests should be presented on Fig. 2 and Table 1 removed.
Table 2 legend: please mention in the 1st sentence that the values shown are P values.
Line 236: change “The main goal of this study was” to “Our main goal was”
Line 267: change “couple” to “two”
Line 272: The use of the word “real” here is a bit confusing, I would revise.
Line 305-309: this long sentence could be split in two.
Line 384: Change “dramatically” to “significantly”
Figure 2, 3: It is confusing for the reader that bar graphs were used Fig. 3 and line graphs Fig. 2, when they both show very similar time series. I would recommend for consistency to change all time series as line graphs, unless there is some specific reason its like this.
Experimental design: I think it would be useful for some of the discussion about pseudo-replication in the response to reviewers document to be briefly mentioned somewhere in the manuscript.
Validity of the findings: Line 102-104: The hypotheses should directly match the tests done (i.e. the actual statistical comparisons). The only factors tested here were temperature and time; no measurements of migration were made. Also, the temperatures were not changed according to any migrations, they just tested how eggs would develop from lobsters staying either inshore or offshore. So the hypotheses should state something like “It was hypothesized that different water temperatures, simulated in the laboratory to be similar to temperatures experienced by lobsters in varying coastal zones (inshore vs offshore), will affect egg biochemistry. This hypothesis was tested to gain information useful for understanding how egg development may be affected by lobsters migrating between those zones”

Line 207: the lack of significant difference in temperature between inshore and offshore water was surprising and may influence the validity of the conclusions, as this study is meant to inform about lobsters migrating between these areas to expose their eggs to different temperatures. Please provide some text about the implications of the non-significant temperature result in the discussion.

Line 324-326: I think this text needs to be clarified/corrected. I guess the “constant” temperature being referred to here is the one used in this experiment, which according to line 248 appears more optimal for growth, but here it is stated as “sub-optimal”. Also, if more protein is used for energy as stated here, then there should be less rather than more protein in the “constant” treatment which is not the case (Fig. 2).

To allow the reader to understand the conclusions better, please somewhere early in the discussion provide a clear statement about whether or not the results supported the specific hypotheses from the introduction.

Line 384-385: Please mention here why “there is still the potential”, rather than just saying that the potential exists. It seems the results may indicate that no evidence could be found to support the hypothesis that “variations in the energetics of embryogenesis influenced by the seasonal movements of some lobsters to and from these two disparate locations”, especially since these disparate locations did not even have significantly different temperatures according to my understanding (line 207). Also I think the word “are” is needed between “embryogenesis” and “influenced”.
Additional comments: I think a concluding sentence in the abstract stating the relevance of the results would be useful.

Line 39: add “potentially” between “thereby” and “exposing”, especially since this study did not appear to find any significant different (Line 207) between the two areas where migrations occur between.

Line 130: Here it says the “constant” treatment was 12 degrees, but in figure 1 it seems this treatment had 16 degrees. Please correct one or the other.

Line 211-217: According to how I understood this, here all the results are from the 2 factor analyses. Firstly the text here details the main effects, then results of the interactions. The interactions are all significant, which makes it redundant to mention the main effects as was done first (i.e. effects of the factors in isolation are not informative). If this understanding is correct, then the text from line 211-215 may not be needed.

Line 300-302: a reference needs to be provided for this statement.

Line 350-378: These paragraphs are on a different topic to the study and are sufficiently unrelated that they confuse the story a bit and I would say they are probably not needed, or could be reduced to a few sentences.


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: DISSECTION OF THREE QUANTITATIVE TRAIT LOCI FOR GRAIN SIZE ON THE LONG ARM OF CHROMOSOME 10 IN RICE (ORYZA SATIVA L.)
Review round: 1
Reviewer: 1

Output:
Basic reporting: The structure of the manuscript conforms with PeerJ standards and those of the discipline. Raw data are supplied, as required by the journal.

1. However, there are many parts of the manuscript that I found difficult to read and understand, due to grammatical errors, misused words and awkward phrasings. I therefore suggest obtaining assistance from an English-language scientific editorial service.  To illustrate what I mean, here are some examples. All of the examples given are from the introduction, but  there are similar issues elsewhere in the manuscript.

Line 34: Instead of “All of them are complex traits”, I would say “All of these traits are complex” or “These traits are all complex”

Lines 45-47: Would this be better as “Causal genes have been isolated for 16 quantitative trait loci (QTL) affecting these traits. These loci are distributed across eight of the 12 chromosomes of the rice genome.”

Lines 48-50: I had to pause and reread this sentence to deduce that the ‘opposite directions’ referred to a comparison between effects on GL and effects on GW (at both loci) and not to a comparison between the effects of GW7/GL7 and GS9.

Lines 51-52 and 54-55: The comparisons should be phrased as “larger effects on X and Y than on Z”

Lines 54-55:  It should say “have large effects on TGW and GL and no effect on GW”

Lines 58-59 refer to “pathways . . . . , such as ubiquitin-proteosome, phytohormones …….” Some of these may be pathways but others (e.g. phytohormones, transcriptional regulatory factors) are not. This needs to be rephrased.

Line 60: “substantially improved understanding of” or “greatly increased the information available on” would be better. “Extremely” is too extreme. Rather than saying that information has been improved, say that understanding has been improved or that information has been increased.

2. The review of the literature on rice QTL for grain size traits seems good. However, to provide a more complete (and interesting!) rationale for the work, I suggest including some of the context about hybrid rice, which is introduced only in the Discussion.

3. The focus of the Discussion should be on the new results that are presented in this manuscript. Comments on the significance, meaning and novelty of those results can and should be supported by comparisons to other literature, but it always needs to be clear how the points discussed are relevant to the new results. Also, it was not always clear which statements are based on this work and which on the literature. This may improve with language editing.

4. The figures are appropriate but I think that they could be improved. While I don’t think PeerJ specifies line widths or fonts for figures, the use of thicker lines and a sans serif font can make figures easier to read. In Fig. 1, the blue shading of the 5 Mb scale bar is not necessary and is confusing, especially since other shades of blue are used in the legend. I assume that the black rectangles represent centromere positions, but shouldn’t this be specified? Figure 2 is very helpful, but why is one arrow dashed? All of the others are solid. Fig. 4 contains frequency histograms for continuously varying traits. There should be no gaps between the bars, and the the ‘hatching’ of the blue sections of the bars is not necessary. For easier comparison among the 15 charts in Fig. 4,  the numbers of lines could be converted to proportions of lines and all charts could be drawn on the same scale (same maximum value on the vertical axes).
Experimental design: This manuscript reports original primary research that is within the scope of the journal. One of its strengths is the rigorous approach taken in materials development.

1. While I understood the research question, I thought it could have been better defined the end of the introduction. The statement about genetic frameworks being lacking puzzled me. With many QTL having been mapped and 16 of them cloned, it seems to me that the genetic framework is pretty good!

2. The statistical methodology could be described in more detail. What was the model that was used in SAS proc GLM?
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: DISSECTION OF THREE QUANTITATIVE TRAIT LOCI FOR GRAIN SIZE ON THE LONG ARM OF CHROMOSOME 10 IN RICE (ORYZA SATIVA L.)
Review round: 1
Reviewer: 2

Output:
Basic reporting: This paper did QTL analysis using 12 populations in rice, trying to dissect a previously identified QTL region on the long arm of chr10. Three QTL were further refined within this region, with their genetic effects examined in two seperate population groups: F10 pops and F10:11 NIL sets.

This writing is clear and professional, with sufficient background and reference cited. Tables and figures are presented in a clear way. Results were described clearly and reasonable conclusions were drawn.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: L57: grammar mistake: These QTL were found to be...
L65: Please specify what specific trait each group of these QTL involved in
L69: occurred --> been observed
L78: delete "the"
L83: I don't think this is new insight, maybe you want to limit this conclusion to your specific trait and the specific QTL identified here. Also since there is possible very strong GXE between qGS10.1 and qGS10.2, it might worth mentioning.
L102: data not shown), missing a right parenthesis
L105: two others --> the other two
L108: situated --> located
L115: were followed --> followed; same for L121
L125: Which method did you use for QTL mapping? interval mapping or composite interval mapping? please specify.
L176: were used --> used
L184: I don't think you "demonstrated" the observation, maybe using some word less absolute such as "suggested" is more appropriate. same for L191.
L192: I feel expressions such as "a bit", "little" should be avoided in scientific writing.
L206: Since there is such a big discrepancy between your two populations, is it possible to test either of your two hypotheses? I actually doubt phenotypic measurement error could lead to such a big discrepancy.


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: DISSECTION OF THREE QUANTITATIVE TRAIT LOCI FOR GRAIN SIZE ON THE LONG ARM OF CHROMOSOME 10 IN RICE (ORYZA SATIVA L.)
Review round: 2
Reviewer: 1

Output:
Basic reporting: the submitted change fixed all the concerns that I brought up and I agree to accept the current version.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: no comment


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

Input:
Title: FISH NURSERY VALUE OF ALGAE HABITATS IN TEMPERATE COASTAL REEFS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript entitled " Fish nursery value of different algae habitats within temperate coastal reefs " by Hinz et al., analyses the main environmental and biological factors in shallow rocky bottoms that determines the success of recruitment of some key species of fish. The topic is interesting and the paper is a good contribution to an important aspect of the ecology of fish assemblages, essential for its adequate management and protection. The experimental design is adequate, the ms is well structured and organized and the results and conclusions are pertinent and relevant.
Therefore, I consider that the work is worthy to be published.

There are however some minor considerations that must be taken into account.

In the material and methods section, the explanation for the interpretation of PCA graphs is perhaps too detailed for a quite generalized analyses, it could be summarized and referenced.

By the contrary, the sampling design is a little bit ambiguous and could be better explained. While the general idea of the design is clear, however the sentence in line 154-156 “Some of the sampling stations were sampled in both spring and winter although focusing on settlers of different species.”, suggest that not all the samples have been used for the same analyses. This aspect should be better explained.

There are some typing mistakes, for example “Cystoceira” at line 125

There are some missing references, for example in lines 230, 246…

Some expressions should be reconsidered, for example, in line 308 “Cystoseira spp. (1-ET) decreased scientifically in height between spring and summer surveys”. It is clear that science has nothing to do with the growth of Cystoseira. Authors must better explain that the results are statistically significant.

At the end of the discussion, some considerations are confusing. It is clear that the suitability of a habitat for the maintenance of a species should not only consider the settlement of juveniles or the abundance of adults of any age, and should also consider its suitability for recruitment, but one thing should not exclude the other.

The work can have an impact since it is clear and concise and provides relevant information to understand the recruitment processes in fish populations in shallow rocky habitats
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: FISH NURSERY VALUE OF ALGAE HABITATS IN TEMPERATE COASTAL REEFS
Review round: 1
Reviewer: 2

Output:
Basic reporting: This article is an interesting approach to the role of habitat to three benthic fish settlement and juvenile survival.
The introduction is well written and focused correctly in the main issues of the study. Nevertheless, there are some aspects that should be clarified with a more detailed explanation or including some references of previous studies for a better comprehension.
Line 71: In the sentence “There survival of juvenile settlers from recruitment trough the adult life stages..” there is a little confusion between settlement and recruitment, since as it is seems that the juvenile settlers come from recruitment.
In the line 385 the authors use the term juvenile settlement, which is confusing since “juveniles” are considered already recruits, and thus have overcome the settlement and post-settlement mortality.
The article is well written, but with some typographical errors which should be corrected:
Line. 22- This instead of these
Line 89, 221, 352, 524-    space needed
Line 125-  dobble point at last of the sentence
Line 241-  (reference) need to insert a reference for this sentence
Line 319-  “scientifically” does not make sense here

The literature references are in general good and well cited. Nevertheless, there are some sentences that should be reinforced or make more clear
Line 61- I would cite also Thierret et al., 2016
Line 68- I would see a reference of some study stating this point
Line 103- Idem
Line 136- In the method section, I would see more information abou the timing period of the studies species, since there are several studies describing diferent periods of settlement, showing a certain variability in the settlement period. This is a major issue since the design of sampling for this study is based on the settlement period of the studies species.
For Diplodus vulgaris the authors do not specify the settlement period of this specie. The autors sample in spring in aims to describe the Distribution of settled individuals on the different habitat types, and also in the discussion (line 399) the authors describe a settlement period for this species during spring (but not specifying which month). But there are several studies describing the settlement period for D. vulgaris in winter, with 2 pulses, the first in November-December and the second in January-February (Vigliola et al, 1998; GarciaRubies & Macpherson 1995, Harmelin-Vivien et al.1995), also an study that the authors cite describe a settlement period for this specie from winter until march (Biaggi et al., 1998).
The same happens for Symphodus ocellatus while some authors (Raventos et al., 2009; 2005) described the settlement period in May and mid-July, not in late summer as other studies do. Other studies that the authors cite for S. ocellatus and Coris julis (Harmelin et al., 1995) do not study this species.
Thus, I believe that this point should be more discussed and clarified.
All the citations should be carefully checked, since there are some citations that do not appear in the Reference section, e.g.: Atlin et al., 2015 (line 139), Kabasakal, 2001 (line 144), Sinopoli et al., 2001 (line 145).
In the line 241 there is some reference missing.

Refecrences:
García-Rubies, A. and Macpherson, E., 1995. Substrate use and temporal pattern of recruitment in juvenile fishes of the Mediterranean littoral. Marine biology, 124(1), pp.35-42.
Harmelin-Vivien, M.L., Harmelin, J.G. and Leboulleux, V., 1995. Microhabitat requirements for settlement of juvenile sparid fishes on Mediterranean rocky shores. In Space Partition within Aquatic Ecosystems (pp. 309-320). Springer, Dordrecht.
Raventós, N. and Macpherson, E., 2005. Effect of pelagic larval growth and size-at-hatching on post-settlement survivorship in two temperate labrid fish of the genus Symphodus. Marine Ecology Progress Series, 285, pp.205-211.
Raventós, N. and Macpherson, E., 2005. Environmental influences on temporal patterns of settlement in two littoral labrid fishes in the Mediterranean Sea. Estuarine, Coastal and Shelf Science, 63(4), pp.479-487.
Vigliola, L., Harmelin-Vivien, M.L., Biagi, F., Galzin, R., Garcia-Rubies, A., Harmelin, J.G., Jouvenel, J.Y., Le Direach-Boursier, L., Macpherson, E. and Tunesi, L., 1998. Spatial and temporal patterns of settlement among sparid fishes of the genus Diplodus in the northwestern Mediterranean. Marine Ecology Progress Series, 168, pp.45-56.
Experimental design: This study contains a high amount of work, which is well designed and described.
I have only one aspect that maybe the authors can clarify. This is the algal sampling and the relationship between different algae morphotypes and juvenile fish abundances. It is not clear for me how they correlated the algal cover of the three 0.25 m2 quadrats with the 30m2 fish transects. Did the authors considered that the algal coves is homogeneous in all the transect? If not, did they observed some variability in a small scale inside the transect? This may explain the differences with other studies such as Cheminée et al 2013, 2017 and Thierret et al., 2016, which were performed in the same area of study. Maybe a deeper discussion on this aspect could clarify this point.
Validity of the findings: The findings of this study are interesting, especially the diet study of fish, that brings new knowledge for these species. For the relationship with juvenile fish and algae, in which did not find a clear pattern, the authors could better clarify comparing with the results of other similar studies performed in the area.
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: FISH NURSERY VALUE OF ALGAE HABITATS IN TEMPERATE COASTAL REEFS
Review round: 1
Reviewer: 3

Output:
Basic reporting: Some spelling and editing mistakes (also in attachments and references), but no big worries. The references are OK perhaps you can have a look on the studies on tropical seaweed beds as fish nurseries by Tano et al.  and also some studies from the great barrier reef.  Structure is OK, a bit wordy in the introduction and in the results. The hypothesis could be more elaborated and stated more clearly. The study has nothing to add when it comes to  different aspects when it comes to algae species composition and different algae habitats their potential exchangeability in a changing world as this has not been analysed/presented. However, the ecological functions of a algae habitat is studied and can be discussed. So the authors could make the outcome of the study stronger by elaborate such message.
Experimental design: The research question could be tighten and more clearly stated (see above). The research is relevant and meaningful and do fill an knowledge gap, and the investigation seems of high standard. However, some clarifications when it comes to choice of studied species and description of sites should be clarified for increased quality (see specific comments). The lack of specific algae species information and other juveniles then specific studied fish species are to be discussed and explained, as well as the choice of study depths. Also seasonal and diurnal  variations should be managed.
Validity of the findings: In line with comments above the findings are relevant and interesting however, there is a lack of further discussion putting the results into a wider perspective. What will happen  with this fish species if the Cystoseira dominated habitats (or "Cystoseira-like" habitats ) will continue to decrease?
Additional comments: The study is interesting and well performed and do fill a gap of importance in the temperate marine areas. The importance of vegetated rocky shores are stated but not understood as being under studied. The study do contribute with important findings however, the paper can be and should be revised for clarity (aim, methods and discussion) for its fully potential.


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: FISH NURSERY VALUE OF ALGAE HABITATS IN TEMPERATE COASTAL REEFS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The corrections made by the authors clarify my doubts about the contents of the article and correct typographical and grammatical errors.
Experimental design: The corrections made by the authors clarify my doubts about the contents of the article and correct typographical and grammatical errors.
Validity of the findings: The corrections made by the authors clarify my doubts about the contents of the article and correct typographical and grammatical errors.
Additional comments: The corrections made by the authors clarify my doubts about the contents of the article and correct typographical and grammatical errors.


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: FOOT PRESSURE DISTRIBUTION IN WHITE RHINOCEROSES (CERATOTHERIUM SIMUM) DURING WALKING
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: see below
Validity of the findings: see below
Additional comments: This paper examines foot pressure distribution in white Rhinos with a focus on the relative pressure produced under the footpad
Overall I think this paper presents valuable data which would be of interest to biomechanists studying foot pressure distributions.
However, there appears to be some information missing from the methods, and a lack of statistical testing of the results.
I have added some detailed comments below.

Intro
The intro seems to state that this is an exploratory study, which is fine, but it does include hypotheses which are tested. Which seems to contract the intro.
I think the authors might also comment further on the comparison between elephants and rhinos. Infact I think the study could be highly hypothesis driven if the question compares the foot pressure distributions on 5 toed elephants with three toed rhinos. Does the reduced number of toes increase peak pressures?

Methods
The methods seems to be missing a section describing how many trials were collected from each individual and foot.
From the figures we can see that a large number of trials were included - but i could not find where the exact sample size was reported.

Results
Given that there appears to be a moderately large sample size, it seems strange that no statistical analysis is attempted. The results report which pressures were the highest etc, but no indication if these were significantly higher.
Even if the statistical results are not significant, I think its still important to report them.
I suggest a linear mixed effect analysis, or ANOVA, include both ROI and foot (and speed?), with subject as a random effect.

Finally i think a better comparison could be made between elephants and rhinos. Perhaps you could allow for the different force plates used in each study by comparing the relative peak pressures to
those of the central ROI (which i think is usually the lowest?). I.e. how many percent higher is the peak ROI in elephant, compared with its central ROI, in comparison with the rhino? Does the reduction in toe number result in higher relative stresses around these digits?


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: FOOT PRESSURE DISTRIBUTION IN WHITE RHINOCEROSES (CERATOTHERIUM SIMUM) DURING WALKING
Review round: 1
Reviewer: 2

Output:
Basic reporting: The basic reporting is good. In a few cases sentences become too long for clarity (e.g., the second sentence of the introduction that begins with "Extant rhinoceroses" and spans too many ideas for a single sentence). In some places semicolons are incorrectly used in place of a comma: see lines 56, 80, 94, 197, and 228. There should be an additional semiocolon after the closed parenthetical on line 131 to complete the list. Outside of these small corrections, the language is clear throughout. The literature appears to be suitably addressed. It would be interesting to know about the evolutionary history of foot pathology in other very large terrestrial mammals (e.g. the extinct brontotheres and chalicotheres, or other extinct proboscideans), but that curiosity is obviously outside the scope of this study and the authors need not address it (I am unaware of any existing research to cite).

In Figure 1B, the labels for regions of interest are too small to be clear, even very zoomed-in. The dots should be made clearer (even if larger is not possible, they should be outlined in a brighter, contrasting color). I suggest the labels for each dot be shown outside of the foot area, with a line connecting each label box to the appropriate dot on the pressure diagram. Alternatively, separate this into a pressure diagram (as an example of data) and a separate line drawing of the feet (which can more clearly show each ROI location).

Raw data pressure data is not provided as far as I can tell, nor is code to extract ROI or COP from said data. Images of pressure and COP trajectories are provided (though these might be easier to interpret if also made into supplemental videos), as well as peak pressures, walking speed, etc at each ROI for the images provided. The data included are sufficient to evaluate the descriptions and conclusions of the paper, so I leave it to the editor whether this is sufficient sharing of raw data.

The article is a self-contained and useful contribution to the literature, with a clearly defined scope and relevant results.
Experimental design: The research question is well-defined and meaningful. The methods are clear and sufficiently described. I have no suggested changes here.
Validity of the findings: The authors clearly recognize the statistical limitations of this study due to small sample size, and they do not overstep the limits of a qualitative interpretation. Speculation is clearly noted as such, and I appreciate the power analysis indicating the number of specimens that would be necessary for certain analyses.
Additional comments: Is there any research comparing osteopathology in captive vs. wild rhinoceros? The authors do note that the evolutionary history of pathology in rhinoceros feet complicates simple husbandry questions such as whether it is even possible to prevent disease, but if such a reference exists it would be valuable to include (or to point out that, if it does not exist, it would be a valuable contribution to this exploration of rhinoceros foot health).

This is an interesting study and I look forward to seeing it published.


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

Input:
Title: FOOT PRESSURE DISTRIBUTION IN WHITE RHINOCEROSES (CERATOTHERIUM SIMUM) DURING WALKING
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: In my opinion, the authors have fully addressed the concerns from the previous round of review. I am inclined to agree with their comments about statistical hypothesis-testing and whether it is appropriate to conduct formal tests on their data, given the sample size. I think this study has clear value as an exploratory analysis, and look forward to seeing it published.


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

Input:
Title: CELLS RELEASED FROM S. EPIDERMIDIS BIOFILMS PRESENT INCREASED ANTIBIOTIC TOLERANCE TO MULTIPLE ANTIBIOTICS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript entitled "Cells released from S. epidermidis biofilms present
increased antibiotic tolerance to multiple antibiotics"  (#34906) compared the antimicrobial tolerance profiles of Brc, planktonic or biofilm cultures of S. epidermidis to different antibiotics. The data is consistent and it will provide good support for further works in the future. Moreover, the text is clear, well structured and provides sufficient basement for the purpose. Therefore, I support the article publishing with minor revisions as followed:

Brc is used as a plural acronym. So, in the line 192 it is necessary to change the verb "presents" to "present".

 "...we concluded that Brc presents an increased tolerance to several antibiotics..."
Experimental design: The experimental design is solid and consistent with the purpose of the work. However, how Brc were obtained is not very clear. So, I would like to suggest I would suggest a more detailed statement about it.

Line 108 "Brc were collected by careful aspiration of the biofilm bulk fluid 24 hours after media replacement, as described above."
Validity of the findings: The provided data is robust, statistically valid and well controlled. However, there are some typos in the text and figures regarding the symbols used to demonstrate statistical difference.

In the legend of Figure 1. is missing to cite what means "***", and there is a "g" instead gama.

"...Brc are represented with * (* p <0.05; ** p <0.01), and between Brc and their planktonic
counterparts with γ (gγγ p <0.001)."

The same happens in the legend of Figure 2.

"...and between Brc and their planktonic counterparts with γ (g p <0.05; gγ p <0.01)."
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: CELLS RELEASED FROM S. EPIDERMIDIS BIOFILMS PRESENT INCREASED ANTIBIOTIC TOLERANCE TO MULTIPLE ANTIBIOTICS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The article is well written and well illustrated in its figures.
Authors should carefully review bibliographic references, as is the case already in the first reference in which some authors' names are missing.
Experimental design: no comment'
Validity of the findings: no comment'
Additional comments: This article deals with a very important and interesting subject in microbiology, and in research on antimicrobial agents and bacterial resistance.
However the article is a simple article, and would fit well as brief communication. Their data is very well grounded and clear but although very important they indicate a lot to be initial data than would be a larger project.
It could also address a little more grounding in the introduction of respect for the importance of studying brc, as well as the importance of making an antimicrobial profile of the different types of S. epiderminidis cells


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: CELLS RELEASED FROM S. EPIDERMIDIS BIOFILMS PRESENT INCREASED ANTIBIOTIC TOLERANCE TO MULTIPLE ANTIBIOTICS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors readily accepted the reviewers' suggestions and made the necessary changes to improve the manuscript. Therefore, I recommend the publication of this paper.
Experimental design: No comments.
Validity of the findings: No comments.
Additional comments: No additional comments


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

Input:
Title: RETROTRANSPOSON-BASED GENETIC VARIATION OF POA ANNUA POPULATIONS FROM CONTRASTING CLIMATE CONDITIONS
Review round: 1
Reviewer: 1

Output:
Basic reporting: In this work, it was used inter primer binding sites (iPBS) technique for study the genetic polymorphism within selected Poa annua populations in terms of climatic conditions characteristic for the sampling sites.
Experimental design: On the content of the manuscript and the quality of the analysis - there are no comments.
Validity of the findings: However, the authors did not provide any electrophoresis pictures, therefore, the manuscript cannot be considered for publication in the current form, since the primary data is not presented.
Additional comments: Authors need to add a few pictures of electrophoresis for several iPBS primers for all Poa annua samples to assess the quality of the primary data. Otherwise, the article should be rejected.
Based on the quality of these PCR-fingerprinting pictures, it will be possible to evaluate the quality of the subsequent analysis and the work as a whole.


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: RETROTRANSPOSON-BASED GENETIC VARIATION OF POA ANNUA POPULATIONS FROM CONTRASTING CLIMATE CONDITIONS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript is considered with interesting information. Its writing is clear, and its possible publication is recommended. The information considered as the introduction, and discussion is appropriate. Materials and Methods are clear. The results are adequate, and the Tables and Figures are also appropriate. Regarding the bibliographic citations, it is suggested to attend the following observations because it is not referenced correctly:

a) there are bibliographic citations referred to throughout the text that is not in the list of references and vice versa.
b) there are citations throughout the text constituted by the three authors; however, some include the full last name of these three authors followed by the year, and in other cases only the first author is cited followed by "et al.". This latest form of citation is the one recommended in scientific literature.
c) some citations from the list of references have the year in which they were published in cursive letters and there is no reason for that.
d) check that book citations in the references list, include the pages consulted.
Experimental design: The objective of the research is clear and demonstrated with the experimental design developed. The results were analyzed in an appropriate way, which allowed the biological interpretation of them. However, the authors do not indicate in the Material and Methods sections if repetitions of the PCR reactions or the electrophoretic runs were performed to check their repeatability. It is recommended indicate this aspect.
Validity of the findings: The validation; In this case, the interpretation for the results is clear through the various analyzes carried out. The discussion was conducted correctly by comparing the results obtained in the present investigation with information from other researchers.
However, the conclusions included a bibliographical citation: the conclusions do not include citations.
Additional comments: Too many bibliographic citations were included, which makes the reading and fluency of the article is not adequate. Is it possible to suggest to the authors that they only consider those most relevant citations?


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: RETROTRANSPOSON-BASED GENETIC VARIATION OF POA ANNUA POPULATIONS FROM CONTRASTING CLIMATE CONDITIONS
Review round: 1
Reviewer: 3

Output:
Basic reporting: The basic reporting of the results in the manuscript is OK. I have a feeling that it can be improved. The language seem to be a bit of a challenge and it need to be improved to clarify many aspects of the MS.
Experimental design: The experimental design was set up very well. Enough coverage of the regions with good sample sized. It includes samples from both the native and introduced ranges which is good for a population genetic analysis and adaptation study.
Validity of the findings: The study has good results but the interpretation and validation can be improved. It doesn’t seem like the authors understand fully the kind of question they are trying to answer. So far the MS has no focal point. I feel like certain analysis and not necessary but this would be clearer if the authors can clearly state the questions and hypotheses they are testing here.
Additional comments: The manuscript entitled “Retrotransposon-based genetic variation of Poa annua populations from contrasting climate conditions” is a very interesting study which highlights both aspects of population genetics, ecology and adaptation. The authors used a good DNA marker for the investigation. The analyses were done adequately and the authors have a good understanding of the data. However, the MS lacks a focal point. Therefore, the results are hard to validate. Moreover, the DISCUSSION session needs a lot of improvement. The tables and figures also need to be improved. The English language seem to be a challenge and it needs to be improved to clarify most claims made by the authors. I feel like this is a very important study to illustrate aspects of adaptation during colonization and range expansion. But the authors didn’t discuss much about adaptation. There are a lot of grammatical errors which need to be corrected.


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: RETROTRANSPOSON-BASED GENETIC VARIATION OF POA ANNUA POPULATIONS FROM CONTRASTING CLIMATE CONDITIONS
Review round: 2
Reviewer: 1

Output:
Basic reporting: All comments have been addressed
Experimental design: The study of electrophoresis pictures of PCR samples, I can say. Of course, the author basically received adequate PCR with these primers and DNA samples. However, not everything is perfect, due to which the analysis could have this data may contain errors.
The authors could improve the quality of separation of PCR bands with a long gel and image acquisition not with a digital camera, but with more sensitive equipment, or through a red filter and a long exposure.
Validity of the findings: no comments
Additional comments: No additional comments


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

Input:
Title: RETROTRANSPOSON-BASED GENETIC VARIATION OF POA ANNUA POPULATIONS FROM CONTRASTING CLIMATE CONDITIONS
Review round: 2
Reviewer: 2

Output:
Basic reporting: My suggestions were taken care of correctly. It is only suggested to check the following: on line 260 the word " warm " is repeated.
Experimental design: No comments
Validity of the findings: No comments
Additional comments: It is only suggested to check the following: on line 260 the word " warm " is repeated.


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: RETROTRANSPOSON-BASED GENETIC VARIATION OF POA ANNUA POPULATIONS FROM CONTRASTING CLIMATE CONDITIONS
Review round: 2
Reviewer: 3

Output:
Basic reporting: The basic reporting of the results in the manuscript has been improved to my satisfaction, except for the Fig 2 which needs to be clarified. The ∆K figure is WRONG. The authors must submit the appropriate figure if this paper has to be published.
Experimental design: The experimental design was set up very well. Enough coverage of the regions with good sample sized. It includes samples from both the native and introduced ranges which is good for a population genetic analysis and adaptation study.
Validity of the findings: The study has good results and interpretation of the results has been made clear.
Additional comments: The manuscript entitled “Retrotransposon-based genetic variation of Poa annua populations from contrasting climate conditions” is a very interesting study which highlights both aspects of population genetics, ecology and adaptation. The authors used a good DNA marker for the investigation. The analyses were done adequately and the authors have a good understanding of the data. The improved MS now has a focal point. Moreover, the DISCUSSION section has been improvement adequately. The tables and figures legends also have been improved.


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: RETROTRANSPOSON-BASED GENETIC VARIATION OF POA ANNUA POPULATIONS FROM CONTRASTING CLIMATE CONDITIONS
Review round: 3
Reviewer: 1

Output:
Basic reporting: The basic reporting of the whole manuscript has been improved to my satisfaction and the paper is now clear and concise.
Experimental design: The experimental design was set up very well. Enough coverage of the regions with good sample size. It includes samples from both the native and introduced ranges which is good for a population genetic analysis and adaptation study.
Validity of the findings: The results are valid with the use of an up to date technique to answer the question.
Additional comments: The manuscript entitled “Retrotransposon-based genetic variation of Poa annua populations from contrasting climate conditions” has been improved adequately by addressing all the comments. I am satisfied with the way everything has been presented. Kindly address the very minor comments I have. Well done and all the best.


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: ORDER, PLEASE! UNCERTAINTY IN THE ORDINAL-LEVEL CLASSIFICATION OF CHLOROPHYCEAE
Review round: 1
Reviewer: 1

Output:
Basic reporting: The authors sequenced the chloroplast genome of 18 incertae sedis or deeply branching Chlorophyceae species and used a chloroplast phylogenomic approach to solve the phylogenetic relationships between Chlorophyeceae orders. Despite that three different and complementary state-of-the-art analyses were performed, the results did not converge on a single topology, but two mutually exclusive and equally likely phylogenetic scenarios were recovered. Both scenarios are thoroughly discussed, in the light as well of chloroplast genomes organization and evolution.

The manuscript is well written in clear, unambiguous and professional English and the structure conforms to PeerJ standards. Objectives, methods and results are clearly presented and the discussion is well reasoned. The figures are of high quality, appropriately described and labelled and they support and complement the text. The raw data and the custom script are available and accessible.
Experimental design: This manuscript presents original primary research within the scope of PeerJ journal. The research question is clearly defined and circumscribed and aimed at closing an existing knowledge gap. The dataset generated is meaningful and comprises of a well-balanced taxon sampling. The authors methodology is rigorous and state-of-the-art approaches were used in conformity with the prevailing technical standards in the field. The description of materials and methods are exhaustive and, together with the raw data and the scripts made available, ensure the replicability of this study.
Validity of the findings: The dataset is robust and the taxon sampling is exhaustive. Some among the best softwares available have been used for a rigorous and comprehensive data analysis (ML, Bayes, coalescence-based). However, the analyses did not converge toward a consensus topology. This is often the case in many phylogenetic studies, where similar datasets result in contrasting and alternative conclusions. Nevertheless, the authors discuss properly the likelihood of the two scenarios, and they link them with chloroplast genome organization, both based on the published literature and on the novel results presented in the manuscript. The conclusions are well stated and they link with the original question investigated.
Additional comments: I found the manuscript well written, well-structure and the methods are robust and comprehensive. I only have minor comments.

1.	In my version of the manuscripts, caption of Figure 2, Figure 3, Figure 4, Table 1 and Table 2 report “Microsoft Word – 105” at the beginning of each paragraph. I guess is a typographic misprint.

2.	I am aware that Chlorophyceae chloroplast genomes often harbor a substantial fraction of repetitive regions, which impair the performance of assemblers. I would like to suggest you, for your future studies, to use a better suited software for assembling your reads (e.g.: SPAdes, Bankevich, 2012), instead of Geneious. You may get less contigs (or even one contig) for each chloroplast genome.
You may like as well to identify additional chloroplast non-coding contigs, if present, by binning your assembly according to the k-mer composition (e.g.: with MyCC, Lin, 2016). Using this strategy, you will probably recover as well the mitochondrial genome, depending on the sequencing depth of the libraries.


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: ORDER, PLEASE! UNCERTAINTY IN THE ORDINAL-LEVEL CLASSIFICATION OF CHLOROPHYCEAE
Review round: 1
Reviewer: 2

Output:
Basic reporting: The authors investigated in this study the relationship of the green algal class Chlorophyceae at ordinal level using concatenated data set of 58 protein-coding genes. They used these plastid-coding genes to test the robustness of five 'traditional' orders (Sphaeropleales, Volvocales, Oedogoniales, Chaetophorales, Chaetopeltidales). The analyses for these tests were conducted using amino acid and nucleotide sequences.

The manuscript is mainly well-written, but starting with the title it contains sometimes sloppy terms. The background literature is not sufficient cited. The used traditional orders are not introduced, which were originally based solely on morphological and ultrastructural features. Molecular phylogenetic analyses revised these orders, but they were never formally described and alternative grouping in orders equally valid. Most phylogenetic studies using nuclear genes such as 18S and ITS are not cited (Nakada et al. 2008, Pröschold et al. 2001). The naming of the different lineages in the figures is mixed and confusing. Sometimes the authors used family names, sometimes names based on the PhyloCode and sometimes simply clades named after a representative, all not properly defined (for example, in Fig. 1, no introduction is given about the origin of these trees). The photograph shown in Fig. 1 are of low quality and should be replaced.

The main topic of this study is the ordinal structure of the Chlorophyceae. The authors demonstrated conflicting scenarios in their phylogenetic analyses using different methods and different alignments (amino acid and nucleotide) and discussed these results in the manuscript. However, no possible explanation for these conflicts is given.
Experimental design: The new chloroplast genomes of 18 relevant taxa close some gaps in phylogeny. All raw data are available as supplement. All analyses are of high standard, but several additional analyses are needed. For example, the authors found discrepancies between the phylogenies using amino acid and nucleotide alignment. Are these caused by the saturation of the third codon bases? This should be tested. It should also be mentioned which gene or taxon causes conflicts. Alternative tree topologies should be tested using approximately unbiased tests, which are implemented for example in CONSEL. In addition, it should also be demonstrated if adding of 18S data would increase the support of the topology.
A minor point: Bayesian support below 0.95 mean no support. Therefore, values below this level should be excluded in the figures and replaced by a '-'.
Validity of the findings: As mentioned above, the authors demonstrated that there is a conflict in the robustness of the orders of the Chlorophyceae. The validity of these findings is not discussed in context of other nuclear genes (18S, ITS, 28S) and non-molecular data (morphology, ultrastructure, physiology). It should also be explained why single-gene topologies are different of those of concatenated data sets. It is possible that some plastid-coding genes need to adapt faster than others or be exchanged by horizontal gene transfer as described for prokaryotes? Some explanations and conclusions should be given for these conflicts in the tree topologies.
Additional comments: By addressing the points highlighted above, the manuscript can be accepted for publication, however, present form I recommend 'Major Revisions'.


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

Input:
Title: ORDER, PLEASE! UNCERTAINTY IN THE ORDINAL-LEVEL CLASSIFICATION OF CHLOROPHYCEAE
Review round: 1
Reviewer: 3

Output:
Basic reporting: This excellent paper analyses phylogenetic relationships within the Chlorophyceae based on a taxon-rich dataset of chloroplast genomes. The dataset includes 18 newly sequenced species, and fills several important sampling gaps, in particular in the Sphaeropleales and Volvocales. The analyses support several previously inferred phylogenetic relationships within the Chlorophyceae, but a number of relationships remain unresolved, including the positions of the Treubarinia and Sphaeropleaceae.

The text is clearly written and the figures are of high quality. The introduction provides a good overview of the background and relevant literature is cited. Methods and Results are clear. Some parts of the Discussion are repeated from the Results, and could be moved to the Results section.
Experimental design: The research is novel, and research questions are well defined and relevant. It is clearly stated how the research fills several important sampling gaps for the phylogeny of Chlorophyceae, notably in the Sphaeropleales and Volvocales.

The phylogenetic analyses are of a high technical standard, and the methods are clearly described.

Details on algal culturing conditions, DNA extraction, library preparation, sequencing and annotation are not provided, but can be found in Fučíková et al. (2016), as indicated in the text. For clarity, or for those that have no access to the publication these methods could be provided in supplementary material.

One of the aims of the paper was to assess the presence/absence of genes to study the evolution of gene loss. However, it is difficult to prove the absence of a gene in incompletely assembled genomes, as the authors write in the manuscript. However, additional analyses, such as similarity searches of a gene of interest against all contigs of an assembly, could confirm or provide additional evidence for the absence of a gene.

The evolution of genomic features is based on a single tree topology. However, the phylogenetic analyses present several alternative phylogenetic hypotheses. I think the choice of topology for the analysis of genomic features should be explained more clearly.

The rationale for the SVDquartets is not entirely clear from the text. In the Methods section it is written that SVDquartets allows for site-specific rate variation in tree inference, while in the discussion it is written that SVDquartets aims to account for incomplete lineage sorting and the resulting conflict among signals from different genes. This should be clarified. Also, how relevant is incomplete lineage sorting in phylogenetic analyses inferred from plastid genome data, where all genes are (normally) linked? The authors write “Even though plastid genes are often assumed to evolve as a single locus, among-gene conflict has been demonstrated in previous studies (e.g., Fučíková, Lewis & Lewis, 2016; McManus et al., 2018). However, in these cited papers, I didn't find compelling evidence for unlinked loci in plastid genomes, and among-gene conflict does not necessarily imply that these genes are unlinked. I think think this should be better explained.
Validity of the findings: The phylogenetic data are robust, and the conclusions are well stated, and linked to the original research question.
Additional comments: Minor comments.
Abstract: “the only class whose monophyly remains uncontested as gene and taxon sampling improves.” The Chlorophyta includes several other (smaller) classes (Pedinophyceae, Chlorodendrophyceae, Mamiellophyceae, …) that are monophyletic. Perhaps specify 'core Chlorophyta'.

Fig. 3. The graphical legend (bottom left) doesn’t show the symbol for medium internode certainty.

Line 153. Sequences of 58 protein-coding chloroplast genes were aligned using the translation-aided algorithm in Geneious v.10. I suppose this is based on a given amino acid cost matrix. Please indicate the parameters 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: ORDER, PLEASE! UNCERTAINTY IN THE ORDINAL-LEVEL CLASSIFICATION OF CHLOROPHYCEAE
Review round: 2
Reviewer: 1

Output:
Basic reporting: The author made some changes in the ms and responded to my comments. However,
the naming of the lineages is still not changed and difficult to follow for a non-phycologist. Therefore, I strongly recommend to define the lineages by simply naming them according to representatives. As many publication have demonstrated, most classical orders are not monophyletic. One option to present a 18S tree only and name here the clades and orders. This tree could then replace Fig. 1.
Experimental design: no comment
Validity of the findings: All analyses are of high standard, but several additional analyses are needed. For example, the authors found discrepancies between the phylogenies using amino acid and nucleotide alignment. Are these caused by the saturation of the third codon bases? This should be tested.
Response by the authors: Saturation implies that the rate of evolution is so high that no historical signal is present. Saturated sites represent noise and thus are expected to reduce support overall. They should not strongly support any particular clade, nor should they conflict strongly with sites that have strong signal. One concern is that saturated 3rd position sites are not as affected by stabilizing selection and thus might have a different base composition than 1st or 2nd position sites. This is effectively handled by partitioning, which allows 3rd position sites to have their own nucleotide frequencies. We have explored the question of information content in 3rd vs 1st,2nd sites, but prefer to not complicate this paper by including the results. Briefly, we conducted analyses using Phycas that used a flat prior on resolution class, where resolution class is the number of internal nodes (resolution class 1 is the star tree, while the highest resolution class consists of all fully-resolved trees). If site have no information about tree topology, the posterior should also be flat across resolution classes (highest possible entropy). If sites have lots of information, all of the posterior probability is concentrated in the fully-resolved class (lowest possible entropy). This analyses shows that our 3rd position sites have more information than 2nd position sites for all genes, and, in several genes, 3rd position site have more information than even 1st position sites. Thus, the 3rd positions are not saturated, and, even if they were, the codon-level partitioning should have accommodated any deviation in nucleotide frequencies relative to 1st and 2nd position sites.

Comment: This response should be in the manuscript because that could be one reason for the discrepancies between nt and aa tree. If the 3rd position is more informative than the first two, what would be happened if you analyze these positions only. I personally do not believe that this is true. For example, when I checked randomly rbcL or psaA, most 3rd codon positions were saturated.

It should also be mentioned which gene or taxon causes conflicts.
Response by the authors: We address the ‘straggler’ taxa in our Discussion. The gene issue is visualized in the Treespace supplements. Fig. S6, and other supplementary graphs in the Treespace subfolder (esp. for groupings 1 and 4) show that the concatenation yields a very different tree from all single gene trees – that is the main problem, but not entirely unexpected. But among single gene trees there doesn’t seem to be one gene that would be completely apart and at odds with the rest.

Comment: To address this point, the authors also should analyze their data sets by non-tree based methods (i.e. TIGER, Cummins & McInerney 2011: Syst. Biol. 60: 833-844). This would discover if fast-evolving genes cause trouble by the best tree finding.
Additional comments: I still miss some explanations for the discrepancies demonstrated in the ms. I would like to see some explanations for different tree topologies. Is that caused by different evolutionary rates of genes or by biases in the analyzing methods?


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: ORDER, PLEASE! UNCERTAINTY IN THE ORDINAL-LEVEL CLASSIFICATION OF CHLOROPHYCEAE
Review round: 2
Reviewer: 2

Output:
Basic reporting: This is a solid phylogenetic study. The manuscript is clearly written and nicely illustrated. In my previous review, I only had minor comments, all of which have been satisfactory dealt with. The sequence data on GenBank are not yet available, but I trust that these will be released after publication.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: no comment


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

Input:
Title: ORDER, PLEASE! UNCERTAINTY IN THE ORDINAL-LEVEL CLASSIFICATION OF CHLOROPHYCEAE
Review round: 3
Reviewer: 1

Output:
Basic reporting: The author made some changes in the ms and responded to my comments. However,
the naming issue of my last review still remained, but I leave it now by the editor to decide if the changes made by the authors are enough for publication.
Experimental design: no comment
Validity of the findings: The authors made substantial changes and added explanations.
Additional comments: There are still issues about the naming but I leave it with editor to decide.


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: ORDER, PLEASE! UNCERTAINTY IN THE ORDINAL-LEVEL CLASSIFICATION OF CHLOROPHYCEAE
Review round: 3
Reviewer: 2

Output:
Basic reporting: In my review of the first version of the manuscript, I only had minor comments, all of which have been satisfactory dealt with.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: no comment


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

Input:
Title: DEVELOPMENT AND FEASIBILITY STUDY OF AN APP (LADLE) FOR WEIGHT LOSS AND BEHAVIOUR CHANGE
Review round: 1
Reviewer: 1

Output:
Basic reporting: - Language is appropriate.
- Literature review is sufficient and updated.
- Raw data was shared appropriately. I suggest that results on the repeated measures should be presented as a table rather than in the text (lines 316-318).
- In regards to results, I suggest the authors provide dispersion parameters of the data on weight loss (e.g., standard deviation, 95% CI, interquartile range) - lines 322-324.
Experimental design: - The article is in accordance with  the journal´s aims and scope
- The research question was well contextualized by the authors
- There was not an explicit statement about how the authors obtained informed consent from participants.
- I think the Methods need some clarification in regards to design, power to detect the expected outcomes, outcomes assessment and data analysis (see detailed comments bellow)
Validity of the findings: - It is not possible to infer about robustness of the data as the authors have not provided details on sample size or power and statistical analysis.

-Overall conclusions are supported to the results. However, the authors should acknowledge that comparisons to other studies´retention rates might not be valid since populations and interventions might differ substantially from the one in the present study i.e., was not controlled.
Additional comments: I commend that the article described in detail the rationale and development of an app targeted to people who want to lose weight. However, I think the report of the study itself and its results needs some clarification in regards to the following issues:

- The authors claim that the app is low cost. However, there is no information in the article in regards to production costs.

- In order for the readers to be able to assess the power of the study, I suggest the authors how they managed sample size issues (sample size calculations, for example)

- The authors should clarify exclusion criteria and whether the criteria for ‘staters’ and ‘finishers’ were established prior or after the intervention

- It is not clear whether outcomes on weight loss were assessed by measurements or participant´s self report.

- it is not clear in the Methods (Lines 258-262) that the comparison to other weight loss interventions was conducted in regards to results of previous works. I suggest making a clear statement on this issue to avoid misunderstanding on the design of the study.

- Description of data analysis in the paper has been too simplistic. I suggest that the authors inform the readers about statistical tests that were applied, how missing values were handled, statistical softwares that were used.

- In lines 322-324, the authors present the results on absolute weight loss. To allow a more comprehensive understanding of these numbers, I suggest that the authors provide dispersion parameters of the data (e.g., standard deviation, 95% CI, interquartile range)

-The authors should acknowledge that comparisons to other studies´retention rates might not be valid since populations and interventions might differ substantially from the one in the present study.


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

Input:
Title: DEVELOPMENT AND FEASIBILITY STUDY OF AN APP (LADLE) FOR WEIGHT LOSS AND BEHAVIOUR CHANGE
Review round: 1
Reviewer: 2

Output:
Basic reporting: The article is well written and clear with good use of English.

The literature review is a little superficial, focussing largely on a systematic review from 5 years ago, rather than reviewing more recent studies. The discussion does not describe the limitations of this evaluation and it is important to do this.

I think there is a typo either in the abstract or the method. In the abstract, it says the intervention has 36 hours of audio lessons. In the method it says 36 short audio lessons of 5 minutes. I suspect the abstract is wrong.
Experimental design: This is a really interesting intervention and it is important that more apps are developed by experts in psychology of eating behaviour and obesity and are properly evaluated. However, I have some reservations about the comparisons made in this evaluation. The choice of studies seems to be somewhat arbitrary. This is a research study rather than a service evaluation (i.e. participants are recruited by advert, must meet strict inclusion criteria, consent to participate), but the comparisons made with commercial weight loss programmes use a service evaluation (Ahern, 2011) and a service evaluation non-inferiority analysis that does not give key information used in the comparison (Madigan 2014; see Table 1). A number of other service evaluations have been conducted of Slimming World which provide the information required (e.g. Stubbs 2015 https://www.ncbi.nlm.nih.gov/pubmed/26359180) and several large trials have been conducted using Weight Watchers (Ahern 2017 https://www.thelancet.com/journals/lancet/article/PIIS0140-6736(17)30647-5/fulltext) and Slimming World (Aveyard 2016 https://www.thelancet.com/journals/lancet/article/PIIS0140-6736(16)31893-1/fulltext). What is the rationale for not using the intervention arms from the trials? In addition, I would have expected to see a comparison with the Power + trial (Little 2016 https://www.thelancet.com/journals/landia/article/PIIS2213-8587(16)30099-7/fulltext) which uses a web based intervention with nurse support. I would like to see a more comprehensive and appropriate use of comparators, but as a minimum I would like to see a justification of the comparison studies chosen in the methods.

The choice of what criteria are compared should also be justified. Currently they compare % of completers, but completion is defined differently across the studies. Eg. In this study completion is defined as 1/3 of the course, whereas in Ahern 2011, this is defined as 100% of the course and in Logue 2014 it is 1/2 the course. If you apply the Ahern 2011 criteria to this study, the completion rate is 9%, if you apply the Logue 2014 criteria it is 25%. While this is described in the paper, it is not acknowledged in the discussion of findings. Was the completion criteria defined prior to the data analysis or after examining distributions?

Weight change for all who engaged is not provided for comparison with other studies.
The discussion also focusses on % of completers losing 5%. It ignores the differences in median weight loss, which appear to be more substantial.
Validity of the findings: The findings of the evaluation are reasonably well described. The comparisons with other studies are somewhat haphazard and superficial. I would expect more rigour in terms of study selection and I would want more discussion of the limitations of comparisons across studies, including definitions of outcomes, demographic make up, recruitment methods, etc.
Additional comments: I really like the intervention and I think it has a lot of potential. I would like to see this evaluation published. However, the comparisons of effectiveness/completion rates with other studies needs to be revised to be more transparent and to acknowledge limitations.


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

Input:
Title: DEVELOPMENT AND FEASIBILITY STUDY OF AN APP (LADLE) FOR WEIGHT LOSS AND BEHAVIOUR CHANGE
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: To make it clear to the reader the design of the study and the rationale for not having a comparison group, I would suggest that the authors consider evaluating whether the study would be better described as a feasibility study. Under a broad definition, a feasibility study helps investigators prepare for full-scale research leading to intervention. I would suggest referring to the following paper to evaluate this suggestion Am J Prev Med. 2009 May; 36(5): 452–457.doi: 10.1016/j.amepre.2009.02.002
Validity of the findings: No comment
Additional comments: In my opinion, the authors have been able to perform substantial improvement and the paper is much clearer in the present version. However, I still have some comments:

1. To make it clear to the reader the design of the study and the rationale for not having a comparison group, I would suggest that the authors consider evaluating whether the study would be better described as a feasibility study. Under a broad definition, a feasibility study helps investigators prepare for full-scale research leading to intervention. I would suggest referring to the following paper to evaluate this suggestion Am J Prev Med. 2009 May; 36(5): 452–457.doi: 10.1016/j.amepre.2009.02.002

2. I suggest adding to the limitations that recent evidence suggests that the role of breakfast in weight loss is questionable – this evidence was not available by the time of the development of the behaviour change intervention. BMJ 2019;364:l42

3. Lines 390-405: Please insert citations for all the studies that are being compared to the present one.